WO2000017344A1 - RESPONSE REGULATOR $i(CaSSK1) - Google Patents
RESPONSE REGULATOR $i(CaSSK1) Download PDFInfo
- Publication number
- WO2000017344A1 WO2000017344A1 PCT/US1999/022218 US9922218W WO0017344A1 WO 2000017344 A1 WO2000017344 A1 WO 2000017344A1 US 9922218 W US9922218 W US 9922218W WO 0017344 A1 WO0017344 A1 WO 0017344A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cassklp
- casskl
- cell
- albicans
- drug
- Prior art date
Links
- 230000004044 response Effects 0.000 title claims abstract description 39
- 241000222122 Candida albicans Species 0.000 claims abstract description 107
- 238000000034 method Methods 0.000 claims abstract description 88
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 33
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 31
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 31
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 64
- 239000012634 fragment Substances 0.000 claims description 63
- 108020004414 DNA Proteins 0.000 claims description 56
- 239000003795 chemical substances by application Substances 0.000 claims description 41
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 40
- 230000000694 effects Effects 0.000 claims description 37
- 230000014509 gene expression Effects 0.000 claims description 33
- 239000013598 vector Substances 0.000 claims description 33
- 239000002773 nucleotide Substances 0.000 claims description 32
- 125000003729 nucleotide group Chemical group 0.000 claims description 32
- 150000001875 compounds Chemical class 0.000 claims description 31
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 29
- 239000000203 mixture Substances 0.000 claims description 27
- 239000003814 drug Substances 0.000 claims description 26
- 238000003752 polymerase chain reaction Methods 0.000 claims description 26
- 229940079593 drug Drugs 0.000 claims description 25
- 150000001413 amino acids Chemical class 0.000 claims description 16
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 15
- 201000003984 candidiasis Diseases 0.000 claims description 15
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 15
- 230000002401 inhibitory effect Effects 0.000 claims description 15
- 201000010099 disease Diseases 0.000 claims description 14
- 238000009396 hybridization Methods 0.000 claims description 13
- 206010007134 Candida infections Diseases 0.000 claims description 11
- 108020004511 Recombinant DNA Proteins 0.000 claims description 11
- 239000000427 antigen Substances 0.000 claims description 10
- 108091007433 antigens Proteins 0.000 claims description 10
- 102000036639 antigens Human genes 0.000 claims description 10
- 230000026731 phosphorylation Effects 0.000 claims description 10
- 238000006366 phosphorylation reaction Methods 0.000 claims description 10
- 108091033319 polynucleotide Proteins 0.000 claims description 10
- 102000040430 polynucleotide Human genes 0.000 claims description 10
- 239000002157 polynucleotide Substances 0.000 claims description 10
- 238000011282 treatment Methods 0.000 claims description 10
- 108091034117 Oligonucleotide Proteins 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 239000002299 complementary DNA Substances 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 230000002829 reductive effect Effects 0.000 claims description 8
- 230000001419 dependent effect Effects 0.000 claims description 7
- 230000028993 immune response Effects 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- 241000124008 Mammalia Species 0.000 claims description 6
- 230000007423 decrease Effects 0.000 claims description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 5
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims description 5
- 238000009007 Diagnostic Kit Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 238000002560 therapeutic procedure Methods 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 230000001737 promoting effect Effects 0.000 claims description 2
- 230000004936 stimulating effect Effects 0.000 claims description 2
- 108010033276 Peptide Fragments Proteins 0.000 claims 1
- 102000007079 Peptide Fragments Human genes 0.000 claims 1
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 238000010276 construction Methods 0.000 abstract description 7
- 238000012512 characterization method Methods 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 description 109
- 210000004027 cell Anatomy 0.000 description 103
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 61
- 102000004169 proteins and genes Human genes 0.000 description 55
- 235000018102 proteins Nutrition 0.000 description 54
- 230000006870 function Effects 0.000 description 41
- 239000002609 medium Substances 0.000 description 33
- 239000013612 plasmid Substances 0.000 description 33
- 102000004196 processed proteins & peptides Human genes 0.000 description 33
- 230000037361 pathway Effects 0.000 description 32
- 239000000523 sample Substances 0.000 description 31
- 229920001184 polypeptide Polymers 0.000 description 30
- 239000007787 solid Substances 0.000 description 26
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 24
- 229920001817 Agar Polymers 0.000 description 23
- 239000008272 agar Substances 0.000 description 23
- 238000004458 analytical method Methods 0.000 description 21
- 230000012010 growth Effects 0.000 description 20
- 210000001519 tissue Anatomy 0.000 description 20
- 108700026244 Open Reading Frames Proteins 0.000 description 17
- 235000001014 amino acid Nutrition 0.000 description 17
- 238000001514 detection method Methods 0.000 description 17
- 208000015181 infectious disease Diseases 0.000 description 17
- 108091008020 response regulators Proteins 0.000 description 17
- 229940024606 amino acid Drugs 0.000 description 16
- 230000015572 biosynthetic process Effects 0.000 description 16
- 239000007788 liquid Substances 0.000 description 16
- 230000001018 virulence Effects 0.000 description 16
- 238000003556 assay Methods 0.000 description 15
- 108010072039 Histidine kinase Proteins 0.000 description 14
- 101100022564 Schizosaccharomyces pombe (strain 972 / ATCC 24843) mcs4 gene Proteins 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 13
- 229940095731 candida albicans Drugs 0.000 description 13
- 101150032598 hisG gene Proteins 0.000 description 13
- 108700028369 Alleles Proteins 0.000 description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 12
- 239000012528 membrane Substances 0.000 description 12
- 229910052757 nitrogen Inorganic materials 0.000 description 12
- 210000002966 serum Anatomy 0.000 description 12
- 241000239290 Araneae Species 0.000 description 10
- -1 STEll Proteins 0.000 description 10
- 238000002105 Southern blotting Methods 0.000 description 10
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 10
- 239000003446 ligand Substances 0.000 description 10
- JTJMJGYZQZDUJJ-UHFFFAOYSA-N phencyclidine Chemical compound C1CCCCN1C1(C=2C=CC=CC=2)CCCCC1 JTJMJGYZQZDUJJ-UHFFFAOYSA-N 0.000 description 10
- 230000007704 transition Effects 0.000 description 10
- 239000013615 primer Substances 0.000 description 9
- 229960005486 vaccine Drugs 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 108020004705 Codon Proteins 0.000 description 8
- 101150019756 HST7 gene Proteins 0.000 description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 230000036542 oxidative stress Effects 0.000 description 8
- 239000002245 particle Substances 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 230000004083 survival effect Effects 0.000 description 8
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 7
- 108091054455 MAP kinase family Proteins 0.000 description 7
- 102000043136 MAP kinase family Human genes 0.000 description 7
- 230000000295 complement effect Effects 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 239000008121 dextrose Substances 0.000 description 7
- 239000012636 effector Substances 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- 230000009545 invasion Effects 0.000 description 7
- 210000000056 organ Anatomy 0.000 description 7
- 230000002018 overexpression Effects 0.000 description 7
- 230000035882 stress Effects 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- 230000009466 transformation Effects 0.000 description 7
- 239000003981 vehicle Substances 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 241000276498 Pollachius virens Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000007792 addition Methods 0.000 description 6
- CKLJMWTZIZZHCS-REOHCLBHSA-L aspartate group Chemical group N[C@@H](CC(=O)[O-])C(=O)[O-] CKLJMWTZIZZHCS-REOHCLBHSA-L 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 230000000877 morphologic effect Effects 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 235000002639 sodium chloride Nutrition 0.000 description 6
- 101150117862 CPP1 gene Proteins 0.000 description 5
- 101100204221 Candida albicans STE7 gene Proteins 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 239000004677 Nylon Substances 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000004520 electroporation Methods 0.000 description 5
- 230000007613 environmental effect Effects 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 210000003734 kidney Anatomy 0.000 description 5
- 229920001778 nylon Polymers 0.000 description 5
- 230000008723 osmotic stress Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000000699 topical effect Effects 0.000 description 5
- 238000013519 translation Methods 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108091035707 Consensus sequence Proteins 0.000 description 4
- 241000168726 Dictyostelium discoideum Species 0.000 description 4
- 230000005526 G1 to G0 transition Effects 0.000 description 4
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 4
- 101100246753 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) pyrF gene Proteins 0.000 description 4
- 108091092724 Noncoding DNA Proteins 0.000 description 4
- 108700005075 Regulator Genes Proteins 0.000 description 4
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 206010042938 Systemic candida Diseases 0.000 description 4
- 101150050575 URA3 gene Proteins 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 4
- 229940009098 aspartate Drugs 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 208000019164 disseminated candidiasis Diseases 0.000 description 4
- 229930182830 galactose Natural products 0.000 description 4
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 238000004811 liquid chromatography Methods 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 230000000858 peroxisomal effect Effects 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 230000037452 priming Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000009885 systemic effect Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 210000005253 yeast cell Anatomy 0.000 description 4
- YHVHQZYJGWGAKN-ZUWUZHNASA-N (3s,6s,9r,12s)-6-(4-aminobutyl)-12-benzyl-9-(1h-indol-3-ylmethyl)-3-[(1r)-1-phenylmethoxyethyl]-1,4,7,10,13-pentazacycloicosane-2,5,8,11,14-pentone Chemical compound O([C@H](C)[C@H]1C(NCCCCCCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N1)=O)CC1=CC=CC=C1 YHVHQZYJGWGAKN-ZUWUZHNASA-N 0.000 description 3
- 101150034399 CPH1 gene Proteins 0.000 description 3
- 101150009468 CST20 gene Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 101100085205 Dictyostelium discoideum ptpB gene Proteins 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 101000794228 Homo sapiens Mitotic checkpoint serine/threonine-protein kinase BUB1 beta Proteins 0.000 description 3
- 102100030144 Mitotic checkpoint serine/threonine-protein kinase BUB1 beta Human genes 0.000 description 3
- 238000000636 Northern blotting Methods 0.000 description 3
- 108010055012 Orotidine-5'-phosphate decarboxylase Proteins 0.000 description 3
- 101150063139 PTP2 gene Proteins 0.000 description 3
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 3
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 3
- 102100037248 Prolyl hydroxylase EGLN2 Human genes 0.000 description 3
- 101710170760 Prolyl hydroxylase EGLN2 Proteins 0.000 description 3
- 101100222708 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) CPR1 gene Proteins 0.000 description 3
- 101100267932 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) YRB1 gene Proteins 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 229940121375 antifungal agent Drugs 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000000680 avirulence Effects 0.000 description 3
- 238000001574 biopsy Methods 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 230000028564 filamentous growth Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000028644 hyphal growth Effects 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000005075 mammary gland Anatomy 0.000 description 3
- 239000003094 microcapsule Substances 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 230000028160 response to osmotic stress Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000010361 transduction Methods 0.000 description 3
- 230000026683 transduction Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 2
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 2
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 2
- 108020005544 Antisense RNA Proteins 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 241000701822 Bovine papillomavirus Species 0.000 description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 101100043662 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) steA gene Proteins 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108060003393 Granulin Proteins 0.000 description 2
- 102000035473 Histidine kinase domains Human genes 0.000 description 2
- 108091010875 Histidine kinase domains Proteins 0.000 description 2
- 101000628647 Homo sapiens Serine/threonine-protein kinase 24 Proteins 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 206010028034 Mouth ulceration Diseases 0.000 description 2
- 241000221961 Neurospora crassa Species 0.000 description 2
- 206010030216 Oesophagitis Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 108091027981 Response element Proteins 0.000 description 2
- 241000714474 Rous sarcoma virus Species 0.000 description 2
- 101150032846 STE12 gene Proteins 0.000 description 2
- 101100454130 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) KSS1 gene Proteins 0.000 description 2
- 101100390137 Schizosaccharomyces pombe (strain 972 / ATCC 24843) fab1 gene Proteins 0.000 description 2
- 102100026764 Serine/threonine-protein kinase 24 Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- KYOBSHFOBAOFBF-UHFFFAOYSA-N UMP Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1C(O)=O KYOBSHFOBAOFBF-UHFFFAOYSA-N 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 206010046914 Vaginal infection Diseases 0.000 description 2
- 201000008100 Vaginitis Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 230000008649 adaptation response Effects 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000843 anti-fungal effect Effects 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 238000012230 antisense oligonucleotides Methods 0.000 description 2
- 230000035578 autophosphorylation Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229920002988 biodegradable polymer Polymers 0.000 description 2
- 239000004621 biodegradable polymer Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000036755 cellular response Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 229960002086 dextran Drugs 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 230000006353 environmental stress Effects 0.000 description 2
- 208000006881 esophagitis Diseases 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 210000001508 eye Anatomy 0.000 description 2
- 238000005189 flocculation Methods 0.000 description 2
- 230000016615 flocculation Effects 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 238000010562 histological examination Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000003018 immunosuppressive agent Substances 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000000155 isotopic effect Effects 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- KYOBSHFOBAOFBF-ZAKLUEHWSA-N orotidine-5'-monophosphate Chemical compound O[C@@H]1[C@@H](O)[C@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1C(O)=O KYOBSHFOBAOFBF-ZAKLUEHWSA-N 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 229920001308 poly(aminoacid) Polymers 0.000 description 2
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000003906 pulsed field gel electrophoresis Methods 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000003938 response to stress Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 101150080291 ste7 gene Proteins 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 230000007279 water homeostasis Effects 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- JPKJQBJPBRLVTM-OSLIGDBKSA-N (2s)-2-amino-n-[(2s,3r)-3-hydroxy-1-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2r)-1-(1h-indol-3-yl)-3-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-1-oxobutan-2-yl]-6-iminohexanamide Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCC=N)[C@H](O)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C=O)C1=CC=CC=C1 JPKJQBJPBRLVTM-OSLIGDBKSA-N 0.000 description 1
- ICLYJLBTOGPLMC-KVVVOXFISA-N (z)-octadec-9-enoate;tris(2-hydroxyethyl)azanium Chemical compound OCCN(CCO)CCO.CCCCCCCC\C=C/CCCCCCCC(O)=O ICLYJLBTOGPLMC-KVVVOXFISA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- SNBCLPGEMZEWLU-QXFUBDJGSA-N 2-chloro-n-[[(2r,3s,5r)-3-hydroxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methyl]acetamide Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CNC(=O)CCl)[C@@H](O)C1 SNBCLPGEMZEWLU-QXFUBDJGSA-N 0.000 description 1
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 description 1
- FVFVNNKYKYZTJU-UHFFFAOYSA-N 6-chloro-1,3,5-triazine-2,4-diamine Chemical group NC1=NC(N)=NC(Cl)=N1 FVFVNNKYKYZTJU-UHFFFAOYSA-N 0.000 description 1
- XZIIFPSPUDAGJM-UHFFFAOYSA-N 6-chloro-2-n,2-n-diethylpyrimidine-2,4-diamine Chemical compound CCN(CC)C1=NC(N)=CC(Cl)=N1 XZIIFPSPUDAGJM-UHFFFAOYSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 108020004491 Antisense DNA Proteins 0.000 description 1
- 101100532518 Arabidopsis thaliana SAHH1 gene Proteins 0.000 description 1
- 108010024976 Asparaginase Proteins 0.000 description 1
- 102000015790 Asparaginase Human genes 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 101150020019 CLA4 gene Proteins 0.000 description 1
- 102100031277 Calcineurin B homologous protein 1 Human genes 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108090000489 Carboxy-Lyases Proteins 0.000 description 1
- 102000004031 Carboxy-Lyases Human genes 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 241000839426 Chlamydia virus Chp1 Species 0.000 description 1
- 206010061764 Chromosomal deletion Diseases 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 101100457919 Drosophila melanogaster stg gene Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102100037642 Elongation factor G, mitochondrial Human genes 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 102000005731 Glucose-6-phosphate isomerase Human genes 0.000 description 1
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 102000000587 Glycerolphosphate Dehydrogenase Human genes 0.000 description 1
- 108010041921 Glycerolphosphate Dehydrogenase Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101150096276 HOG1 gene Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- 101000777252 Homo sapiens Calcineurin B homologous protein 1 Proteins 0.000 description 1
- 101000943802 Homo sapiens Cysteine and histidine-rich domain-containing protein 1 Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 238000012695 Interfacial polymerization Methods 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- FDIKHVQUPVCJFA-YFKPBYRVSA-N L-Phosphohistidine Chemical group OP(=O)(O)N[C@H](C(=O)O)CC1=CN=CN1 FDIKHVQUPVCJFA-YFKPBYRVSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 229910009891 LiAc Inorganic materials 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 239000004907 Macro-emulsion Substances 0.000 description 1
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010059724 Micrococcal Nuclease Proteins 0.000 description 1
- 102000013967 Monokines Human genes 0.000 description 1
- 108010050619 Monokines Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 208000007027 Oral Candidiasis Diseases 0.000 description 1
- 101100491257 Oryza sativa subsp. japonica AP2-1 gene Proteins 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010034016 Paronychia Diseases 0.000 description 1
- 241000029132 Paronychia Species 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108010085186 Peroxisomal Targeting Signals Proteins 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 102000001105 Phosphofructokinases Human genes 0.000 description 1
- 108010069341 Phosphofructokinases Proteins 0.000 description 1
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 1
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 1
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010011939 Pyruvate Decarboxylase Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 101150033582 RSR1 gene Proteins 0.000 description 1
- 208000035415 Reinfection Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 101150020516 SLN1 gene Proteins 0.000 description 1
- 235000003534 Saccharomyces carlsbergensis Nutrition 0.000 description 1
- 241001123227 Saccharomyces pastorianus Species 0.000 description 1
- 101100395426 Schizosaccharomyces pombe (strain 972 / ATCC 24843) sty1 gene Proteins 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 1
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 238000010162 Tukey test Methods 0.000 description 1
- 241000287411 Turdidae Species 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 101150102488 YPD1 gene Proteins 0.000 description 1
- 101100127670 Zea mays LA1 gene Proteins 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 229940127024 acid based drug Drugs 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical class C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 108010004469 allophycocyanin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229940025131 amylases Drugs 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000002141 anti-parasite Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003816 antisense DNA Substances 0.000 description 1
- 230000009118 appropriate response Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 229960003272 asparaginase Drugs 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- DQXBYHZEEUGOBF-UHFFFAOYSA-N but-3-enoic acid;ethene Chemical compound C=C.OC(=O)CC=C DQXBYHZEEUGOBF-UHFFFAOYSA-N 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229960002713 calcium chloride Drugs 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 101150069072 cdc25 gene Proteins 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000025084 cell cycle arrest Effects 0.000 description 1
- 230000006369 cell cycle progression Effects 0.000 description 1
- 230000009134 cell regulation Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000006552 constitutive activation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 230000021953 cytokinesis Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000459 effect on growth Effects 0.000 description 1
- 101150016954 efg1 gene Proteins 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000029578 entry into host Effects 0.000 description 1
- 235000021321 essential mineral Nutrition 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- ZFKJVJIDPQDDFY-UHFFFAOYSA-N fluorescamine Chemical compound C12=CC=CC=C2C(=O)OC1(C1=O)OC=C1C1=CC=CC=C1 ZFKJVJIDPQDDFY-UHFFFAOYSA-N 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000009395 genetic defect Effects 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 101150100117 gfm1 gene Proteins 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 244000052637 human pathogen Species 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 1
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 1
- 229940031574 hydroxymethyl cellulose Drugs 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 239000000815 hypotonic solution Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 239000000367 immunologic factor Substances 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 229940125721 immunosuppressive agent Drugs 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000001325 log-rank test Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000005497 microtitration Methods 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000009826 neoplastic cell growth Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000000426 osmoregulatory effect Effects 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 235000016236 parenteral nutrition Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 210000002824 peroxisome Anatomy 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 229960002816 potassium chloride Drugs 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 229950008679 protamine sulfate Drugs 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 108010030416 proteoliposomes Proteins 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229960004249 sodium acetate Drugs 0.000 description 1
- 229960002668 sodium chloride Drugs 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000001540 sodium lactate Substances 0.000 description 1
- 235000011088 sodium lactate Nutrition 0.000 description 1
- 229940005581 sodium lactate Drugs 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 229940035044 sorbitan monolaurate Drugs 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000000021 stimulant Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000010399 three-hybrid screening Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 229940117013 triethanolamine oleate Drugs 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229960001322 trypsin Drugs 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
- C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to nucleic acid molecules encoding response regulator CaSskl protein including mutants, variants, fragments and derivatives thereof, and to vectors and host cells comprising such nucleic acid molecules; methods of using response regulator; method for screening for inhibitors of response regulator; and kits comprising the compositions or polypeptides of the invention.
- the sensor histidine kinases and their corresponding response regulators which were first described in prokaryotes in phosphotransfer pathways referred to as ' two-component systems ' , enable cells to detect environmental changes and elicit an appropriate response
- the prototypical prokaryotic two- component regulator system is comprised of two proteins, a histidine protein kinase (or sensor protein) and a response regulator (or effector protein) , which is associated with an internal response.
- the sensor kinase when activated by an environmental signal, autophosphorylates a histidine residue, which then serves as a phosphodonor to a conserved aspartate residue in the response regulator. This phophorylation modulates the activity of the effector protein so as to elicit an adaptive response to the stimulus .
- the prokaryotic response regulators are characterized by a conserved domain of approximately 125 amino acids, usually attached via a linker sequence to a domain with an effector function.
- the effector domain generally acts as a transcription factor.
- a different type of effector domain is attached to a response regulator (Hoch and
- a response regulator domain is located at the C-terminus of Slnlp, a transme brane hybrid protein that serves as an osmosensor (Ota and Varshavsky, 1993, Science 262, 566-569) which, after autophosphorylation of a histidine residue in its histidine kinase domain and transfer to an aspartate in its response regulator domain, phosphorylates a histidine residue of Ypdlp (Posas et al . , 1996, supra) . This protein, in turn, phosphorylates Ssklp, the other respnse regulator (Maeda et al .
- the Mcs4 response regulator from Schizosaccharomyces pombe is an essential component of the Wakl-Wisl-Styl MAP kinase pathway, through which Mcs4 coordinates cell cycle progression with the cellular respnse to environmental stresses such as osmotic and oxidative stress, heat shock and nutritional limitations (Cottarel, 1997, Genetics 147, 1043-1051; Shieh et al . , 1997, Genes Dev 11, 1008-1022; Shiozaki et al . , 1997, Mol Biol Cell 8, 409-419) .
- Candida albicans Three hybrid histidine kinase genes have been reported in Candida albicans, including CaSLNl (Nagahashi et al . , 1998, Microbiology 144 , 425-432), CaNIKl/COSl (Nagahashi et al . , 1998, supra; Srikantha et al . , 1998, Microbiology 144, 2715-2729; Alex et al . , 1998, Proc Natl Acad Sci USA 93, 7069-7073) and CaHKl (Calera et al . , 1998, Yeast 14, 665-674).
- the CaSLNl and CaNIKl encoded proteins, CaSlnlp and CaNiklp, show structural and functional homology with the proteins Slnlp and Nikl from S. cerevisiae and N. crassa , respectively.
- the CaHKl encoded protein, Cahklp while homologous at its C-terminus with the histidine kinase domains of many sensor proteins, at its N-terminus, only shows homology with a putative histidine kinase from S. pombe (GenBank Accession No. Z98978) with unknown function.
- Ssklp, Skn7p and Mcs4 have a large N-terminal domain, and the conserved aspartate residues in their response regulators are located towards the C-terminus and are not followed by a domain with an effector function.
- CaSSKl pathogenic yeast C. albicans
- CaSsklp encoded protein
- Candida albicans is the most frequently isolated opportunistic fungal pathogen in humans.
- Candidiasis is a general- term for a variety of local and systemic processes caused by colonizationor infection of the host by species of the yeast Candida.
- Candidiasis occurs worldwide; superficial infections of the skin, mouth and other mucus membranes are universal .
- Invasive systemic disease has become a problem due to the use of high doses of antibiotics that destroy normal bacterial flora, immunosuppressive agents, and agents toxic to bone marrow, e.g. during cancer therapy.
- Candidiasis is also seen among immunocompromised individuals such as AIDS patients, organ transplant patients, patients receiving parenteral nutrition, and cancer patients receiving parenteral and chemotherapy.
- disease ranges from aggressive local infections such as peiodontitis, oral ulceration, or esophagitis in HIV-infected patients, to complex and potentially lethal infections of the bloodstream with subsequent dissemination to brain, eye, heart, liver, spleen, kidneys, or bone.
- the infection typically begins at an epithelial site, evades local defenses, and invades the bloodstream in the face of immunosuppression.
- disease caused by C. albicans ranges from mild, easily treated, superficial disease (e.g., thrush in newborn infants; paronychia in workers whose hands are immersed in water) to more severe, chronic or recurrent infections (e.g., candidal vaginitis) .
- Candida species occur in two forms that are not temperature- or host-dependent .
- the usual colonizing forms are yeasts that may assume a pseudomycelial configuration, especialy during tissue invasion. Pseudomyceliae result from sequential budding of yeasts into branching chains of elongated organisms.
- C. albicans A number of factors have been associated with the virulence properties of C. albicans, such as adherence to host cells and the ability to undergo the transition from yeast to hyphal growth. This switch is induced in vi tro by many environmental conditions including temperature, pH and the presence of serum. Diploid strains of Saccharomyces cerevisiae also switch their pattern of growth from unicellular yeast to chains of elongated cells that remain attached to each other (pseudohyphae) under conditions of nitrogen starvation (Gimeno, et al . , 1992, Cell 68, 1077- 1090) .
- This switch requires STE20, STEll , STE7 , KSS1 and STE12 , genes of a conserved MAP kinase pathway, but also depends on PHD1 , a gene which functions in a STE12- independent pathway (Banuett, 1998, Mol Biol Rev 62, 249- 274) .
- the homolog genes CST20 Kelvan and Fink, 1996, Proc Natl Acad Sci USA 93, 13223-13228; Leberer et al .
- the CPP1 gene which encodes a phosphatase similar to the MAP kinase phosphatase Msg5p of S. cerevisiae, modulates the activity of the CPHl- pathway likely by dephosphorylating Ceklp (Csank et al . , 1997, Mol Biol Cell 8, 2539-2551).
- the C. albicans genes CaCLA4 (a CLA4 homolog) (Leberer et al . , 1997, Curr Biol 1 , 539-546), EFG1 (a PHD1 homolog) (Lo et al .
- CaSSKl encodes a protein (CaSsklp) which is a structural homolog of Ssklp and Mcs4 from S. cerevisiae and S. pombe, respectively.
- Ssklp is a response regulator of the "two-component" cascade that regulates the HOG pathway of S. cerevisiae (Maeda et al .
- Mcs4 is an element of the Styl pathway of Schizosaccharomyces pombe (Cottarel, 1997, Genetics 147, 1043-1051; Shieh et al . , 1991 , Genes Dev 11, 1008-1022). Unlike Ssklp which only functions in osmosensing in S. cerevisiae, Mcs4 plays a role in regulating the adaptive responses to different stresses including osmotic and oxidative stress, and coordinates the responses to these environmental stimuli with the cell cycle (Cottarel, 1997, supra; Shieh et al . , 1997, supra) .
- CaSSKl fails to complement the lack of either SSK1 or mcs4 + in S. cerevisiae and S. pombe, respectively (Calera and Calderone, 1999, supra) . This indicated that CaSSKl may have other functions in C. albicans rather than modulating the response to osmotic or oxidative stress. In this paper, we present data indicating that CaSSKl is involved in the morphogenesis and virulence of C. albicans .
- CaSSKl a response regulator two- component gene from Candida albicans
- the CaSSKl gene was cloned following a PCR based approach as described below.
- CaSSKl has an open reading frame of 2022 base pairs (bp) .
- bp base pairs
- STRE stress response elements
- CaSSKl is located on chromosome 1 and is expressed in either yeast or mycelial phases of C. albicans .
- CaSSKl encodes a 674 amino acid protein (CaSsklp) with an estimated molecular mass of 73.5 kDa and a basic isoelectric point (pi 9.5). It has a tripeptide (NKA) located in its C-terminus, which resembles the peroxisomal signalling target type 1 sequence (PSTl) of most of the peroxisomal matrix proteins .
- a homology search of CaSsklp with other proteins in databases showed that the C-terminus of CaSsklp exhibits the greatest similarity with the C-terminus of Ssklp and Mcs4 from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively.
- the response regulator domain of CaSsklp contains the motifs that are characteristic of all response regulators, including the conserved aspartate and lysine residues as well as the putative aspartate, which is phophorylated by a phosphohistidine residue. Finally, in spite of the structural similarities among CaSsklp, Ssklp and Mcs4, CaSsklp does not seem to exhibit functional homology with these proteins .
- the Accession No. for the described sequence is AF084608, as filed in the EMBL/GenBank/DDBJ database.
- DNA fragment of about 3058 bp (SEQ ID NO:l) containing a 5' upstream promotor region of 486 bp, an open reading frame of 2022 bp encoding CaSsklp, and a 3' downstream non-coding region.
- the DNA fragment is useful as a diagnostic agent, an agent for preparation of CaSsklp, and a therapeutic agent.
- the inhibition can be at the DNA level by introducing mutations into the gene encoding CaSSKl , by inhibiting transcription of the gene, by inhibiting translation of the RNA encoding CaSsklp, or by inhibiting the function of CaSsklp.
- Candida albicans said method comprising administering to an individual in need of such treatment an effective amount of an inhibitor of CaSsklp expression or function in a pharmaceutically acceptable diluent or excipient.
- FIG. 1A and IB (A) Northern blot of total RNA. RNA samples obtained from cultures grown in medium 199 for 3 hours at 28'C (pH 3.5) (lane Y) and at 37 °C (pH 7.5) (lane M) , to induce yeast and hyphal growth, respectively. (B) CHEF gel analysis. It was performed with duplicate samples . The chromosomal bands were visualized with ethidium bromide (left panel) and a Southern blot of the gel (right panel) was hybridized with a 1.85 kb EcoRI-Bglll fragment of the CaSSKl gene as a probe. C. albicans chromosomal designations (on the left side) are those proposed by Wickes et al . (1991, Infect . Immun . 59, 1762-1771).
- FIG. 2A and 2B (A) Complementation analysis of a S. pombe mcs4-13 cdc25-22 double mutant with CaSSKl (pBREP42-CaSSKl) . This S. pombe strain was also transformed with the plasmids pREP42-mcs4 as a positive control and the plasmids pREP42 and pBREP42 as negative controls . Transformants were grown in EMM minimal medium at either 25°C or 30°C for four days. (B) Complementation analysis of a S. cerevisiae SSKl SLN1 double mutant with CaSSKl (pBRS415-CaSSKl) .
- This strain was also transformed with the plasmids pRS415- SSK1 as a positive control and the plasmids pBRS415 and pBRS415-p/t as negative controls. Transformants (1.5 X 10 4 cells per spot) were grown in presence of either galactose or glucose for two days at 30°C.
- FIG. 3A, 3B, and 3C (a) Schematic representation of the construction of the cassette used to disrupt CaSSKl and, (b) the cassette CaSSKl -URA3 -hi sG used to reintroduce one wild type CaSSKl allele.
- (c) is shown the corresponding southern blot analyses of strains CSSKll-1, CSSK12-1, CSSK21-1 and CSSK22-1 obtained during the disruption process and a revertant strain (CSSK23-1) . Genomic DNA from these strains was Bglll digested, and hybridized with a 1.4 kb Nsil-EcoRI fragment located at the 5 ' -end of the CaSSKl gene as a probe. The exact size and genotype of the expected hybridizing DNA fragments are indicated on the right .
- Figure 4. Broth cultures of Ura3 + Acasskl mutants
- strains CSSKll-1, CSSK21-1 and CSSK23-1 Stationary-phase cells from all strains were inoculated (10 7 cells/ml) in M199 (pH 7.5), grown at 37°C for 3 hours and photographed.
- the CSSK21 null strains flocculated extensively forming clumps of cells that sedimented rapidly to the bottom of the tubes after the cell suspension was shaken. The formation of clumps was not observed in the heterozygote and reconstituted strains.
- FIG. 7 Survival of mice following infection with C. albicans Acasskl mutants. Only the survival curves of mice infected with CAF2 , CSSKll-1, CSSK21-1 and CSSK23-1 are shown. Similar results were observed with mice infected with strains CSSK21-2 and CSSK23-2.
- FIG. 8 Hypothetical scheme of the location of CaSsklp in a putative two-component pathway of C. albicans based on the phenotypic characterization of the Acahkl , Acasskl and Acanikl/cosl mutants.
- the dashed arrows indicate the putative transduction of the signal by CaNiklp/Coslp and the solid arrows the transduction of the signal by Cahklp.
- the present invention relates to a DNA or cDNA segment which encodes CaSSKlp from Candida albicans .
- a PCR reaction using an appropriate pair of degenerate primers was performed and a 447 bp PCR product was identified as a partial DNA sequence of CaSSKl .
- This PCR fragment was then used to isolate the entire CaSSKl gene.
- the CaSSKl DNA fragment has an open reading frame of 2022 bp in addition to the 5' promoter region and 3' nontranslated region.
- the complete 3058 bp CaSSKl DNA fragment is specified in SEQ ID NO:l.
- one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from (a) a nucleotide sequence comprising a sequence encoding a full length CaSsklp polypeptide having the sequence specified in SEQ ID NO:l, (b) a nucleotide sequence which encodes the complete amino acid sequence in SEQ ID NO: 2, or the complete amino acid sequence encoded by the cloned DNA in GenBank accession no. AF084608.
- isolated nucleic acid molecules of the invention include DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode CaSsklp or fragments thereof.
- the genetic code and species-specific codon preferences are well known in the art.
- Nucleic acid molecules of the present invention may be in the form of RNA, such as mR ⁇ A, or in the form of D ⁇ A, including, for instance, cD ⁇ A and genomic D ⁇ A obtained by cloning or produced synthetically.
- the D ⁇ A may be double-stranded or single-stranded.
- Single-stranded D ⁇ A or R ⁇ A may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the antisense strand.
- isolated nucleic acid molecule is intended a nucleic acid molecule, D ⁇ A or R ⁇ A, which has been removed from its native environment.
- recombinant D ⁇ A molecules contained in a vector are considered isolated for the purposes of the present invention.
- Further examples of isolated D ⁇ A molecules include recombinant D ⁇ A molecules maintained in heterologous host cells or purified (partially or substantially) D ⁇ A molecules in solution.
- Isolated R ⁇ A molecules include in vivo or in vitro R ⁇ A transcripts of the D ⁇ A molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
- the present invention is further directed to nucleic acid molecules encoding portions or fragments of the nucleotide sequences described herein.
- Fragments include portions of the nucleotide sequence of SEQ ID NO:l or at least 10 contiguous nucleotides in length selected from any two integers , one of which representing a 5' nucleotide position and a second of which representing a 3' nucleotide position, where the first nucleotide for each nucleotide sequence is position 1. That is, every combination of a 5 ' and 3' nucleotide position that a fragment at least 10 contiguous nucleotide bases in length or any interger between 10 and the length of an entire nucleotide sequence of CaSSKl minus 1.
- the invention includes polynucleotides comprising fragments specified by size, in nucleotides, rather than by nucleotide positions.
- the invention includes any fragment size, in contiguous nucleotides, selected from intergers between 1- and the entire length of an entire nucleotide sequence minus 1.
- Preferred sizes include 20-50 nucleotides, 50-300 nucleotides useful as primers and probes.
- Regions from which typical sequences may be derived include but are not limited to, for example, regions encoding specific epitopes or domains within said sequence, such as the region comprising nucleotides 1599-2115 of SEQ ID NO: 1.
- the invention provides isolated nucleic acid molecules comprising polynucleotides which hybridize under stringent hybridization conditions to a polynucleotide sequence of the present invention described above, or a specified fragment thereof.
- stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate) , 50 mM sodium phosphate (pH 7.6) , 5X Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured sheared salmon sperm DNA, followed by washing the filters in 0. IX SSC at about 65°C.
- sequences encoding the polypeptides of the present invention or portions thereof may be fused to other sequences which provide additional functions known in the art such as a marker sequence, or a sequence encoding a peptide which facilitates purification of the fused polypeptide, peptides having antigenic determinants known to provide helper T-cell stimulation, peptides encoding sites for post- tranlational modifications, or amino acid sequences which target the fusion protein to a desired location, e.g. a heterologous leader sequence.
- the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the CaSsklp polypeptides.
- Variant may occur naturally, such as a natural allelic variant.
- allelic variant is intended one of several alternate forms of a gene occupying a given locus of a chromosome of an organism.
- Non-naturally occuring variants may be produced by known mutagenesis techniques.
- Such variants include those produced by nucleotide substitution, deletion, or addition of one or more nucleotides in the coding or noncoding regions or both. Alterations in the coding regions may produce conservative or nonconservative amino acid substitutions, deletions, or additions. Especially preferred among these are silent substitutions, additions, and deletions which do not alter the properties and activities of CaSsklp polypeptides disclosed herein or portions thereof . Also preferred in this regard are conservative substitutions .
- Nucleic acid molecules with at least 90-99% identity to a nucleic acid shown in SEQ ID NO:l is another aspect of the present invention. These nucleic acids are included irrespective of whether they encode a polypeptide having CaSsklp activity.
- a polypeptide having CaSsklp activity is intended polypeptides exhibiting activity similar, but not identical, to an activity of the CaSsklp of the invention, as measured in the assays described below.
- the biological acitivity or function of the polypeptides of the present invention are expected to be similar or identical to polypeptides from other organisms that share a high degree of structural identity/similarity.
- the present invention relates to a recombinant DNA molecule that includes a vector and a DNA sequence as described above.
- the vector can take the form of a plasmid, phage, cosmid, YAC, eukaryotic expression vector such as a DNA vector, Pichia pastoris, or a virus vector such as for example, baculovirus vectors, retroviral vectors or adenoviral vectors, and others known in the art.
- the cloned gene may optionally be placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences , or sequences which may be inducible and/or cell type-specific.
- Suitable promoters will be known to a person with ordinary skill in the art.
- the expression construct will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
- the vectors preferred for use include pGEX-YT3 , YPB11-ADH to name a few.
- the construct into the host cell can be effected by calcium phosphate transfection, electroporation, infection, and other methods known in the art and described in standard laboratory manuals such as Current Protocols in Molecular Biology, Ausubel, F. M. et al . (Eds), Wiley & Sons, Inc. or Sherman et al . , 1986, Methods in Yeast Genetics . Cold Spring Harbor Laboratory Press, New York. All documents cited herein supra and infra are hereby incorporated in their entirety by referece thereto.
- the present invention relates to host cells stably transformed or transfected with the above-described recombinant DNA constructs.
- the host cell can be prokaryotic (for example, bacterial) , lower eukaryotic (for example, yeast or insect) or higher eukaryotic (for example, all mammals, including but not limited to rat and human) . Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control sequences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites.
- Transfer vectors compatible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers . These markers may be used to obtain successful transformants by selection. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985) for general cloning methods .
- the DNA sequence can be present in the vector operably linked to a sequence encoding an IgG molecule, an adjuvant, a carrier, or an agent for aid in purification of CaSsklp, such as glutathione S- transferase, or a series of histidine residues also known as a histidine tag.
- the recombinant molecule can be suitable for transfecting eukaryotic cells, for example, mammalian cells and yeast cells in culture systems. Saccharomyces cerevisiae, Saccharomyces carlsbergensis , and Pichia pastoris are the most commonly used yeast hosts, and are convenient fungal hosts. Control sequences for yeast vectors are known in the art.
- Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) , such as HEK293 cells , and NIH 3T3 cells, to name a few.
- Suitable promoters are also known in the art and include viral promoters such as that from SV40, Rous sarcoma virus (RSV) , adenovirus (ADV) , bovine papilloma virus (BPV) , and cytomegalovirus (CMV) .
- suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolic enzymes, such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate.
- Mammalian cells may also require terminator sequences and poly A addition sequences; enhancer sequences which increase expression may also be included, and sequences which cause amplification of the gene may also be desirable. These sequences are known in the art.
- the transformed or transfected host cells can be used as a source of DNA sequences described above. When the recombinant molecule takes the form of an expression system, the transformed or transfected cells can be used as a source of the protein described below.
- the present invention relates to a CaSsklp protein having an amino acid sequence corresponding to SEQ ID NO: 2 (GenBank accession no. AF084608) and encompassing 674 amino acids or any allelic variation thereof or biologically active derivative thereof .
- a polypeptide or amino acid sequence derived from the amino acid sequences mentioned above refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 2-5 amino acids, and more preferably at least 8- 10 amino acids, and even more preferably at least 11- 15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence .
- a "biologically active derivative thereof” is a CaSsklp that is modified by amino acid deletion, addition, substitution, or truncation, or that has been chemically derivatized, but that nonetheless functions in the same manner as the protein of SEQ ID NO: 2.
- substitutions of aliphatic amino acids such as alanine, valine, and isoleucine with other aliphatic amino acids can often be made without altering the structure or function of a protein.
- substitution of aspartic acid for glutamic acid, in regions other than the active site of an enzyme are likely to have no appreciable affect on protein structure or function.
- fragment is meant to refer to any polypeptide subset. Fragments can be prepared by subjecting C.
- albicans proteins to the action of any one of a number of commonly available proteases, such as trypsin, chymotrypsin or pepsin, or to chemical cleavage agents, such as cyanogen bromide.
- variant is meant to refer to a molecule substantially similar in structure and function to either the entire CaSSKl or to a fragment thereof .
- a protein or peptide is said to be 'substantially similar' if both molecules have substantially similar amino acid sequences, preferably greater than about 80% sequence identity, or if the three-dimensional backbone structures of the molecules are superimposable, regardless of the level of identity between the amino acid sequences.
- two molecules possess similar activity they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequences of amino acid residues are not identical.
- 'analog' is meant to refer to a protein that differs structurally from the wild type CaSsklp, but possesses similar activity.
- a recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence; it may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system.
- the polypeptide can be fused to other proteins or polypeptides which increase its antigenicity, such as adjuvants for example.
- the methods of the present invention are suitable for production of any polypeptide of any length, via insertion of the above- described nucleic acid molecules or vectors into a host cell and expression of the nucleotide sequence encoding the polypeptide of interest by the host cell.
- Introduction of the nucleic acid molecules or vectors into a host cell to produce a transformed host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods .
- Such methods are described in many standard laboratory manuals, such as Davis et al . , Basic Methods In Molecular Biology (1986) .
- Transformations into yeast are typically carried out according to the method of Van Solingen et al . , 1977, J. Bact . , 130, 946 and Hsiao et al . 1979, Proc Natl Acad Sci USA 76, 3829-3833.
- the cells may be cultivated under any physiologically compatible conditions of pH and temperature, in any suitable nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals that support host cell growth.
- Recombinant polypeptide-producing cultivation conditions will vary according to the type of vector used to transform the host cells.
- certain expression vectors comprise regulatory regions which require cell growth at certain temperatures, or addition of certain chemicals or inducing agents to the cell growth medium, to initiate the gene expression resulting in the production of the recombinant polypeptide.
- the term "recombinant polypeptide-producing conditions," as used herein, is not meant to be limited to any one set of cultivation conditions .
- Appropriate culture media and conditions for the above-described host cells and vectors are well-known in the art.
- the polypeptide of interest may be isolated by several techniques. To liberate the polypeptide of interest from the host cells, the cells are lysed or ruptured.
- This lysis may be accomplished by contacting the cells with a hypotonic solution, by treatment with a cell wall-disrupting enzyme such as lysozyme, by sonication, by treatment with high pressure, or by a combination of the above methods .
- a cell wall-disrupting enzyme such as lysozyme
- sonication by treatment with high pressure, or by a combination of the above methods .
- Other methods of bacterial cell disruption and lysis that are known to one of ordinary skill may also be used.
- the polypeptide may be separated from the cellular debris by any technique suitable for separation of particles in complex mixtures. The polypeptide may then be purified by well known isolation techniques .
- Suitable techniques for purification include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC) , high performance LC (HPLC) , fast performance LC (FPLC) , hydroxylapatite chromatography and lectin chromatography.
- LC liquid chromatography
- HPLC high performance LC
- FPLC fast performance LC
- the recombinant or fusion protein can be used as a diagnostic tool and in a method for producing antibodies against CaSsklp, detectably labeled and unlabeled, or as a bait protein in the yeast 2-hybrid assay to isolate proteins which interact with CaSsklp.
- the transformed host cells can be used to analyze the effectiveness of drugs and agents which inhibit CaSsklp function, such as host proteins or chemically derived agents or natural or synthetic drugs and other proteins which may interact with the cell to down- regulate or alter the expression of CaSsklp, or its cofactors.
- drugs and agents which inhibit CaSsklp function such as host proteins or chemically derived agents or natural or synthetic drugs and other proteins which may interact with the cell to down- regulate or alter the expression of CaSsklp, or its cofactors.
- the present invention relates to monoclonal or polyclonal antibodies specific for the above-described recombinant proteins (or polypeptides) .
- an antibody can be raised against a peptide described above, or against a portion thereof of at least 10 amino acids, perferrably, 11-15 amino acids.
- Persons with ordinary skill in the art using standard methodology can raise monoclonal and polyclonal antibodies to the protein (or polypeptide) of the present invention, or a unique portion thereof .
- Material and methods for producing antibodies are well known in the art (see for example Goding, in, Monoclonal Antibodies: Principles and Practice, Chapter 4, 1986) .
- the level of expression of CaSsklp can be detected at several levels.
- assays for the detection and quantitation of CaSSKl RNA can be designed, and include northern hybridization assays, in si tu hybridization assays, and PCR assays, among others. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985) , or Current Protocols in Molecular Biology, Ausubel, F. M. et al . (Eds), Wiley & Sons, Inc. for general description of methods for nucleic acid hybridization.
- Polynucleotide probes for the detection of CaSSKl RNA can be designed from the sequence available at accession number AF084608.
- RNA isolated from samples can be coated onto a surface such as a nitrocellulose membrane and prepared for northern hybridization.
- tissue sample can be prepared for hybridization by standard methods known in the art and hybridized with polynucleotide sequences which specifically recognize CaSSKl RNA .
- the presence of a hybrid formed between the sample RNA and the polynucleotide can be detected by any method known in the art such as radiochemistry, or immunochemistry, to name a few.
- probes are fairly long and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in the corresponding nucleic acid sequences. In other cases, it may be desirable to use two sets of probes simultaneously, each to a different region of the gene. While the exact length of any probe employed is not critical, typical probe sequences are no greater than 500 nucleotides, even more typically they are no greater than 250 nucleotides; they may be no greater than 100 nucleotides, and also may be no greater than 75 nucleotides in length. Longer probe sequences may be necessary to encompass unique polynucleotide regions with differences sufficient to allow related target sequences to be distinguished. For this reason, probes are preferably from about 10 to about 100 nucleotides in length and more preferably from about 20 to about 50 nucleotides.
- the D ⁇ A sequence of CaSSKl can be used to design primers for use in the detection of CaSSKl using the polymerase chain reaction (PCR) or reverse transciption PCR (RT-PCR) .
- the primers can specifically bind to the CaSSKl cD ⁇ A produced by reverse transcription of CaSSKl R ⁇ A, for the purpose of detecting the presence, absence, or quantifying the amount of CaSSKl R ⁇ A by comparison to a standard.
- the primers can be any length ranging from 7-40 nucleotides, preferably 10-15 nucleotides, most preferably 18-25 nucleotides homologous or complementary to a region of the CaSSKl sequence. Reagents and controls necessary for PCR or RT-PCR reactions are well known in the art.
- the amplified products can then be analyzed for the presence or absence of CaSSKl sequences, for example by gel fractionation, by radiochemistry, and immunochemical techniques. This method is advantageous since it requires a small number of cells.
- a determination whether the cell is overexpressing or underexpressing CaSSKl can be made by comparison to the results obtained from a normal cell using the same method.
- Decreased CaSSKl may be an indication of reduced virulence of the infecting yeast, or an indication that tissue-specific or site- specific expression of the gene is reduced.
- the present invention relates to a diagnostic kit for the detection of
- kits comprising a package unit having one or more containers of CaSSKl oligonucleotide primers for detection of CaSSKl by PCR or RT-PCR or CaSSKl polynucleotides for the detection of CaSSKl RNA in cells by in si tu hybridization or northern analysis, and in some kits including containers of various reagents used for the method desired.
- the kit may also contain one or more of the following items: polymerization enzymes, buffers, instructions, controls, detection labels.
- Kits may include containers of reagents mixed together in suitable proportions for performing the methods in accordance with the invention. Reagent containers preferably contain reagents in unit quantities that obviate measuring steps when performing the subject methods .
- the present invention provides a method for identifying and quantifying the level of CaSSKl present in a particular biological sample. Any of a variety of methods which are capable of identifying (or quantifying) the level of CaSSKl in a sample can be used for this purpose.
- Diagnostic assays to detect CaSSKl may comprise a biopsy or in si tu assay of cells from an organ or tissue sections, as well as an aspirate of cells from a tumour or normal tissue.
- assays may be conducted upon cellular extracts from organs, tissues, cells, urine, or serum or blood or any other body fluid or extract.
- the assay will comprise, contacting the sample to be assayed with a CaSsklp ligand or substrate, natural or synthetic, or an antibody, polyclonal or monoclonal, which recognizes CaSsklp, or antiserum capable of detecting CaSsklp, and detecting the complex formed between CaSsklp present in the sample and the CaSsklp ligand, substrate, or antibody added.
- CaSsklp ligands or substrates include for example, a downstream component in the CaSSKl pathway, a substrate for phosphorylation by CaSsklp, or an CaSsklp interacting protein, in addition to natural and synthetic classes of ligands and their derivatives which can be derived from natural sources such as animal or plant extracts.
- CaSsklp ligands or anti-CaSsklp antibodies, or fragments of ligand and antibodies capable of detecting CaSsklp may be labeled using any of a variety of labels and methods of labeling for use in diagnosis and prognosis of disease associated with candidiasis.
- types of labels which can be used in the present invention include, but are not limited to, enzyme labels, radioisotopic labels, non- radioactive isotopic labels, and chemiluminescent labels .
- suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5- steroid isomerase, yeast-alcohol dehydrogenase, alpha- glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase, etc .
- radioisotopic labels examples include 3 H, ni ln, 125 I, 32 P, 35 S, 14 C, 57 To, 58 Co, 59 Fe, 75 Se, 152 Eu, 90 Y, 67 Cu, 21 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, X1 C, 19 F, 123 I, etc.
- suitable non-radioactive isotopic labels include 157 Gd, 5 Mn, 162 Dy, 52 Tr, 46 Fe, etc.
- suitable fluorescent labels include a 152 Eu label, a fluorescein label, an isothiocyanate label , a rhodamine label , a phycoerythrin label , a phycodyanin label, an allophycocyanin label, a fluorescamine label, etc.
- chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, etc.
- suitable labels which may be employed in accordance with the present invention.
- the binding of these labels to ligands and to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J. H. , et al . , 1976 ( Clin . Chim. Acta 70, 1-31), and Schurs, A. H. W.
- the detection of the antibodies (or fragments of antibodies) of the present invention can be improved through the use of carriers.
- Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides , agaroses, and magnetite.
- the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
- the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to CaSsklp.
- the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
- the surface may be flat such as a sheet, test strip, etc.
- the ligands or antibodies, or fragments of antibodies or ligands of CaSsklp discussed above may be used to quantitatively or qualitatively detect the presence of CaSsklp. Such detection may be accomplished using any of a variety of immunoassays known to persons of ordinary skill in the art such as radioimmunoassays, immunometic assays, etc. Using standard methodology well known in the art, a diagnostic assay can be constucted by coating on a surface (i.e. a solid support) for example, a microtitration plate or a membrane (e.g. nitrocelluolose membrane) , antibodies specific for CaSsklp or a portion of CaSsklp, and contacting it with a sample from a person suspected of having a
- CaSSKl related disease The presence of a resulting complex formed between CaSsklp in the sample and antibodies specific therefor can be detected by any of the known detection methods common in the art such as fluorescent antibody spectroscopy or colorimetry.
- detection methods common in the art such as fluorescent antibody spectroscopy or colorimetry.
- a good description of a radioimmune assay may be found in Laboratory Techniques and Biochemistry in Molecular
- Sandwich assays are described by Wide at pages 199-206 of Radioimmune Assay Method, edited by Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970.
- the diagnostic methods of this invention are predictive of patients suffering from candidiasis, periodontitis, oral ulceration, or esophagitis in HIV- infected patients, candidal vaginitis, and bloodstream candidiasis .
- the protein can be used to identify inhibitors of CaSsklp activity. Using a phophorylation asssay such as those known in the art, natural and synthetic agents and drugs can be discovered which result in a reduction or elimination of CaSsklp autophosphorylation or phosphorylation activity. Knowledge of the mechanism of action of the inhibitor is not necessary as long as a decrease in the activity of CaSsklp is detected.
- Inhibitors may include agents or drugs which either bind or sequester CaSsklp substrate (s) or cofactor (s), or inhibit the CaSsklp itself, directly, for example by irreversible binding of the agent or drug to CaSsklp, or indirectly, for example by introducing an agent which binds the CaSsklp substrate. Agents or drugs related to this invention may result in partial or complete inhibition of CaSsklp activity.
- Inhibitors of CaSsklp may be used in the treatment or amelioration of candidiasis and diseases associated with candidiasis
- Agents which decrease CaSSKl RNA include, but are not limited to, one or more ribozymes capable of digesting CaSSKl RNA, or antisense oligonucleotides capable of hybridizing to CaSSKl RNA such that the translation of CaSSKl RNA is inhibited or reduced resulting in a decrease in the level of CaSSKlp .
- These antisense oligonucleotides can be administered as DNA, as DNA entrapped in proteoliposomes containing viral envelope receptor proteins (Kanoda, Y. et al . , 1989, Science 243, 375) or as part of a vector which can be expressed in the target cell such that the antisense DNA or RNA is made.
- the DNA can be injected along with a carrier.
- a carrier can be a protein such as a cytokine, for example interleukin 2, or polylysine-glycoprotein carrier.
- cytokine for example interleukin 2, or polylysine-glycoprotein carrier.
- carrier proteins and vectors and methods of using same are known in the art.
- the DNA could be coated onto tiny gold beads and said beads introduced into the skin with, for example, a gene gun (Ulmer, J. B. et al . , 1993, Science 259, 1745).
- antibodies, or compounds capable of reducing or inhibiting CaSsklp, that is reducing or inhibiting either the expression, production or activity of CaSsklp, such as antagonists can be provided as an isolated and substantially purified protein, or as part of an expression vector capable of being expressed in the target cell such that the
- CaSsklp-reducing or inhibiting agent is produced.
- co-factors such as various ions, i.e. Ca2 + or factors which affect the stability of the enzyme can be administered to modulate the expression and function of CaSsklp.
- These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal, intraperitoneal, oral, rectal, or parenteral (e.g. intravenous , subcutaneous , or intramuscular) route.
- CaSsklp-inhibiting compounds may be incorporated into biodegradable polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the CaSsklp-inhibiting compound is slowly released systemically.
- the biodegradable polymers and their use are described, for example, in detail in Brem et al . (1991) J. Neurosurg. 74, 441-446. These compounds are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of CaSsklp.
- agents which are capable of negatively affecting the expression, production, stability or function of CaSsklp are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of CaSsklp.
- An amount is said to be sufficient to "effect" the inhibition or induction of CaSsklp if the dosage, route of administration, etc. of the agent are sufficient to influence such a response .
- the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of agent which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient) , although a lower or higher dosage may be administered.
- a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient .
- Such an agent is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
- An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
- the compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle.
- a pharmaceutically acceptable carrier vehicle e.g., water, alcohol, and water.
- suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin are described, for example, in Remington's Pharmaceutical Sciences [16th ed. , Osol, A. ed., Mack Easton PA. (1980)].
- a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of the above-described compounds together with a suitable amount of carrier vehicle.
- Control release preparations may be achieved through the use of polymers to complex or absorb the compounds .
- the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release.
- Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
- microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacrylate) - microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres , microemulsions, nanoparticles , and nanocapsules or in macroemulsions .
- colloidal drug delivery systems for example, liposomes, albumin microspheres , microemulsions, nanoparticles , and nanocapsules or in macroemulsions .
- kits for use in the diagnostic or therapeutic methods described above.
- Kits according to this aspect of the invention may comprise one or more containers, such as vials, tubes, ampules, bottles and the like, which may comprise one or more of the compositions of the invention.
- kits of the invention may comprise one or more of the following components, one or more compounds or compositions of the invention, and one or more excipient, diluent, or adjuvant.
- the present invention describes a C. albicans strain which contains a deletion of CaSSKl response regulator gene.
- the strain ⁇ casskl is avirulent and does not form hyphae on solid inducing media.
- ⁇ casskl similar to wild type, is able to form hyphae in liquid media, however, unlike wild type, the hyphae flocculate.
- the ⁇ casskl strain may function as a gene or gene product delivery sytem.
- an antigen of interest could be delivered to an organ which is naturally invaded by ⁇ casskl in a patient, animal or plant where the antigen can provide benefit .
- the antigen can be introduced into ⁇ casskl in a second plasmid.
- a second plasmid could be used to provide a source of vaccine antigen for pathogens found in organs naturally invaded by C. albicans such as a systemic invasion, or kidney, lung, central nervous system, eye, to name a few.
- expression of spermicides by ⁇ casskl transformed with a desired gene could provide a cheap and infrequent method of contraception.
- ⁇ casskl represents a safe delivery vehicle and is advantageous because it can carry one or more compounds and can be genetically engineered to carry- one or more nucleic acid molecules capable of effecting gene therapy and/or of encoding one or more proteins and/or RNA molecules.
- the compound of interest can be carried by ⁇ casskl within the yeast cell, on the yeast membrane surface, spanning the yeast membrane, withing the yeast periplasm, and combinations thereof . At least some of the compound of interest remains associated with the yeast at least until the yeast reaches its target, or site of action (e.g. the bloodstream, interstitial tissue, or a cell) , at which point it is also possible that a compound carried by the yeast may be released.
- a compound capable of protecting an animal or plant from disease is a compound that when administered to an animal or plant can prevent a disease from occuring and/or cure or alleviate disease symptoms or cause.
- diseases from which to protect an animal or plant include, but are not limited to, infections, genetic defects and other metabolic disorders .
- Such classes of diseases can lead to abnormal cell growth (e.g., benign or malignant neoplasia, hyperplastic syndromes) , degenerative processes, and/or immunological defects as well as to a number of other disorders .
- ⁇ casskl vehicles of the present invention preferably include compounds capable of stimulating an immune response, compounds capable of suppressing an immune respone, toxic compounds, compounds capable of inhibiting transcription of a gene, compounds capable of inhibiting translation of a gene, compounds capable of inhibiting the ability of an infectious agent to produce progeny, compounds capable of replacing a defective gene, compounds capable of replacing a defective protein (including nucleic acid molecules capable of encoding such proteins and mimetopes of such proteins) and/or biological response modifiers (e.g., cytokines, such as lymphokines and monokines, as well as other growth modulating factors), and mixtures thereof.
- cytokines such as lymphokines and monokines, as well as other growth modulating factors
- Such compounds include, but are not limited to, antibiotics, antibodies, antifungal compounds, antigens, antiparasite compounds, antisense compounds, antiviral compounds, chemotherapeutic agents , cytokines, growth modulating factors (including both growth stimulants and suppressants), herbicides, hormones, immunosuppressants, nucleic acid-based drugs (e.g., DNA- or RNA-based drugs), nucleic acid molecules comprising coding regions, nucleic acid molecules comprising regulatory sequences, nucleoside analogs, other oligonucleotides, peptide analogs, peptides, pesticides, prodrugs (e.g., compounds that are activated at the site of action), other proteins, ribozymes, steroids, toxins, and/or vitamins.
- nucleic acid-based drugs e.g., DNA- or RNA-based drugs
- nucleic acid molecules comprising coding regions
- nucleic acid molecules comprising regulatory sequences, nucleoside analogs, other oligonu
- Cell types naturally targeted by ⁇ casskl include, but are not limited to, cells of granulocytic- monocytic lineage, B lymphocytes and T lymphocytes as well as cells in the epithelial regions of an animal .
- the ⁇ casskl can be engineered such that it targets specific cell types by positioning a cell- binding element (e.g., antibodies, receptors, and receptor ligands) in the membrane of ⁇ casskl.
- a cell- binding element e.g., antibodies, receptors, and receptor ligands
- Such an element is preferably absent from, or found in only small amounts, on cell types that are not targeted for delivery.
- the present invention includes the delivery of a composition comprising ⁇ casskl of the present invention to an animal or to cell in culture.
- Such compositions can be delivered to an animal either in vivo or ex vivo, or can be delivered to cells in vitro .
- Such administration can be systemic, mucosal, and/or proximal to the location of the targeted cell type.
- routes to administer yeast in vivo include aural, bronchial, genital, inhalatory, nasal, ocular, oral, parenteral, rectal, topical, transdermal , and urethral routes .
- Ex vivo delivery refers to a method that includes the steps of contacting a population of cells removed from an animal with a composition comprising ⁇ casskl of the present invention under conditions such that the yeast is adsorbed by targeted cell types and returning the contacted cells to the animal .
- a delivery method is particularly useful in the treatment of cells involved in hematopoiesis and the immune response as well as in the treatment of tumors .
- In vitro delivery refers to the delivery of ⁇ casskl of the present invention to a population of cells (which can also include tissues or organs) in culture .
- a preferred single dose of a yeast vehicle of the present invention is from about 1 x 10 5 to about 5 x 10 7 yeast cell equivalents per kilogram body weight of the organism being administered the composition.
- the ⁇ casskl strain of the invention can be used directly in vaccine formulations, or lyophilized, as desired, using lyophilization protocols well known to the artisan. Lyophilized compositions will typically be maintained at about 4°C. When ready for use the lyophilized composition is reconstituted in a stabilizing solution, e.g., saline or comprising Mg ++ and HEPES, with or without adjuvant, as further described below.
- ⁇ casskl vaccine of the invention contains as an active ingredient an immunogenically effective amount of a nonvirulent C. albicans as described herein.
- the ⁇ casskl may be introduced into a host, particularly humans, with a physiologically acceptable carrier and/or adjuvant.
- Useful carriers are well known in the art, and include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
- the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration, as mentioned above.
- the compositions may contain pharmaceutically accepatable auxilliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.
- Administration of the ⁇ casskl disclosed herein may be carried out by any suitable means, including both parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection) , by in ovo injection in birds, orally and by topical application of the yeast (typically carried in the pharmaceutical formulation) to an airway surface.
- Topical application of the yeast to an airway surface can be carried out by intranasal administration (e.g. by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally) .
- Topical application of the yeast strain to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the yeast as an aerosol suspension, and then causing the subject to inhale the respirable particles.
- respirable particles of a pharmaceutical formulation including both solid particles and liquid particles
- Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed.
- the host becomes at least partially or completely immune to C. albicans infection, or resistant to developing moderate or severe C. albicans infection.
- the vaccine composition containing the ⁇ casskl of the invention are administered to a person susceptible to or otherwise at risk of C. albicans infection to enhance the individual ' s own immune response capabilities .
- Such an amount is defined to be a "immunogenically effective dose".
- the precise amount again depends on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1 x 10 5 to about 5 x 10 7 yeast cell equivalents per kilogram body weight of the organism being administered the composition.
- the vaccine formulations should provide a quantity of ⁇ casskl of the invention sufficient to effetively protect the patient against serious or life- threatening C. albicans infection.
- ⁇ casskl vaccines of the invention may be desirable to combine with vaccines which induce protective responses to other agents .
- Single or multiple administration of the vaccine compositions of the invention can be carried out. Multiple administration may be required to elicit sufficient levels of immunity. Levels of induced immunity can be monitored by measuring amount of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection. It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are obvious and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.
- Candida albicans (strain SC5314) was routinely grown in YPD complex medium (1% yeast extract, 2% peptone, 2% glucose) , at 28 °C with shaking at 250 rpm for 14 hours. Cells were harvested from liquid medium by centrifugation at 4,000 x g for 10 minutes at 4°C. For RNA isolation, the same strain was grown in medium 199 containing Earle's salts and glutamine but lacking sodium bicarbonate (Gibco BRL) and buffered with 155 mM Tris-HCl either at pH 7.5 or 3.5.
- YPD complex medium 1% yeast extract, 2% peptone, 2% glucose
- Cultures were inoculated to a density of 10 7 cells ml -1 into prewarmed medium and incubated at 28 °C or 37 °C with vigorous agitation for 3 hours. In all cases, stationary-phase cells grown at 28 °C in YPD were used as an inoculum.
- Schizosaccharomyces pombe strain JM1303 (mcs4-13 cdc25-22 ura4-Dl8) (Shieh et al . , 1997, Genes Dev. 11, 1008-1022) was routinely grown in YE (3% dextrose, 0.5% yeast extract, 2% agar) at 25°C or in EMM minimal medium
- Saccharomyces cerevisiae strain TM187 (MATa. Ieu2 ura3 trpl slnl : :hisG sskl : : TRPl + pSSP25) (Maeda et al . , 1994) was routinely grown in CAD (0.67% YNB, 0.5% casamino acids, 2% dextrose) at 30°C.
- Escherichia coli strain DH5 ⁇ which was used for transformation and plasmid amplification, was grown at 37 °C in LB broth supplemented with 0.1 mg of ampicillin per ml of medium when necessary.
- E. coli strain LE392 was used for propagation of bacteriophage ⁇ EMBL3.
- the Candida albicans strains used in this work are listed in Table 1. All strains were routinely grown either in YPD complex medium (1% yeast extract, 2% peptone and 2% dextrose) or SD minimal medium (0.67% yeast nitrogen base, 2% dextrose) at 28°C.
- strains were also grown on solid Spider medium (1% nutrient broth, 1% mannitol, 0.2% K 2 HP0 4 ) , solid synthetic low-ammonium- dextrose (SLAD) medium (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate, 2% dextrose and 50 ⁇ M ammonium sulfate as sole nitrogen source) , serum [10% fetal bovine serum (Gibco BRL)] and Medium 199 (M199) containing Earle's salts and glutamine but lacking sodium bicarbonate (Gibco BRL) and buffered with 155 mM Tris-HCl at pH 7.5 or pH 4.0. All liquid media were sterilized by filtration.
- SLAD solid synthetic low-ammonium- dextrose
- M199 Medium 199
- Solid media were prepared by adding 1.5% agar as a final concentration (2% for SLAD plates) at 50°C after autoclaving. Prewarmed liquid media were inoculated to a density of 10 7 cells/ml and incubated either at 28°C or 37°C with vigorous agitation. On solid media, the appropriate dilutions of cells were plated to obtain approximately 60-80 colonies per plate. In all cases, stationary-phase cells grown at 28°C in YPD were used as an inoculum. Table 1
- Plasmid DNA was extracted from E. coli DH5 ⁇ cells according to the manufacturer's instructions (Plasmid
- albicans strains was obtained according to the method described by Sherman et al . (1986, supra) .
- For southern blot analyses 4 ⁇ g of DNA per lane was loaded in a 0.8% agarose gel and transferred by capillarity to positively-charged nylon membranes (Hybond-N+ , Amersham) by standard protocols (Sambrook et al . , 1989, supra) .
- Probes were Digoxigenin- labelled by random priming (DIG DNA Labeling Kit, Boehringer Mannheim) , and hybridized and detected according to the chemiluminescent method described in the manufacturer's recommendations (DIG Nucleic Acid Detection Kit, Boehringer Mannheim) . Construction of the CaSSKl probe and screening of a ⁇ EMBL3 Candida albicans genomic library
- a fragment of the CaSSKl gene was isolated by PCR amplification with the degenerate primers, 5'-AA(T/C) GT(A/G/T) TT(G/A) AT(A/T) GT(A/G/T) GAA GA(T/C) AA-3' and 5 ' -CAT (A/G)CA (A/T)CC CCA TTC (T/A/G)GT (T/A)AT TTT-3 ' .
- These primers were designed according to a pair of amino acid sequences conserved between Ssklp from S. cerevisiae and Mcs4 from S. pombe. These sequences, VLIVEDN (SEQ ID NO: 3) and KITE GCM
- SEQ ID NO: 4 are located at positions 505-512 and 642- 649 in Ssklp and at positions 363-370 and 500-507 in Mcs4, respectively, being separated in both cases by 145 amino acids .
- PCR amplification was performed with genomic DNA from C. albicans strain SC5314 as template. Thirty-two cycles of PCR were performed with the following steps: 45 seconds at 94'C; 1 minute at 50 °C and 2.5 minutes at 72 °C. The reaction yielded specifically a fragment with the predicted size of approximately 435 bp.
- the PCR product was subcloned into pCR2.1 (Invitrogen) to generate pSSKl, sequenced and the 447 bp DNA fragment identified as a partial C.
- albicans DNA homolog of SSKl and mcs4 ⁇ The PCR product was excised from pSSKl by BcoRI digestion, digoxigenin- labelled by non-radioactive random priming (DIG DNA Labelling Kit, Boehringer Mannheim) and, in turn, used as probe to screen a ⁇ EMBL3 C. albicans genomic library (Monod et al . , 1994, Mol . Microbiol . 13, 557-368), according to the protocol described by Sambrook et al . (1989, supra) . The detection of the positive clones was done according to the colorimetric method described in the manufacturer's recommendations (DIG Nucleic Acid Detection Kit, Boehringer Mannheim).
- the entire ORF of CaSSKl was amplified from pER-CaSSKl by PCR using the 5' oligonucleotide 5'- AAATAGGATCCCATGAATTTTCTCTATAAC-3 ' (SEQ ID NO: 5) and the 3' oligonucleotide 5 ' -CGTTGGATCCTTTCAAGCTTTGTTTAATCTTCG -3' (SEQ ID NO: 6), both of them containing the BamHl site (bolded) and confirmed by DNA sequencing.
- the resulting 2.04 kb product was digested with BamHI and cloned into the BamHI site of pBREP42 to generate pBREP42 -CaSSKl .
- the pBREP42 plasmid is a low copy number version of the pREP42 plasmid (Maundrell, 1993, Gene 123, 127-130), which was constructed by introducing the 5.50 kb PvuII-PvuII cassette [ura4 + -nmt promoter -MCS-nmt poly.A signal -arsl] from pREP42 into the pBR322 digested with EcoRV/PshAI .
- the pREP42-mcs4 plasmid (Shieh et al . ,
- TM187 strain To ensure a correct level of expression of CaSSKl in S. cerevisiae, we first constructed a low copy version of the pRS415 plasmid (Sikorski and Hieter, 1989, Genetics 122, 19-27) (which we named as pBRS415) and then, both the promotor and terminator of the SSKl gene from S. cerevisiae were introduced into pBRS415 to generate pBRS415-p/t.
- the pBRS415 plasmid was constructed by introducing a 4.17 kb Seal-Sail fragment which contains the cassette [LEU2-APSH4-CEN6] from pRS415 (Stratagene) in the pBR322 digested with Seal/ Sail .
- the pBRS415-p/t plasmid was generated by introducing sequentially a 0.72 kb fragment that contains the promotor of SSKl and the 0.36 kb terminator sequence of SSKl , linked by a BamHI restriction site.
- the promotor sequence was obtained by PCR using the oligonucleotides 5 ' -AGGTCGACCCACTGCTGGATC-3 ' (SEQ ID NO: 7) and 5 ' -AGCGGATCCTTCCACAGTAACGC-3 ' (SEQ ID NO: 8).
- the terminator sequence was also obtained by PCR using the oligonucleotides 5 ' -TTTGGATCCACCGTGTAGAGGACATTATG- 3 ' (SEQ ID NO: 9 ) and 5 ' -ATAGGTACCCAGCTGGTG AATCCAAGCCCG- 3' (SEQ ID NO: 10) .
- the 2.04 kb PCR product that contains the entire ORF of CaSSKl was digested with BamHI and cloned into the only BamHI site of pBRS415-p/t to generate pBRS415- CaSSKl .
- the pSSK1222 plasmid (Maeda et al . , 1994, Nature, 369, 242-245) was used as a control.
- Each of these plasmids (pSSK1222, pBRS415, pBRS415-p/t and pBRS415-CaSSKl ) was used to transform S. cerevisiae strain TM187. Southern and Northern blot analysis Genomic DNA from C.
- albicans was obtained according to the method described by Sherman et al . (1986, Methods in Yeast Genetics . Cold Spring Harbor Laboratory Press, New York) . Four micrograms of DNA per lane were typically loaded for Southern analyses. DNA was transferred by capillarity to positively-charged nylon membranes (Hybond-N+, Amersham) by standard protocols (Sambrook et al . , 1989, supra) and hybridized with the same probe that was used for screening the genomic library.
- RNA ladder 0.24-9.5 kb RNA ladder (Gibco-BRL) was used for sizing in formaldehyde agarose gels.
- Chromosomal DNA was resolved by pulsed-field gel electrophoresis according to the protocol described by Wickes et al . (1991, Infect . Immun. 59, 1762-1771), in a Bio-Rad CHEF DRII unit with a linear ramp switch time of 300 seconds to 1400 seconds for 144 hours at 13 °C.
- Gels were stained for 30 minutes with ethidium bromide (0.5 ⁇ g ml "1 in water) , destained in distilled water for 30 minutes and then photographed. The gels were blotted onto nylon membranes by alkaline transfer for 16 hours.
- the 1.85 kb EcoRI-Bglll fragment was radioactively labelled by random priming and used as a probe. Hybridization was carried out as described above.
- Homology searches, sequence analysis and multiple augments Homology searches were performed using the BLAST network service (Altschul et al . , 1990, J " . Mol . Biol . 215, 403-410). Motif searches were performed using the PROSITE and BLOCKS network databases . The computer analysis of sequences was performed using the DNA Strider 1.2. Sequences were aligned by the CLUSTAL program (Higgings and Sharp, 1988, Gene 73, 237-244).
- plasmids used for C. albicans transformation were constructed, which carries a cassette designed to delete most of the ORF of CaSSKl .
- a 3.80 kb Stul-Bglll DNA fragment from pMB7 (Fonzi, and irwin, 1993, Genetics 134, 717-728), which contains the hisG-URA3-hisG cassette, was used to replace most of the ORF of CaSSKl (Fig. 3A) .
- the pBRl plasmid was linearized by digestion with Aatll which cuts once in the plasmid outside of the cassette, and approximately 2 ⁇ g of
- DNA was used to transform the Ura ⁇ C. albicans strain CAI4 by electroporation (Thompson, et al . , 1998, Yeast 14, 565- 571) . Electroporation was chosen for transformation experiments since the most commonly used LiAc procedure (Gietz, et al . , 1991, Nucleic Acids Res . 20, 1425) consistently failed to yield transformants, even when a high amount of DNA (up to 30 ⁇ g) was used.
- Transformed cells were selected as Ura + on SD minimal medium and spontaneous Ura " ⁇ derivates from a Ura + independent clone were selected on SD medium containing 5 ' -fluoroorotic acid (1 mg/ml) and uridine (25 ⁇ g/ml) .
- the transformants were then used to delete the second allele of CaSSKl .
- the pBR2 vector was designed.
- a 2.37 kb Xbal-Bglll DNA fragment from pMB7 that contains the URA3-hisG cassette was introduced into the only Spel/BamHI sites of pBR- CaSSKl, which are located downstream of CaSSKl , generating the CaSSKl -URA3-hisG cassette (Fig. 3B) .
- the pBR2 plasmid was linearized and used to transform a
- Ura Acasskl null strain as described above.
- the construction of the expression plasmids pLJ19 and pCCal (kindly provided by Dr. D. Harcus) , which carry the CPH1 and CPP1 genes of C. albicans respectively under the control of the ADHl promoter, was previously described (Csank et al . , 1997, Mol . Biol . Cell 8, 2539-2551; Csank et al, 1998, Infect . Immun . 66, 2713-2721) .
- the encoding region of CaHOGl flanked by Bglll sites was amplified by PCR with the 5' oligonucleotide 5 ' -GCAGATCTGAAAATGTCTGCAGATGGAG-3 ' (SEQ ID NO: 11) and the 3' oligonucleotide 5'- TTAGATCTTTGAAGATTAAGCTCCGTTGGC-3' (SEQ ID NO: 12) using the pBSK8 plasmid containing the CaHOGl gene as a template .
- flanking regions of the PCR product were confirmed by sequencing, digested with Bglll and inserted into the Bglll site of the plasmid pYPBl-ADHpt (kindly provided by D. Harcus) containing the ADHl promoter, the C. albicans URA3 as a selectable marker, and an autonomously replicating sequence (Csank et al . , 1997, 1998, supra; Leberer et al . , 1997, Curr. Biol . 7, 539-546). Determination of generation time. Preinoculum cultures of each strain were always prepared in YPD medium for 24 h at 28° C.
- the optical density of precultures was determined at 600 nm (OD 600 ) , and 250 ml flasks containing 50 ml of prewarmed fresh medium (YPD or M199 pH 4.0) was inoculated to a final OD 600 of 0.1 with the appropriate preinoculum and incubated at 28° C and 200 rpm. The OD 600 was measured every 1.5 hours until the stationary phase of the growth curve was reached.
- the generation times calculated for each strain are the averages of three independent experiments .
- the C. albicans strains used in these experiments included a parental control with only one functional URA3 allele (strain CAF2 ) and five strains in which either one (CSSKll-1, CSSK23-1 and CSSK23-2) or both alleles (CSSK21-1 and CSSK21-2) have been deleted.
- CSSK23 strains were included in all experiments to ensure that all phenotypic traits observed with the CSSK21 strains were due solely to the CaSSKl mutation rather than to unrelated mutations that may have occurred during construction of the Acasskl null strains.
- Example 1 Isolation of a putative response regulator gene of C. albicans (CaSSKl)
- the CaSSKl gene was cloned following a PCR-based approach as described in the Material and Methods section. A PCR reaction using an appropriate pair of degenerate primers was performed and a 447 bp PCR product was identified as a partial DNA sequence of CaSSKl . This PCR fragment was then used to isolate the entire CaSSKl gene.
- 6 clones were selected for further analysis. These ⁇ EMBL3 clones revealed that they each carried the same C. albicans DNA fragment as we observed upon restriction analysis with Bg-lII. Also, each one contained the two expected hybridizing fragments as shown by Southern analysis. After subcloning and sequencing both fragments from one of the clones, we obtained the entire open reading frame (ORF) of CaSSKl .
- Example 2 Structure of the C. albicans CaSSKl gene and its encoded protein CaSSKl has a 2022 bp ORF.
- the 5 ' noncoding region upstream from the ATG codon ending at position -89, there is a long AT-rich sequence (84% A+T) in which two putative TATAAA elements were found between positions -17 to -22 and -28 to -33, upstream from the start codon, respectively.
- two CAAT motifs were found at -309 and -329 positions, respectively.
- the 2022 bp ORF of CaSSKl encodes a protein of 674 amino acids with a predicted molecular mass of 73.5 kDa and a pi of 9.5.
- the codon usage frequency in CaSSKl corresponds to the low expressed genes in C. albicans (Lloyd and Sharp, 1992, Nucleic Acids Res . 20, 5289-5295).
- the PTSl motifs have been involved in targeting proteins to peroxisomes and are located in the extreme C-termini of the majority of the peroxisomal matrix proteins. Their specificity is remarkable considering the small size and relaxed consensus sequence.
- the C-terminal PTSl has been studied by extensive mutational analyses (Elgersma et al . , 1996, supra) . These studies revealed a high degeneracy of the PTSl consensus sequence and suggests that additional domains in these proteins may be of importance to determine whether or not a certain PTSl is recognized by the components of the peroxisomal import machinery (Elgersma et al . , 1996, supra) . However, whether or not this motif is used as a PTSl sequence in CaSsklp remains to be elucidated.
- the C-terminal sequences from both prokaryote and eukaryote response regulators show highly conserved residues.
- these residues include a pair of aspartates near the N- terminus (one aspartate in eukaryotes) , an aspartate motif which accepts a phosphate and a motif near the C- terminus which contains a key lysine (Parkinson, 1993, Cell 73, 857-871) .
- the Asp 556 should be the predicted site of phosphorylation.
- the Asp 513 should be one of the pair of aspartates that are conserved among prokaryotic response regulators but apparently not in eukaryotic cells, which appears as ED instead of DD.
- the Lys 638 should be the other conserved residue.
- Example 3 Complementation analysis of a S. pombe mcs4 mutant with CaSSKl
- Mcs4 functions in adapting cells to oxidative, heat, and nutritional stresses by coordinating environmental responses with meiosis and mitosis (Cottarel, 1997, Genetics 147, 1043-1051; Shieh et al . , 1997, supra; Shiozaki et al . , 1997, Mol . Biol . Cell 8, 409-419). Since Mcs4 and CaSsklp share structural similarity, it could be also possible that CaSsklp plays a similar function in C. albicans .
- Example 4 Complementation analysis of a S. cerevisiae SSKl mutant with CaSSKl It has been reported that Ssklp acts downstream of the Slnlp-Ypdlp osmosensor pathway of S. cerevisiae (Posas et al . , 1996, Cell 86, 865-875) . Thus, considering the high similarity that CaSsklp shares with the C-terminus of Ssklp, and that homologues of Slnlp and Ypdlp from S. cerevisiae also exist in C.
- CaSsklp is a component of the putative homologous CaSlnlp-CaYpdlp- CaSsklp pathway of C. albicans , which also could be involved in osmoregulation.
- CaSSKl was expressed in a ssklA S. cerevisiae strain. However, since the deletion of SSKl in S.
- Example 5 Chromosomal deletion of CaSSKl It was previously shown that CaSSKl was unable to rescue the lack of SSKl or mcs4 + in S. cerevisiae or S. pombe respectively, which suggested that despite their structural homology, they may not be functional homologs (Calera and Calderone, 1999, Yeast 15) . Thus, to further investigate the function of CaSSKl in the growth, morphogenesis and virulence of C. albicans, we used the urablaster technique (Fonzi and Irwin, 1993, supra) to obtain Acasskl mutants.
- the hisG- URA3-hisG cassette was used to replace a 1.47 kb fragment of CaSSKl that includes the encoding region for the conserved putative aspartate residue which is phosphorylated (Fig. 3A) .
- Ura + transformants were selected on SD minimal medium and several isolates were tested by southern blot to confirm this replacement. DNA from a CSSK11 representative isolate exhibited two hybridizing bands, a
- CSSK12-1 and CSSK12-2 A representative CSSK12 intrachromosomal recombinant strain is shown by southern blot (Fig. 3C) .
- Example 6 CaSSKl is not involved in a response to osmotic or oxidative stress.
- CaSSKl does not function in regulating the response to either osmotic or oxidative stress (Calera and
- Example 7 CaSSKl is required for hyphal formation on solid media.
- the Acasskl null and heterozygote Ura3 + strains were grown in several media (solid and liquid) that induce this morphological switch, including Spider, serum and M199 (pH 7.5) medium (Fig. 5) .
- Colonies from the wild type (CAF2) and the heterozygote strains (CSSK11) developed radial filaments emerging from the edge of the colonies after two days in M199 (pH 7.5) and after three days in Spider medium.
- the Acasskl null mutants when grown in agar with 10% serum showed a severe reduction in hyphal development in comparison with the CAF2 , forming irregular smooth colonies with yeast growth in the center of the colonies from which some filaments emerged.
- the heterozygote strains formed colonies intermediate between those of the Acasskl null strains and the wild type (Fig. 5) .
- Example 8 Avirulence of Acasskl mutants .
- the ability of Acasskl C. albicans null strains to establish infection in a murine model of hematogenously disseminated candidiasis was investigated.
- two factors that may also affect the virulence of the mutants such as their generation time and orotidine 5 ' -monophosphate (OMP) decarboxylase activity.
- OMP orotidine 5 ' -monophosphate
- survival of mice infected with CSSK21, CSSK11 and CSSK23 was greater than that of mice infected with CAF2.
- mice succumbed to CAF2 infection by 72 h.
- the avirulence of the Acasskl null strains indicate that CaSSKl is required for the pathogenesis of C. albicans . Furthermore, several experiments indicated that the virulence of the CSSKll and CSSK23 strains was due to the presence of one functional allele, while the avirulence of the CSSK21 strains was due to the absence of any CaSSKl allele: 1) The same results in survival and tissue counts were obtained with two independent Acasskl null strains (CSSK21-1 and CSSK21-2) . 2) The virulence of the CSSK21 strains was restored by the reintroduction of a parental copy of CaSSKl in them both (CSSK23-1 and CSSK23-2) .
- albicans homologues have been identified, including CST20, HST7 , CEK1 and CPH1 (Csank et al . , 1998, supra; Kohler and Fink, 1996, supra; Leberer et al . , 1996, supra; Liu et al . , 1994, supra) , which function in a C. albicans STE12-homolog MAPK cascade, as well as EFGl (Lo et al . , 1997, Cell 90, 939-949; Stoldt et al . , 1997, EMBO J. 16, 1982-1991) which, like its S.
- CaHKl Calera and Calderone, 1999, Microbiol . 145; Calera et al . , 1998, Yeast 14, 665-674) and CaNIKl/COSl (Alex et al . , 1998, Proc . Natl . Acad . Sci . USA 95, 7069-7073).
- CaSSKl the only response regulator kinase gene described so far in C. albicans (Calera and Calderone, 1999, Yeast 15) .
- CaSSKl does not restore the normal growth of either Asskl or Amcs strains of S. cerevisiae or S. pombe respectively, suggesting that CaSSKl may have another role in C. albicans rather than in adaptation of cells to stress (Calera and Calderone, 1999, Yeast 15, supra) .
- the phenotypic characterization of Acasskl mutants supports our previous observation and confirms that CaSSKl is not functionally related to either SSKl or mcs4 + .
- CaSSKl is essential for hyphal formation on solid inducing media and virulence, even though it is not required for formation of hyphae in liquid media.
- Efglp must be the final element of a morphogenesis pathway (Lo et al . , 1997, supra; Stoldt et al, 1997 , supra) .
- the Aefgl mutants are unable to form true hyphae either on solid or liquid media (Lo et al . , 1997, supra) .
- the Aefg mutants grow as a yeast in liquid media even in the presence of serum (Lo et al . , 1997 , supra) .
- the phenotype of the Acasskl null strains resembles the defect in hyphal formation on solid media observed for mutants in genes of the CPHl filamentation pathway but, in contrast, the mutants in genes of the CPHl pathway are able to form hyphae on solid serum, are not totally avirulent, and fail to hyper-invade the agar under low nitrogen availability (Csank et al . , 1998, supra; Kohler and Fink, 1996, supra; Leberer et al . 1996, supra; Liu et al . , 1994, supra) .
- the hyper-invasive phenotype of the Acasskl null strains resembles that of the Acppl mutants, the Acasskl null strains show this phenotype only under low nitrogen while the Acppl mutants show derepressed hyper- invasion of agar under both normally noninducing and inducing conditions in a wide variety of rich and defined solid media (Csank et al . , 1997, supra) .
- CaSSKl affects the activity of the CPHl pathway
- the overproduction of downstream components of the CPHl pathway in a Acasskl background should rescue the lack of CaSSKl .
- in preliminary experiments we were unable to rescue the normal filamentous growth of a Acasskl null mutant by overexpressing either CHP1 or CPP1 . Similar results were previously described by others in a
- the flocculation displayed by the Acasskl null strains under conditions of germ tube formation occurs in a manner similar to the Acahkl mutants (Calera and Calderone, 1999, Microbiology 145) . Both the Acasskl and Acahkl strains show similar growth rates and are totally avirulent in a murine model of hematogenously disseminated candidiasis (Calera et al . , 1999, Infect . Immun. 67, 4280-4284).
- CaSsklp should function as a response regulator in this putative cascade, similar to other response regulators (Banuett 1998, supra) , it must lie downstream of a sensor histidine kinase component, such as Cahklp or CaNiklp/Coslp.
- albicans i.e., the morphological transition from yeast to hyphae and changes in the expression of a hyphal surface compound (s) that occur during hyphal growth.
- a "two-component" cascade may have been adopted by C. albicans to modulate its morphological switching and that other undiscovered elements or pathways must exist that regulate the transition from yeast to hyphae in liquid media in C. albicans .
- two-component signal tranduction cascades have not been found in mammalian cells which emphasize the interest of both CaSslp and Cahklp as targets for the development of antifungals .
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99949857A EP1115855A1 (en) | 1998-09-23 | 1999-09-23 | RESPONSE REGULATOR CaSSK1 |
AU62636/99A AU6263699A (en) | 1998-09-23 | 1999-09-23 | Response regulator (cassk1) |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10152998P | 1998-09-23 | 1998-09-23 | |
US60/101,529 | 1998-09-23 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2000017344A1 true WO2000017344A1 (en) | 2000-03-30 |
WO2000017344A8 WO2000017344A8 (en) | 2000-05-11 |
Family
ID=22285122
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/022218 WO2000017344A1 (en) | 1998-09-23 | 1999-09-23 | RESPONSE REGULATOR $i(CaSSK1) |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1115855A1 (en) |
AU (1) | AU6263699A (en) |
WO (1) | WO2000017344A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2506178A (en) * | 2012-09-25 | 2014-03-26 | Vib Vzw | Mutant yeast strain with decreased glycerol production |
-
1999
- 1999-09-23 WO PCT/US1999/022218 patent/WO2000017344A1/en not_active Application Discontinuation
- 1999-09-23 EP EP99949857A patent/EP1115855A1/en not_active Withdrawn
- 1999-09-23 AU AU62636/99A patent/AU6263699A/en not_active Abandoned
Non-Patent Citations (3)
Title |
---|
CALERA, JOSE A. (1) ET AL: "Identification of a putative response regulator two-component phosphorelay gene ( CaSSK1 ) from Candida albicans.", YEAST, (SEPT. 15, 1999) VOL. 15, NO. 12, PP. 1243-1254., XP000865799 * |
SHIGEHISA NAGAHASHI ET AL.: "Isolation of CaSLN1 and CaNIK1, the genes for osmosensing histidine kinase homologues, from the pathogenic fungus Candida albicans", MICROBIOLOGY, vol. 144, February 1998 (1998-02-01), pages 425 - 432, XP002083238 * |
TATSUYA MAEDA ET AL.: "A two-component system that regulates an osmosensing MAP kinase cascade in yeast", NATURE, vol. 369, no. 6477, 19 May 1994 (1994-05-19), LONDON GB, pages 242 - 245, XP002129354 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2506178A (en) * | 2012-09-25 | 2014-03-26 | Vib Vzw | Mutant yeast strain with decreased glycerol production |
Also Published As
Publication number | Publication date |
---|---|
WO2000017344A8 (en) | 2000-05-11 |
AU6263699A (en) | 2000-04-10 |
EP1115855A1 (en) | 2001-07-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1023311A1 (en) | Novel bacterial polypeptides and polynucleotides | |
JP2000508178A (en) | New compound | |
US20050255478A1 (en) | Staphylococcus epidermidis nucleic acids and proteins | |
WO1998019689A1 (en) | Novel coding sequences | |
JP2000502898A (en) | Components of the Staphylococcus signal transduction system | |
US7666404B2 (en) | Glanders/meliodosis vaccines | |
US5965402A (en) | DNA encoding phoH polypeptides | |
US5869290A (en) | Cayae1 polynucleotides | |
US5854020A (en) | TCSTS polynucleotides | |
US6716625B1 (en) | Histidine kinases of Aspergillus and other fungal species, related compositions, and methods of use | |
WO2000017344A1 (en) | RESPONSE REGULATOR $i(CaSSK1) | |
EP0822987A2 (en) | Polynucleotides and aminoacid sequences from staphylococcus aureus | |
US6284515B1 (en) | Signal recognition particle polypeptides and polynucleotides | |
US5989864A (en) | DNA encoding spo-rel polypeptides | |
US6258578B1 (en) | His5 | |
US5972651A (en) | Ffh | |
US6174705B1 (en) | ARO1 dehydroquinate synthase of candida albicans | |
US5889171A (en) | TarF | |
EP0885965A2 (en) | Histidine kinase polypeptides | |
EP0954525A1 (en) | Novel compounds | |
JP2000502561A (en) | Staphylococcus aureus new TCSTS response regulator | |
US6346393B1 (en) | Polynucleotides encoding a ribonuclease s | |
JP2000502563A (en) | Two-component signal transduction system from Staphylococcus aureus | |
CA2236473A1 (en) | Novel pcra | |
JP2002504321A (en) | New pgsA |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
AK | Designated states |
Kind code of ref document: C1 Designated state(s): AU CA JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: C1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
CFP | Corrected version of a pamphlet front page | ||
CR1 | Correction of entry in section i |
Free format text: PAT. BUL. 13/2000 UNDER (51) REPLACE THE EXISTING SYMBOLS BY "C12N 15/11, 15/54, 15/81, 1/19, C07K14/40, C12N 9/12, C12Q 1/48, G01N 33/573, C07K 16/14, 16/40, C12Q 1/68, A61K 35/72, 39/00 // (C12N 1/19, C12R 1:725)" |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 1999949857 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1999949857 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1999949857 Country of ref document: EP |