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Biomedical subjects

D E Taylor

Publications and source records attributed to D E Taylor.

At least 91 records · Page 5Linked to original sources
PubMed1996

Sequencing, expression, and genetic characterization of the Helicobacter pylori ftsH gene encoding a protein homologous to members of a novel putative ATPase family.

In this study, we isolated and sequenced a Helicobacter pylori gene, designated ftsH, coding for a 632-amino-acid protein which displayed striking similarity throughout its full length to FtsH proteins identified in Escherichia coli, Lactococcus lactis, and Bacillus subtilis. H. pylori FtsH also possessed approximately 200-amino-acid region containing a putative ATPase module which is conserved among members of the AAA protein family (AAA, ATPase associated with diverse cellular activities). The H. pylori ftsH product was overexpressed in E. coli and reacted immunologically with an anti-E. coli FtsH serum (T. Tomoyasu, K. Yamanaka, K. Murata, T. Suzaki, P. Bouloc, A. Kato, H. Niki, S. Hiraga, and T. Ogura, J. Bacteriol. 175:1352-1357, 1993). FtsH was also shown to be present in the membrane fraction of H. pylori, suggesting that it is membrane bound. Disruption of the ftsH gene led to the loss of viability of H. pylori, demonstrating that this gene is essential for cell growth. Overproduction of both H. pylori FtsH and E. coli FtsH together tremendously reduced the growth rate of the E. coli host cells, whereas the growth of the E. coli cells carrying the wild-type E. coli ftsH operon on the chromosome was not significantly affected by overproduction of H. pylori FtsH itself. This result suggests that the abnormal growth of cells results from interaction between H. pylori FtsH and E. coli FtsH.

Adenosine Triphosphatases↗
PubMed1996

Use of near-infrared spectroscopy to monitor tissue oxygenation.

Near-infrared spectroscopy (NIRS) is a relatively new tool that allows continuous noninvasive monitoring of in vivo oxygenation in selected tissues such as muscle and brain. Since hemoglobin, myoglobin, and cytochrome c oxidase are the only biological compounds to exhibit variable absorption of near-infrared (NIR) light in response to changes in oxygen availability, NIRS can determine changes in tissue oxygenation. NIRS can measure regional blood volume, local oxyhemoglobin and deoxyhemoglobin contents, and reduction-oxidation state of cellular mitochondrial cytochrome a, a3. As a comprehensive monitor of regional oxygen metabolism, NIRS has been applied in certain clinical and research settings. Despite technical limitations and the lack of definite "gold standards" to allow validation of results, NIRS remains a promising technology with applications in both the critical care environment and the research laboratory studying mechanisms of oxygen metabolism.

Blood Volume↗
PubMed1995

Genome map of Campylobacter fetus subsp. fetus ATCC 27374.

A physical map of the chromosome of Campylobacter fetus subsp. fetus was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by SalI, SmaI and NotI. Digestion of the type strain ATCC 27374 with these restriction endonucleases resulted in generating 4-14 fragments. The order of the fragments was deduced from hybridization of these restriction fragments to Southern blots of pulsed-field gel electrophoresis gels generated by the other two enzymes. The estimated genome size was 1160 kb. The position of several homologous and heterologous genes was determined on the circular map. These included the 2.8-kb sapA gene, encoding the 97-kDa surface array protein. Three copies of ribosomal RNA genes for which the 16S, 23S and 5S rRNA appeared to be located in close proximity in each of the three regions. The RNA polymerase genes rpoA, rpoB, and rpoD were mapped and appeared to be situated close together in one region. The flagellin genes (flaAB) of C. jejuni and the gyrase genes gyrA and gyrB of C. perfringens and Bacillus subtilis, respectively, were used to identify the locations of flaAB, the gyrA and the gyrB genes on the ATCC 27374 chromosome.

Base Sequence↗
PubMed1995

Reactive oxygen species produced by liver mitochondria of rats in sepsis.

Reactive oxygen species (ROS) can be generated in experimental shock states through several different mechanisms. We measured ROS production in metabolically active liver mitochondria from rats rendered septic by cecal ligation and puncture. By polarography, the State 4 and State 3 respiration rates of liver mitochondria isolated from septic animals were no different from control organelles. During oxidation of succinate, however, nonenzymatic hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid by mitochondria from septic rats was increased, indicating generation of hydroxyl radical (OH.). Inhibition of electron transport at Complex I with rotenone had no effect on this pattern of OH. production, but rotenone and cyanide abolished the differences in OH. formation between control and septic liver mitochondria. Measurements of H2O2 release suggested that septic mitochondria will increase rates of H2O2 production in the presence of succinate. Additional investigations revealed no difference in the release of iron between septic and control mitochondria. When referenced to respiration rate, both OH. and H2O2 production were greater in septic liver mitochondria. The reproducible effect of sepsis on generation of reactive oxygen species by liver mitochondria utilizing FAD-linked but not NAD-linked substrates suggests that enhanced mitochondrial oxidative stress in sepsis is related to alterations in the activity of Complex II of the electron transport chain.

Animals↗
PubMed1995

Oxidative metabolism in sepsis and sepsis syndrome.

The high mortality associated with sepsis syndrome and multiple organ dysfunction syndrome has persisted despite extraordinary research efforts in the laboratory and the intensive care unit. These syndromes produce systemic tissue damage that is likely to result from widespread inflammation and subsequent endothelial injury. This article reviews the oxidative metabolic effects and responses to sepsis syndrome at several levels: the oxygen transport system, the cell, and the mitochondrion. Specifically, aerobic metabolism of carbon substrates and oxygen is altered in sepsis. As a result of systemic inflammation and nonmetabolic oxygen use, oxidative stress may occur both outside and inside the cell. The consequences of these oxidative processes during sepsis may be ongoing cell damage mediated by reactive oxygen and nitrogen oxide species that culminates in multisystem organ failure.

Energy Metabolism↗
PubMed1995

The biosynthesis of Lewis X in Helicobacter pylori.

The biosynthesis of the Lewis X determinant (Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta-) in three strains of Helicobacter pylori has been investigated. Strains UA 861, UA 802 and UA 1182 contain alpha 1,3 fucosyltransferase and beta 1,4 galactosyltransferase activities that synthesize the Lewis X structure by the transfer of monosaccharides from GDP-fucose and UDP-galactose donors, respectively. The enzyme reaction products that formed were characterized by capillary zone electrophoresis and by 1H-NMR spectroscopy. The biosynthetic pathway is therefore identical to that found in humans. In the three strains, the fucosyltransferase and galactosyltransferase activities differed in various cellular fractions. Km values for their donor and acceptor substrates also differed.

Carbohydrate Sequence↗
PubMed1995

The tellurite-resistance determinants tehAtehB and klaAklaBtelB have different biochemical requirements.

The tehAtehB operon from the Escherichia coli chromosome (32.3 min) mediates resistance to potassium tellurite (K2TeO3) when expressed on a multicopy plasmid such as pUC8 (pTWT100). An MIC of 128 micrograms ml-1 is observed when tehAtehB is expressed in a wild-type host and grown on rich media. In this study, the tehAtehB determinant was transformed into mutants deficient in electron transport processes and/or thiol redox coupling within E. coli. These mutants included ubi, nad, cys, nar, trx, grx, gsh and sod. MICs of tehAtehB transformed into these mutants ranged from 1-16 micrograms K2TeO3 ml-1 compared to 0.03-2 micrograms ml-1 for strains transformed with a control plasmid. The tellurite-resistance determinant locus kilA cloned from the IncP alpha plasmid RK2Ter (pDT1558) was also investigated in these strains. This tellurite-resistance determinant showed little or no dependency on the host genotype. The ability of tehAtehB to mediate resistance in wild-type hosts is limited to rich medium. Rich medium may provide a key unidentified cofactor required by TehATehB that is not provided under minimal conditions. Again, the ability of the kilA determinant to mediate tellurite resistance was independent of medium conditions. These data suggest that either a reducing environment or electron-reducing equivalents are required for tehAtehB to mediate high levels of resistance to potassium tellurite. Therefore, the two resistance determinants studied here possess two very different biochemical mechanisms of resistance. Our data also suggest a mechanism for endogenous resistance to tellurite which involves nitrate reductase, superoxide dismutase, and thiol redox processes.

Culture Media↗
PubMed1995

Nucleotide sequence and mutational analysis indicate that two Helicobacter pylori genes encode a P-type ATPase and a cation-binding protein associated with copper transport.

A 2.7 kb fragment of Helicobacter pylori UA802 chromosomal DNA was cloned and sequenced. Three open reading frames (designated ORF1, ORF2 and ORF3, respectively) were predicted from the DNA sequence, of which ORF1 and ORF2 appeared to be located within the same operon. The deduced 611-amino-acid sequence of ORF1, a P-type ATPase (designated hpCopA), had striking homology (29-38%) with several bacterial P-type ATPase and contained the potential functional domains conserved in P-type ATPases from various sources ranging from bacterial to human. A protein of 66 amino acids (designated hpCopP) encoded by ORF2 shared extensive sequence similarity with MerP, a periplasmic mercuric ion-transporting protein, and contains the heavy metal-binding motif. Disruption of ORF1 with a chloramphenicol-resistance cassette (CAT) rendered the H. pylori mutants more susceptible to cupric ion than their parental strains, whereas there is no significant alteration of susceptibility to Ni2+, Cd2d+ and Hg2+ between the mutants and the parental strains. The results obtained indicate that ORF1 and ORF2 comprise a cation-transporting system which is associated with copper export out of the H. pylori cells.

Adenosine Triphosphatases↗
PubMed1995

Helicobacter pylori expresses a complex surface carbohydrate, Lewis X.

Monoclonal antibodies (MAbs) specific for Lewis X (Lex) reacted with whole cells of Helicobacter pylori NCTC11637, UA799, UA802, UA825, UA861, UA1182, and UA1206 in immunoelectron microscopy and enzyme-linked immunosorbent assay (ELISA) experiments. These MAbs have documented specificity to Lex, whereas MAbs for Lea and Leb were negative in both immunoelectron microscopy and ELISA. H. pylori coccoid forms also reacted with the MAbs, whereas the flagellum lacking the sheath showed no reactivity. The Lex structures were associated with membrane fractions in the ELISA experiments, and silver-stained sodium dodecyl sulfate-polyacrylamide gels confirmed the presence of lipopolysaccharides which reacted with the MAbs in immunoblots. Serum from an H. pylori-infected individual contained immunoglobulins which blocked the binding of the Lex MAbs, indicating that part of the host immune response to H. pylori is to the Lex structure. The ability of this gastric pathogen to mimic an oncofetal antigen (self) could explain the down regulation of anti-H. pylori T-cell response seen in H. pylori-infected individuals.

Animals↗
PubMed1995

Phage inhibition, colicin resistance, and tellurite resistance are encoded by a single cluster of genes on the IncHI2 plasmid R478.

A region of the IncHI2 plasmid R478, encoding the phenotypes of tellurite resistance (Ter), phage inhibition (Phi), and colicin resistance (PacB), was cloned and sequenced. Analysis indicated seven open reading frames (ORFs), whose genes were designated terZ, -A, -B, -C, -D, -E, and -F. Five of these predicted ORFs (A to E) had extensive amino acid homology with the previously reported ORFs of the IncHI2 Ter operon from plasmid pMER610. There were domains of highly conserved amino acid residues within the group TerA, -D, -E, and -F and within TerD, -E, and -Z, but no consensus could be found among all five putative polypeptides. There were also regions of good identity and similarity between individual pairs of ORFs which was not reflected in the multiple alignments. The three phenotypes were expressed in Escherichia coli DH5 alpha by an 8.4-kb EcoRI insert subcloned from a cosmid of R478. The latter insert was clonable only as a double insertion with a 4.5-kb fragment, and forced deletion of the smaller fragment was lethal to cells. This lethality was not dependent on the cloned orientation of either fragment, suggesting that there is a trans-acting element in the 4.5-kb fragment. Tn1000 mutagenesis of one of the double-insert clones, pDT2575, showed that the phenotypes, including multiple colicin resistance, were genetically linked. Transpositions into terD, terC, and terZ reduced or abolished all phenotypes, while inserts into terE and terF had no effect on the phenotypes. Insertions in terA reduced phage inhibition levels only. The presence of the terZ and terF ORFs in pMER610 was confirmed, and derivatives of this plasmid mediated Phi, PacB, and Ter.

Amino Acid Sequence↗
PubMed1995

Characterization of chromosomal DNA profiles from Helicobacter pylori strains isolated from sequential gastric biopsy specimens.

The restriction endonuclease profiles of DNAs from Helicobacter pylori strains isolated from 20 patients in two or more consecutive biopsy specimens over a period of up to 2 years were analyzed by pulsed-field gel electrophoresis with NotI and NruI. H. pylori strains possess a high degree of genomic diversity which was not observed to occur in vivo, and attempts to observe it in vitro were not successful.

DNA, Bacterial↗
PubMed1995

Neither reduced uptake nor increased efflux is encoded by tellurite resistance determinants expressed in Escherichia coli.

Rates of uptake of the TeO3(2-) oxyanion were investigated in Escherichia coli cells containing tellurite resistance determinants from both plasmid (RK2Ter, R478, pMER610, MIP233, pHH1508a, pMUR) and chromosomal (tehAB) sources. The uptake was investigated to determine whether or not reduced uptake or increased efflux is involved in the tellurite resistance mechanism. Reduced TeO3(2-) uptake generated by cultures harboring arsABC from the plasmid R773, which has been previously shown to be an oxyanion efflux transporter, was used as the standard. Uptake curves were found to be essentially identical among E. coli cultures harboring the tellurite resistance plasmids RK2Ter, pMER610, pHH1508a, and pMUR and cultures harboring tellurite-sensitive control plasmids. Cultures harboring clones of the tehAB operon from E. coli showed no change in the TeO3(2-) accumulation. Cultures harboring R478 demonstrated reduced uptake. However, a subclone containing only the tellurite resistance determinant displayed no reduced uptake. This suggests that there may be another determinant on R478 other than the primary tellurite resistance determinant that gives rise to TeO3(2-) efflux. These results demonstrate that neither reduced uptake nor increased efflux is responsible for the tellurite resistance in the resistance determinants investigated here.

Adenosine Triphosphatases↗
PubMed1994

Genome conservation in Helicobacter mustelae as determined by pulsed-field gel electrophoresis.

Genomic DNA from 15 strains of Helicobacter mustelae was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with PacI and SfiI. H. mustelae genome DNA appeared very similar in all strains examined, whether isolated from ferrets or mink or from animals bred in either the USA or in the UK. The H. mustelae genome size was estimated to be 1.7 Mb, similar in size to that of H. pylori. A minor difference in PacI PFGE pattern and genome size was observed between rifampicin-resistant and rifampicin-susceptible derivatives of H. mustelae F251. Another minor difference in genome pattern based on PFGE with SfiI was observed between an H. mustelae strain used to experimentally infect four ferrets which resulted in loss of an SfiI site in strains obtained from the newly infected ferrets. Thus, although minor differences in PFGE pattern were noted, H. mustelae lacks the genomic diversity observed in H. pylori.

Animals↗
PubMed1994

Incidence of tellurite resistance determinants among plasmids of different incompatibility groups.

Twenty tellurite resistance (TeR) plasmids of IncH12, IncH13, and IncHII groups isolated from Enterobacteriaceae hybridized with the pMJ606 (IncHI2) TeR probe, whereas the promiscuous Pseudomonas IncP alpha plasmids hybridized with only the IncP alpha TeR probe (pDT1555). Neither of the TeR probes hybridized with the IncP2 Pseudomonas plasmids, which also specify TeR. None of the plasmids hybridized with pUM3, a plasmid carrying the arsABC genes that encodes resistance to arsenite, arsenate, antimony, and moderate levels of tellurite. These results show that the IncH12 TeR determinant is widespread in the Enterobacteriaceae and support the hypothesis that there are at least five distinct TeR determinants: (1) the telAB genes from the Escherichia coli chromosome; (2) IncHI2; (3) IncP2; (4) arsABC; and (5) the TeR determinants of IncP alpha plasmids.

Drug Resistance, Microbial↗