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V L Chan

Publications and source records attributed to V L Chan.

At least 19 recordsLinked to original sources
PubMed2005

Enzymatic activity of Campylobacter jejuni hippurate hydrolase.

The hippurate hydrolase enzyme of Campylobacter jejuni was expressed in Escherichia coli as a six-histidine-tagged fusion protein. The purified recombinant enzyme was characterized to gain an understanding of the structure and activity of the hippurate hydrolase. The recombinant enzyme had a native molecular mass of 193+/- 11 kDa a reduced molecular mass of 42.4+/- 0.8 kDa, and possessed 1.98+/- 0.68 molecules of zinc per enzyme subunit molecule, suggesting that it was a homotetramer with two associated zinc ions. The enzyme was a metallocarboxypeptidase that was sensitive to silver, copper and ferrous ions, and displayed optimal activity at pH 7.5 and 50 degrees C. It hydrolyzed carboxypeptidase substrates in vitro, displaying its highest activity against N-benzoyl-linked small aliphatic amino acids. A high proportion of the enzyme structure consisted of highly ordered alpha-helix and beta-sheet sequences. An alignment of the amino acid sequence of the hippurate hydrolase enzyme with those of related enzymes with similar activities revealed several conserved amino acids, which might be involved in enzyme catalysis or metal ion binding for the enzyme. Site-directed mutagenesis of the recombinant enzyme demonstrated that the Asp(76), Aps(104), Glu(134), Glu(135), His(161) and His(356) positions were important for the catalytic activity of the enzyme.

Amidohydrolases↗
PubMed2005

Duration of RT-PCR positivity in severe acute respiratory syndrome.

Severe acute respiratory syndrome (SARS) is a highly infectious respiratory infection with a high mortality. The duration of infectivity is unknown. The RT-PCR positivity for SARS-associated coronavirus (SARS-CoV) was followed in 45 virologically confirmed SARS patients. Serial RT-PCRs for SARS-CoV were performed in the nasopharyngeal aspirate, stool and urine of 45 SARS patients who survived until discharge. All patients had at least one site that was positive for SARS-CoV on presentation. Time to RT-PCR conversion was studied in all patients. There were 15 males (33.3%) and 30 females (66.7%), with a mean+/- SD age of 40.7+/-14.7 yrs. The median (range) time of RT-PCR conversion was 30 days (2-81). On discharge from the hospital, 18 (40%) remained RT-PCR positive in at least one site. For patients with positive RT-PCR on discharge, the median (range) time to RT-PCR conversion after discharge was 13 days (2-60). A significant proportion of severe acute respiratory syndrome patients remained RT-PCR positive for severe acute respiratory syndrome-associated coronavirus for a substantial duration after discharge. The clinical significance is unknown and this finding merits further study. It is prudent to advise patients to adhere to strict personal hygiene on discharge until RT-PCR becomes negative.

Adolescent↗
PubMed2004

FlaC, a protein of Campylobacter jejuni TGH9011 (ATCC43431) secreted through the flagellar apparatus, binds epithelial cells and influences cell invasion.

Type III secretion systems identified in bacterial pathogens of animals and plants transpose effectors and toxins directly into the cytosol of host cells or into the extracellular milieu. Proteins of the type III secretion apparatus are conserved among diverse and distantly related bacteria. Many type III apparatus proteins have homologues in the flagellar export apparatus, supporting the notion that type III secretion systems evolved from the flagellar export apparatus. No type III secretion apparatus genes have been found in the complete genomic sequence of Campylobacter jejuni NCTC11168. In this study, we report the characterization of a protein designated FlaC of C. jejuni TGH9011. FlaC is homologous to the N- and C-terminus of the C. jejuni flagellin proteins, FlaA and FlaB, but lacks the central portion of these proteins. flaC null mutants form a morphologically normal flagellum and are highly motile. In wild-type C. jejuni cultures, FlaC is found predominantly in the extracellular milieu as a secreted protein. Null mutants of the flagellar basal rod gene (flgF) and hook gene (flgE) do not secrete FlaC, suggesting that a functional flagellar export apparatus is required for FlaC secretion. During C. jejuni infection in vitro, secreted FlaC and purified recombinant FlaC bind to HEp-2 cells. Invasion of HEp-2 cells by flaC null mutants was reduced to a level of 14% compared with wild type, suggesting that FlaC plays an important role in cell invasion.

Bacterial Adhesion↗
PubMed2004

Detection of species-specific helicobacter ribosomal DNA in intestinal biopsy samples from a population-based cohort of patients with ulcerative colitis.

The inflammatory bowel diseases are considered an abnormal host immune response to an environmental stimulus. Evidence suggests a role for intestinal bacteria in initiating and/or providing an ongoing stimulus for inflammation in inflammatory bowel disease. Helicobacter pylori is the major cause of active chronic gastritis and peptic ulcers in humans and has been linked to gastric carcinoma and lymphoma. Studies in various animal models, particularly mice, have identified enterohepatic Helicobacter species that are capable of causing hepatitis and enterocolitis. We hypothesize that Helicobacter species may have a role in maintaining inflammation in humans with inflammatory bowel disease. In order to investigate this, biopsy specimens were obtained from patients with and without inflammatory bowel disease. DNA was extracted from the tissues and subjected to PCR with primers designed to detect the ribosomal DNA of members of the Helicobacter species. DNA from six biopsy samples from 60 inflammatory bowel disease patients tested positive. This included 5 of 33 ulcerative colitis patients that were positive compared to 0 of 29 age-matched controls (P < 0.04). Sequencing of the bands produced by PCR amplification revealed >or=99% homology with H. pylori. These results indicate that a member of the Helicobacter species may be involved in some cases of ulcerative colitis.

Base Sequence↗
PubMed2004

Readmission rates and life threatening events in COPD survivors treated with non-invasive ventilation for acute hypercapnic respiratory failure.

BACKGROUND: Non-invasive ventilation (NIV) has been shown to reduce intubation and in-hospital mortality in patients with chronic obstructive pulmonary disease (COPD) and acute hypercapnic respiratory failure (AHRF). However, little information exists on the outcomes following discharge. A study was undertaken to examine the rates of readmission, recurrent AHRF, and death following discharge and the risk factors associated with them. METHODS: A cohort of COPD patients with AHRF who survived after treatment with NIV in a respiratory high dependency unit was prospectively followed from July 2001 to October 2002. The times to readmission, first recurrent AHRF, and death were recorded and analysed against potential risk factors collected during the index admission. RESULTS: One hundred and ten patients (87 men) of mean (SD) age 73.2 (7.6) years survived AHRF after NIV during the study period. One year after discharge 79.9% had been readmitted, 63.3% had another life threatening event, and 49.1% had died. Survivors spent a median of 12% of the subsequent year in hospital. The number of days in hospital in the previous year (p = 0.016) and a low Katz score (p = 0.018) predicted early readmission; home oxygen use (p = 0.002), APACHE II score (p = 0.006), and a lower body mass index (p = 0.041) predicted early recurrent AHRF or death; the MRC dyspnoea score (p<0.001) predicted early death. CONCLUSIONS: COPD patients with AHRF who survive following treatment with NIV have a high risk of readmission and life threatening events. Further studies are urgently needed to devise strategies to reduce readmission and life threatening events in this group of patients.

Acute Disease↗
PubMed2004

Spontaneous pneumomediastinum in patients with severe acute respiratory syndrome.

Spontaneous pneumomediastinum (SP) unrelated to assisted ventilation is a newly recognised complication of severe acute respiratory syndrome (SARS). The objective of the present study was to examine the incidence, risk factors and the outcomes of SP in a cohort of SARS victims from a community outbreak. Data were retrieved from a prospectively collected database of virologically confirmed SARS patients. One hundred and twelve cases were analysable, with 13 patients developing SP (11.6%) at a mean +/- SD of 19.6 +/- 4.6 days from symptom onset. Peak lactate dehydrogenase level was associated with the development of SP. SP was associated with increased intubation and a trend towards death. Drainage was required in five cases. For patients who survived, the SP and/or the associated pneumothoraces took a median of 28 days (interquartile range: 15-45 days) to resolve completely. In conclusion, spontaneous pneumomediastinum appeared to be a frequent complication of severe acute respiratory syndrome. Further research is needed to investigate its pathogenesis.

Adult↗
PubMed2001

Early unplanned readmission of patients with newly diagnosed tuberculosis discharged from acute hospital to ambulatory treatment.

OBJECTIVE: The aim of this study was to examine whether patients with newly diagnosed tuberculosis (TB) discharged to ambulatory treatment are at risk of unplanned readmission through the emergency department within 28 days of discharge, and the risk factors associated with such readmission. METHODOLOGY: A cohort of 134 patients admitted to an acute medical department with TB, who were subsequently discharged to ambulatory treatment of TB, were studied by a retrospective record review for unplanned readmission in 28 days. Potential risk factors associated with the readmission were recorded during hospital stay and follow-up visits, including age, sex, length of stay, substance abuse, need of assistance in the activities of daily living (ADL), comorbidities, non-compliance, drug complications and use of non-standard drug regimen. RESULTS: Up to 20.1% of patients were readmitted. Factors independently associated with early unplanned readmission were need of assistance in ADL, drug complications, the need to use a non-standard drug regimen and more than three non-chest comorbidities. CONCLUSIONS: A significant readmission rate was found in these patients and potential risk factors were identified. Ambulatory treatment for TB may not be appropriate for selected patients. Local guidelines for the management of TB patients at high risk of readmission is needed.

Activities of Daily Living↗
PubMed2001

JlpA, a novel surface-exposed lipoprotein specific to Campylobacter jejuni, mediates adherence to host epithelial cells.

A 1116 bp open reading frame (ORF), designated jlpA, encoding a novel species-specific lipoprotein of Campylobacter jejuni TGH9011, was identified from recombinant plasmid pHIP-O. The jlpA gene encodes a polypeptide (JlpA) of 372 amino acid residues with a molecular mass of 42.3 kDa. JlpA contains a typical signal peptide and lipoprotein processing site at the N-terminus. The presence of a lipid moiety on the JlpA molecule was confirmed by the incorporation of [3H]-palmitic acid. Immunoblotting analysis of cell surface extracts prepared using glycine-acid buffer (pH 2.2) and proteinase K digestion of whole cells indicated that JlpA is a surface-exposed lipoprotein in C. jejuni. JlpA is loosely associated with the cell surface, as it is easily extracted from the C. jejuni outer membrane by detergents, such as sarcosyl and Triton X-100. JlpA is released to the culture medium, and its concentration increases in a time-dependent fashion. The adherence of both insertion and deletion mutants of jlpA to HEp-2 epithelial cells was reduced compared with that of parental C. jejuni TGH9011. Adherence of C. jejuni to HEp-2 cells was inhibited in a dose-dependent manner when the bacterium was preincubated with anti-GST-JlpA antibodies or when HEp-2 cells were preincubated with JlpA protein. A ligand-binding immunoblotting assay showed that JlpA binds to HEp-2 cells, which suggests that JlpA is C. jejuni adhesin.

Adhesins, Bacterial↗
PubMed2000

Identification of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, arcobacter butzleri, and A. butzleri-like species based on the glyA gene.

Currently, the detection and identification of Campylobacter and Arcobacter species remains arduous, largely due to cross-species phenotypic similarities and a relatively narrow spectrum of biochemical reactivity. We have developed a PCR-hybridization strategy, wherein degenerate primers are used to amplify glyA fragments from samples, which are then subjected to species-specific oligodeoxyribonucleotide probe hybridizations, to identify and distinguish between Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, Arcobacter butzleri, and an A. butzleri-like species. Evaluation of this strategy with genomic DNA from different type strains suggests that this approach is both specific and sensitive and thus may be applicable in a diagnostic assay to identify and differentiate these highly related species.

Arcobacter↗
PubMed1999

Arginine biosynthesis in Campylobacter jejuni TGH9011: determination of the argCOBD cluster.

Arginine biosynthetic genes from Campylobacter jejuni TGH9011 were cloned by functional complementation of the respective Escherichia coli arginine biosynthetic mutants. Complementation of argA, argB, argC, argD, argE, argF, and argH auxotrophs was accomplished using a pBR322-based C. jejuni TGH9011 plasmid library. By cross-complementation analyses, the first four steps of arginine biosynthesis were shown to be closely linked on the genome. Two additional clones complementing the first (ArgA) and fifth (ArgE) steps in arginine biosynthesis were obtained. Neither recombinant showed linkage to the arg cluster, to each other, nor to other arginine biosynthetic functions by cross-complementation. Genes argF and argH were not linked to other arginine biosynthetic genes by cross-complementation analysis. Restriction enzyme patterns of recombinant plasmids fell into five groups. Group I contained the arg(ABCD) complementing locus. Group II and Group III were the two genetic loci corresponding to the argA and argE complementing genes. Group II contains the hipO gene encoding N-benzoylglycine-amino-acid amidohydrolase, also known as hippurate hydrolase. Group III contains the hipO homolog of C. jejuni. Group IV represents the argF gene. Group V is the argH gene. Functional complementation of mutations in the first four steps of the arginine biosynthetic pathway was obtained on recombinant plasmid pARGC2. The predicted order of gene complementation was argCargA(argBargD). The sequence of the insert in plasmid pARGC2 revealed direct homologs for argC, argB, and argD. However, sequence analysis of the gene complementing ArgA function in two separate E. coli argA mutants determined that the C. jejuni gene was not a canonical argA gene. The gene complementing the argA defect, which we call argO, showed limited homology to the streptothricin acetyltransferase gene (sat) of Escherichia coli. The flanking open reading frames in pARGC2 showed no homologies to arginine biosynthetic genes. The structure of the argCOBD gene arrangement is discussed with reference to the presence and location of other arginine biosynthetic genes on the genome of C. jejuni and other bacterial organisms.

Amino Acid Sequence↗
PubMed1998

Campylobacter upsaliensis: waiting in the wings.

Despite strong epidemiological evidence supporting an important role for Campylobacter upsaliensis as a human enteropathogen, it remains relatively unknown in the realm of clinical microbiology. Clinical studies indicate that infection with this organism usually is associated with benign self-limiting diarrhea. However, more serious illnesses, including spontaneous abortion and hemolytic-uremic syndrome, recently have been associated with human infections. Understanding of the virulence properties and molecular biology of C. upsaliensis is beginning to evolve. There is now a pressing need for controlled, prospective epidemiologic studies in addition to further in-depth investigation of the pathogenesis of this enteric campylobacter to more precisely define its role in human disease. Furthermore, since C. upsaliensis is sensitive to the antibiotics routinely used in Campylobacter selective media, widespread appreciation of the importance of this organism will rely on the development of widely applicable, effective techniques for its isolation.

Adult↗
PubMed1997

Campylobacter hyoilei Alderton et al. 1995 and Campylobacter coli Véron and Chatelain 1973 are subjective synonyms.

The taxonomic affiliation of Campylobacter hyoilei was reevaluated by examining a variety of phenotypic and genotypic criteria. Whole-cell protein electrophoresis and a comparison of 66 phenotypic characters revealed that reference strains of C. hyoilei were indistinguishable from Campylobacter coli strains. These data were confirmed by a DNA-DNA hybridization level of 67% between the type strains of the two species. Several species-specific assays based on PCR amplification or probe hybridization further substantiated that C. coli strains and C. hyoilei strains are indistinguishable. It is therefore concluded that C. hyoilei and C. coli represent the same species and that the former name should be regarded as a junior synonym of the latter name.

Bacterial Proteins↗
PubMed1996

Characterization of Campylobacter upsaliensis fur and its localization in a highly conserved region of the Campylobacter genome.

Despite increasing recognition of the importance of Campylobacter upsaliensis in human disease little is known about either the virulence properties or genetics of this enteric pathogen. The complete coding sequence of a C. upsaliensis gene has yet to be published. We have cloned and sequenced the complete iron-uptake regulatory (fur) gene from the type strain of this species. The C. upsaliensis fur homolog was isolated from a genomic library of C. upsaliensis ATCC 43954 constructed in phage lambdaGEM-11. The open reading frame identified encodes a polypeptide consisting of 156 amino acids. The 5'-flanking region of the C. upsaliensis fur gene contains 3 putative Fur-binding sequences and two catabolite activator-binding sequences indicating the potential for autogenous and cAMP-mediated regulation, respectively. Primer extension analysis identified a single transcription start site 262 nt upstream from the AUG initiation codon. Sequence analysis indicates that the Fur protein of C. upsaliensis is highly homologous (87% amino acid identity) to Campylobacter jejuni Fur. Furthermore, the arrangement of the lysS and glyA genes downstream of fur is precisely conserved in both C. upsaliensis ATCC 43954 and C. jejuni TGH9011. Using the polymerase chain reaction close linkage of fur with lysS and glyA was also observed among multiple isolates of C. upsaliensis, C. jejuni and C. coli suggesting a possible functional relevance for this conserved genetic arrangement in campylobacteria.

Amino Acid Sequence↗
PubMed1996

Pulsed-field gel electrophoresis indicates genotypic heterogeneity among Campylobacter upsaliensis strains.

To determine the genomic relatedness among a selection of animal and human Campylobacter upsaliensis isolates, macrorestriction profiles were generated for 20 C. upsaliensis strains, among 7 serogroups, using pulsed-field gel electrophoresis (PFGE). XhoI, SalI and SacII restriction enzyme profiles indicated genomic heterogeneity among strains. Using XhoI and SacII restriction enzyme digestion, genomic similarities between some pairs of strains were Lior serogroup specific. The genomic sizes of these isolates varied from 1.74 to 2.09 Mb. These results demonstrate molecular heterogeneity of this species similar to that found among Helicobacter pylori isolates. Among C. upsaliensis strains, PFGE is highly discriminatory and should prove a useful molecular typing method for epidemiological purposes.

Animals↗
PubMed1995

Complete sequences and organization of the rrnA operon from campylobacter jejuni TGH9011 (ATCC43431).

The rrnA ribosomal RNA (rRNA) operon of Campylobacter jejuni (Cj) TGH9011 (ATCC43431) was cloned and sequenced to completion. rRNAs were then characterized by primer extension and S1 nuclease mapping analysis. The secondary structure models of Cj 16S and 23S rRNAs were constructed, and the models were compared to the corresponding models from other eubacterial rRNA. The analysis presented a typical 5'-promoter-16S-tRNAs-23S-5S-terminator-3' prokaryotic rRNA operon structure. However, an unusual organization of the intercistronic tRNAs was observed where the two tRNAs, tRNA(Ala) and tRNA(Ile), were present in the order 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', which is opposite of the typical 5'-16S-tRNA(Ile)-tRNA(Ala)-23S-3' structure observed in other bacteria.

Base Sequence↗
PubMed1995

Cloning and transcription regulation of the ferric uptake regulatory gene of Campylobacter jejuni TGH9011.

A Campylobacter jejuni (Cj) TGH9011 (ATCC 43431) gene homologous to the Escherichia coli ferric uptake regulatory gene (fur) has been cloned and characterized. Cj fur encodes a polypeptide consisting of 157 amino acids (aa) (18.1 kDa). The 5'-flanking region of the Cj fur gene contains two putative catabolite activator protein (CAP)-binding sequences and four Fur boxes or Fur-binding sequences (FBS), implicating cAMP and autogenous regulation respectively. A major and a minor transcription start point (tsp) were active in Fe(+) and Fe(-) media and three tsp were suppressed in Fe(+) condition. The major transcript has an unusually short leader sequence. The homology of the Cj Fur to other Proteobacteria Fur proteins is moderately low with identity ranging from 36.3% for Yersinia pestis to 31.8% for Legionella pneumophila. Multiple alignments of the Fur sequences identified three conserved motifs, I [aGLKvTlpR1KiL], II [eiGlATvYR] and III [HHDHlvCldcGeviEf] (uppercase aa are identical in 12 or all 13 Fur sequences and lowercase aa are identical in six or more sequences). A truncated TGFH9011 Fur missing 18 aa of the N terminus but retaining all three conserved motifs was shown to bind all four FBS sequences. The binding and transcription studies support autoregulation of fur expression in Cj.

Amino Acid Sequence↗
PubMed1995

Mutational sensitivity patterns define critical residues in the palm subdomain of the reverse transcriptase of human immunodeficiency virus type 1.

We have analyzed 154 single amino acid replacement mutants within a 40 amino acid region (residues 164-203) of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). This region consists of two antiparallel beta-strands (strands 9 and 10) flanked by two alpha helices (E and F). The structure of this region of the 'palm' subdomain is conserved in a variety of DNA and RNA polymerases, indicating a critical role in enzyme structure and function. Functional assays were performed by screening RT activity of mutants expressed in E. coli. A functionally important region corresponding closely to beta-strands 9 and 10 and the loop joining them was revealed by its mutational sensitivity. Structural analysis of mutants was performed by using Western blots to assay correct folding, which is required for processing to produce the mature p66 and p51 RT species. This analysis indicates that beta-strand 10 is a structurally important region. Combined analysis of these two assays revealed diagnostic patterns of mutational sensitivity which identify key positions in the RT sequence at which a specific amino acid side chain is critical, either for structure or function, as well as residues which are external to the RT structure. This work illustrates the utility of large-scale mutagenesis in relating primary sequence to significant features of protein structure and function.

Amino Acid Sequence↗