Andrey Pavlov - Academia.edu (original) (raw)
Papers by Andrey Pavlov
Journal of Biological Chemistry, 1994
BMC microbiology, Jun 13, 2016
Fungi are organisms with the highest natural capacity to degrade lignocellulose substrates, which... more Fungi are organisms with the highest natural capacity to degrade lignocellulose substrates, which is enabled by complex systems of extracellular enzymes, whose expression and secretion depend on the characteristics of substrates and the environment. This study reports a secretome analysis for white-rot basidiomycete Trametes hirsuta cultivated on a synthetic media and a lignocellulose substrate. We demonstrate that T. hirsuta st. 072 produces multiple extracellular ligninolytic, cellulolytic, hemicellulolytic, peroxide generating, and proteolytic enzymes, as well as cerato-platanins. In contrast to other white rot species described earlier, which mostly secreted glucanases and mannosidases in response to the presence of the lignocellulose substrate, T. hirsuta expressed a spectrum of extracellular cellulolytic enzymes containing predominantly cellobiases and xylanases. As proteomic analysis could not detect lignin peroxidase (LiP) among the secreted lignin degrading enzymes, we attr...
Nucleic Acids Research, 2000
The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses t... more The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses translation of its own mRNA. Previous studies implicated two segments of the untranslated 5′-leader of the mRNA in repressor binding, an RNA hairpin structure and the adjacent RNA to the 3′ side, which contains the Shine-Dalgarno sequence. Here, we show by in vitro gp43-RNA binding assays that both translated and untranslated segments of the mRNA contribute to the high affinity of gp43 to its mRNA target (translational operator), but that a Shine-Dalgarno sequence is not required for specificity. Nucleotide sequence specificity appears to reside solely in the operator's hairpin structure, which lies outside the putative ribosome-binding site of the mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restricted arrangement of purine relative to pyrimidine residues and an invariant 5′-AC-3′ in the unpaired (loop) segment of the RNA structure. We propose a generalized structure for the hairpin that encompasses these features and discuss possible relationships between RNA binding determinants of gp43 and DNA binding by this replication enzyme.
Journal of Molecular Biology, 2006
We determined the sequence of the 152,372-bp genome of ϕYS40, a lytic tailed bacteriophage of The... more We determined the sequence of the 152,372-bp genome of ϕYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of ϕYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria. ϕYS40 encodes a cluster of proteins involved in nucleotide salvage, such as flavin-dependent thymidylate synthase, thymidylate kinase, ribonucleotide reductase, and deoxycytidylate deaminase, and in DNA replication, such as DNA primase, helicase, type A DNA polymerase, and predicted terminal protein involved in initiation of DNA synthesis. The structural genes of ϕYS40, most of which have no similarity to sequences in public databases, were identified by mass-spectrometric analysis of purified virions. Various ϕYS40 proteins have different phylogenetic neighbors, including Myovirus, Podovirus, and Siphovirus gene products, bacterial genes, and in one case, a dUTPase from a eukaryotic virus. ϕYS40 has apparently arisen through multiple acts of recombination between different phage genomes as well as through acquisition of bacterial genes.
Journal of Biological Chemistry, 1997
DNA polymerase of phage T4 (T4 gp43), an essential component of the T4 DNA replicase, is a multif... more DNA polymerase of phage T4 (T4 gp43), an essential component of the T4 DNA replicase, is a multifunctional single-chained (898-amino acid) protein that catalyzes the highly accurate synthesis of DNA in phage replication. The enzyme functions both as a DNA-binding replication protein and as a sequence-specific RNA-binding autogenous translational repressor. We have utilized a phylogenetic approach to study the relationships between the two nucleic acid-binding functions of the protein. We found that autogenous translational control of gp43 biosynthesis has been conserved in phage RB69, a distant relative of T4, although we also found that the RB69 system differs from its T4 counterpart in two regards: (a) nucleotide sequence and predicted secondary structure of the RNA target (translational operator), and (b) RNA specificity of the protein. T4 gp43 is specific to the RNA operator sequence of the T4 genome whereas RB69 gp43 can bind and repress operator RNA from both phages equally well. In studies with T4-RB69 gp43 chimeras, we mapped T4 gp43 RNA-binding specificity to a protein segment that also harbors important determinants for DNA binding and the polymerase catalytic function. Our results suggest that RNA functions as a regulator of both the dosage and activity of this DNA replication enzyme.
Environmental Science & Technology, 1998
An immunoassay is described that measured Cd(II) in aqueous samples at concentrations from approx... more An immunoassay is described that measured Cd(II) in aqueous samples at concentrations from approximately 7 to 500 ppb. The assay utilized a monoclonal antibody that bound tightly to a cadmium-ethylenediaminetetraacetic acid (EDTA) complex but not to metal-free EDTA. A inhibition immunoassay format was employed for this analysis; ionic cadmium was diluted into an excess of EDTA before being incubated with the antibody in the presence of an immobilized Cd(II)-EDTA conjugate. Ca(II), Na(I), and K(I), cations commonly encountered in ambient water samples, did not interfere with the cadmium immunoassay at concentrations approaching their solubility limit. The assay reliably measured Cd(II) in the presence of a 1 mM excess of Fe(III), Mg(II), and Pb(II). Zn(II) and Ni(II) had minimal effect on the assay at levels below 100 µM, and the immunoassay was relatively insensitive to interferences by In(III) and Mn(II) at concentrations up to 10 µM. Hg-(II) had the ability to cause a false positive in the assay, but only at concentrations higher than 1 µM. The assay compared favorably with atomic absorption spectroscopy in its ability to measure cadmium in spiked water samples taken from a Louisiana bayou.
Biosensors and Bioelectronics, 2001
Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reag... more Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA™ 3000. Four different monoclonal antibodies specific for complexes of EDTA-Cd(II), DTPA-Co(II), 2,9-dicarboxyl-1,10-phenanthroline-U(VI), or cyclohexyl-DTPA-Pb(II) were incubated with the appropriate soluble metal-chelate complex. In the microwell assay format, the immobilized version of the metal-chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10-1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal-chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25 9 11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81-7.77% and 3.62-14.16% for within run and between run precision, respectively.
Biopolymers and Cell, 1988
Biochemistry, 2012
We have previously introduced a general kinetic approach for comparative study of processivity, t... more We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510-13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also
Applied and Environmental Microbiology, 2011
Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide,... more Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans -splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the recept...
Proceedings of the National Academy of Sciences, 2002
Helix–hairpin–helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The... more Helix–hairpin–helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH) 2 domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of Taq DNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH 2 terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the p...
Background: Bifidobacteria are frequently proposed to be associated with good intestinal health p... more Background: Bifidobacteria are frequently proposed to be associated with good intestinal health primarily because of their overriding dominance in the feces of breast fed infants. However, clinical feeding studies with exogenous bifidobacteria show they don't remain in the intestine, suggesting they may lose competitive fitness when grown outside the gut. Results: To further the understanding of genetic attenuation that may be occurring in bifidobacteria cultures, we obtained the complete genome sequence of an intestinal isolate, Bifidobacterium longum DJO10A that was minimally cultured in the laboratory, and compared it to that of a culture collection strain, B. longum NCC2705. This comparison revealed colinear genomes that exhibited high sequence identity, except for the presence of 17 unique DNA regions in strain DJO10A and six in strain NCC2705. While the majority of these unique regions encoded proteins of diverse function, eight from the DJO10A genome and one from NCC2705, encoded gene clusters predicted to be involved in diverse traits pertinent to the human intestinal environment, specifically oligosaccharide and polyol utilization, arsenic resistance and lantibiotic production. Seven of these unique regions were suggested by a base deviation index analysis to have been precisely deleted from strain NCC2705 and this is substantiated by a DNA remnant from within one of the regions still remaining in the genome of NCC2705 at the same locus. This targeted loss of genomic regions was experimentally validated when growth of the intestinal B. longum in the laboratory for 1,000 generations resulted in two large deletions, one in a lantibiotic encoding region, analogous to a predicted deletion event for NCC2705. A simulated fecal growth study showed a significant reduced competitive ability of this deletion strain against Clostridium difficile and E. coli. The deleted region was between two IS30 elements which were experimentally
Analytica Chimica Acta
At least 20 metals are known to be toxic and fully half of these, including arsenic, cadmium, chr... more At least 20 metals are known to be toxic and fully half of these, including arsenic, cadmium, chromium, copper, lead, mercury, nickel, selenium, silver, and zinc, are released into the environment in quantities that pose a risk to human health [1]. Metals are classified as persistent ...
Biotechnology and Bioengineering, 2005
Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally at 1008C, encodes three prote... more Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally at 1008C, encodes three protein chaperones, a small heat shock protein (sHsp), a prefoldin (Pfd), and a chaperonin (Cpn). In this study, we report that the passive chaperones sHsp and Pfd from P. furiosus can boost the protein refolding activity of the ATP-dependent Cpn from the same hyperthermophile. The thermo-stability of Taq polymerase was significantly improved by combinations of P. furiosus chaperones, showing ongoing protein folding activity at elevated temperatures and during thermal cycling. Based on these results, we propose that the protein folding apparatus in the hyperthermophilic archaeon, P. furiosus can be utilized to enhance the durability and cost effectiveness of high temperature biocatalysts.
![Research paper thumbnail of [Regulation of aldose reductase activity. Mechanism of action of an activated form of the enzyme]](https://a.academia-assets.com/images/blank-paper.jpg)
](Biokhimii͡a (Moscow, Russia), 1992
Activation of bovine eye lens aldose reductase during its incubation with NADPH and glucose was s... more Activation of bovine eye lens aldose reductase during its incubation with NADPH and glucose was studied. The activated form of the enzyme was isolated, and the rate of glucose reduction measured within a broad range of substrate concentrations. Spectrophotometric titration and equilibrium gel-filtration were used to study the interaction of the enzyme active center with substrates. It was found that the reaction kinetics obeys the mechanism of a quasi-equilibrium binding of substrates with isomerization of the enzyme complexes with nicotinamide dinucleotide phosphates. This activation is accompanied by a transition from non-ordered to highly ordered binding of the substrates. The effect of ligands in the catalytic and inhibitory centers of the activated enzyme on the catalytic reaction was examined. It was found that the activated form of aldose reductase is characterized by a lower affinity of the inhibitory center for the flavonoid, morin. Morin binding not only inhibits the react...
Journal of Biological Chemistry, 1994
BMC microbiology, Jun 13, 2016
Fungi are organisms with the highest natural capacity to degrade lignocellulose substrates, which... more Fungi are organisms with the highest natural capacity to degrade lignocellulose substrates, which is enabled by complex systems of extracellular enzymes, whose expression and secretion depend on the characteristics of substrates and the environment. This study reports a secretome analysis for white-rot basidiomycete Trametes hirsuta cultivated on a synthetic media and a lignocellulose substrate. We demonstrate that T. hirsuta st. 072 produces multiple extracellular ligninolytic, cellulolytic, hemicellulolytic, peroxide generating, and proteolytic enzymes, as well as cerato-platanins. In contrast to other white rot species described earlier, which mostly secreted glucanases and mannosidases in response to the presence of the lignocellulose substrate, T. hirsuta expressed a spectrum of extracellular cellulolytic enzymes containing predominantly cellobiases and xylanases. As proteomic analysis could not detect lignin peroxidase (LiP) among the secreted lignin degrading enzymes, we attr...
Nucleic Acids Research, 2000
The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses t... more The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses translation of its own mRNA. Previous studies implicated two segments of the untranslated 5′-leader of the mRNA in repressor binding, an RNA hairpin structure and the adjacent RNA to the 3′ side, which contains the Shine-Dalgarno sequence. Here, we show by in vitro gp43-RNA binding assays that both translated and untranslated segments of the mRNA contribute to the high affinity of gp43 to its mRNA target (translational operator), but that a Shine-Dalgarno sequence is not required for specificity. Nucleotide sequence specificity appears to reside solely in the operator's hairpin structure, which lies outside the putative ribosome-binding site of the mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restricted arrangement of purine relative to pyrimidine residues and an invariant 5′-AC-3′ in the unpaired (loop) segment of the RNA structure. We propose a generalized structure for the hairpin that encompasses these features and discuss possible relationships between RNA binding determinants of gp43 and DNA binding by this replication enzyme.
Journal of Molecular Biology, 2006
We determined the sequence of the 152,372-bp genome of ϕYS40, a lytic tailed bacteriophage of The... more We determined the sequence of the 152,372-bp genome of ϕYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of ϕYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria. ϕYS40 encodes a cluster of proteins involved in nucleotide salvage, such as flavin-dependent thymidylate synthase, thymidylate kinase, ribonucleotide reductase, and deoxycytidylate deaminase, and in DNA replication, such as DNA primase, helicase, type A DNA polymerase, and predicted terminal protein involved in initiation of DNA synthesis. The structural genes of ϕYS40, most of which have no similarity to sequences in public databases, were identified by mass-spectrometric analysis of purified virions. Various ϕYS40 proteins have different phylogenetic neighbors, including Myovirus, Podovirus, and Siphovirus gene products, bacterial genes, and in one case, a dUTPase from a eukaryotic virus. ϕYS40 has apparently arisen through multiple acts of recombination between different phage genomes as well as through acquisition of bacterial genes.
Journal of Biological Chemistry, 1997
DNA polymerase of phage T4 (T4 gp43), an essential component of the T4 DNA replicase, is a multif... more DNA polymerase of phage T4 (T4 gp43), an essential component of the T4 DNA replicase, is a multifunctional single-chained (898-amino acid) protein that catalyzes the highly accurate synthesis of DNA in phage replication. The enzyme functions both as a DNA-binding replication protein and as a sequence-specific RNA-binding autogenous translational repressor. We have utilized a phylogenetic approach to study the relationships between the two nucleic acid-binding functions of the protein. We found that autogenous translational control of gp43 biosynthesis has been conserved in phage RB69, a distant relative of T4, although we also found that the RB69 system differs from its T4 counterpart in two regards: (a) nucleotide sequence and predicted secondary structure of the RNA target (translational operator), and (b) RNA specificity of the protein. T4 gp43 is specific to the RNA operator sequence of the T4 genome whereas RB69 gp43 can bind and repress operator RNA from both phages equally well. In studies with T4-RB69 gp43 chimeras, we mapped T4 gp43 RNA-binding specificity to a protein segment that also harbors important determinants for DNA binding and the polymerase catalytic function. Our results suggest that RNA functions as a regulator of both the dosage and activity of this DNA replication enzyme.
Environmental Science & Technology, 1998
An immunoassay is described that measured Cd(II) in aqueous samples at concentrations from approx... more An immunoassay is described that measured Cd(II) in aqueous samples at concentrations from approximately 7 to 500 ppb. The assay utilized a monoclonal antibody that bound tightly to a cadmium-ethylenediaminetetraacetic acid (EDTA) complex but not to metal-free EDTA. A inhibition immunoassay format was employed for this analysis; ionic cadmium was diluted into an excess of EDTA before being incubated with the antibody in the presence of an immobilized Cd(II)-EDTA conjugate. Ca(II), Na(I), and K(I), cations commonly encountered in ambient water samples, did not interfere with the cadmium immunoassay at concentrations approaching their solubility limit. The assay reliably measured Cd(II) in the presence of a 1 mM excess of Fe(III), Mg(II), and Pb(II). Zn(II) and Ni(II) had minimal effect on the assay at levels below 100 µM, and the immunoassay was relatively insensitive to interferences by In(III) and Mn(II) at concentrations up to 10 µM. Hg-(II) had the ability to cause a false positive in the assay, but only at concentrations higher than 1 µM. The assay compared favorably with atomic absorption spectroscopy in its ability to measure cadmium in spiked water samples taken from a Louisiana bayou.
Biosensors and Bioelectronics, 2001
Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reag... more Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA™ 3000. Four different monoclonal antibodies specific for complexes of EDTA-Cd(II), DTPA-Co(II), 2,9-dicarboxyl-1,10-phenanthroline-U(VI), or cyclohexyl-DTPA-Pb(II) were incubated with the appropriate soluble metal-chelate complex. In the microwell assay format, the immobilized version of the metal-chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10-1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal-chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25 9 11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81-7.77% and 3.62-14.16% for within run and between run precision, respectively.
Biopolymers and Cell, 1988
Biochemistry, 2012
We have previously introduced a general kinetic approach for comparative study of processivity, t... more We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510-13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also
Applied and Environmental Microbiology, 2011
Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide,... more Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans -splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the recept...
Proceedings of the National Academy of Sciences, 2002
Helix–hairpin–helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The... more Helix–hairpin–helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH) 2 domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of Taq DNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH 2 terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the p...
Background: Bifidobacteria are frequently proposed to be associated with good intestinal health p... more Background: Bifidobacteria are frequently proposed to be associated with good intestinal health primarily because of their overriding dominance in the feces of breast fed infants. However, clinical feeding studies with exogenous bifidobacteria show they don't remain in the intestine, suggesting they may lose competitive fitness when grown outside the gut. Results: To further the understanding of genetic attenuation that may be occurring in bifidobacteria cultures, we obtained the complete genome sequence of an intestinal isolate, Bifidobacterium longum DJO10A that was minimally cultured in the laboratory, and compared it to that of a culture collection strain, B. longum NCC2705. This comparison revealed colinear genomes that exhibited high sequence identity, except for the presence of 17 unique DNA regions in strain DJO10A and six in strain NCC2705. While the majority of these unique regions encoded proteins of diverse function, eight from the DJO10A genome and one from NCC2705, encoded gene clusters predicted to be involved in diverse traits pertinent to the human intestinal environment, specifically oligosaccharide and polyol utilization, arsenic resistance and lantibiotic production. Seven of these unique regions were suggested by a base deviation index analysis to have been precisely deleted from strain NCC2705 and this is substantiated by a DNA remnant from within one of the regions still remaining in the genome of NCC2705 at the same locus. This targeted loss of genomic regions was experimentally validated when growth of the intestinal B. longum in the laboratory for 1,000 generations resulted in two large deletions, one in a lantibiotic encoding region, analogous to a predicted deletion event for NCC2705. A simulated fecal growth study showed a significant reduced competitive ability of this deletion strain against Clostridium difficile and E. coli. The deleted region was between two IS30 elements which were experimentally
Analytica Chimica Acta
At least 20 metals are known to be toxic and fully half of these, including arsenic, cadmium, chr... more At least 20 metals are known to be toxic and fully half of these, including arsenic, cadmium, chromium, copper, lead, mercury, nickel, selenium, silver, and zinc, are released into the environment in quantities that pose a risk to human health [1]. Metals are classified as persistent ...
Biotechnology and Bioengineering, 2005
Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally at 1008C, encodes three prote... more Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally at 1008C, encodes three protein chaperones, a small heat shock protein (sHsp), a prefoldin (Pfd), and a chaperonin (Cpn). In this study, we report that the passive chaperones sHsp and Pfd from P. furiosus can boost the protein refolding activity of the ATP-dependent Cpn from the same hyperthermophile. The thermo-stability of Taq polymerase was significantly improved by combinations of P. furiosus chaperones, showing ongoing protein folding activity at elevated temperatures and during thermal cycling. Based on these results, we propose that the protein folding apparatus in the hyperthermophilic archaeon, P. furiosus can be utilized to enhance the durability and cost effectiveness of high temperature biocatalysts.
Biokhimii͡a (Moscow, Russia), 1992
Activation of bovine eye lens aldose reductase during its incubation with NADPH and glucose was s... more Activation of bovine eye lens aldose reductase during its incubation with NADPH and glucose was studied. The activated form of the enzyme was isolated, and the rate of glucose reduction measured within a broad range of substrate concentrations. Spectrophotometric titration and equilibrium gel-filtration were used to study the interaction of the enzyme active center with substrates. It was found that the reaction kinetics obeys the mechanism of a quasi-equilibrium binding of substrates with isomerization of the enzyme complexes with nicotinamide dinucleotide phosphates. This activation is accompanied by a transition from non-ordered to highly ordered binding of the substrates. The effect of ligands in the catalytic and inhibitory centers of the activated enzyme on the catalytic reaction was examined. It was found that the activated form of aldose reductase is characterized by a lower affinity of the inhibitory center for the flavonoid, morin. Morin binding not only inhibits the react...