Biomedical subjects

Bow Ho

Publications and source records attributed to Bow Ho.

13 recordsLinked to original sources

PubMed2003

Transcriptional regulation of limulus factor C: repression of an NFkappaB motif modulates its responsiveness to bacterial lipopolysaccharide.

Serine proteases play fundamental roles in invertebrate development, hemostasis, and innate immunity. This is exemplified by the limulus Factor C, which is a serine protease that binds a pathogen-associated molecule, lipopolysaccharide (LPS) to trigger a blood coagulation cascade. As a central molecule in the limulus innate immunity and hemostasis, Factor C gene expression has been detected in two major immune defense tissues, the amebocytes and hepatopancreas. Infection of the limulus with live Gram-negative bacteria induces a 2-3-fold increase in mRNA transcripts in both tissues. However, in vitro studies in Drosophila cell lines using Factor C promoter-reporter chimera DNA constructs, and site-directed mutagenesis of the promoter demonstrated that a proximal kappaB binding site, aided by an adjacent dorsal-like binding motif responds dramatically to LPS and dorsal transcription factor overexpression. Electrophoretic mobility shift assay further confirmed a strong interaction of the limulus kappaB motif with Rel proteins. However, deletion constructs of the Factor C promoter harboring different numbers of dorsal-like binding sites upstream of the kappaB motif as well as the electrophoretic mobility shift assay of these motifs with Rel proteins strongly suggest that the up-regulation of Factor C gene expression is attenuated during microbial challenge. The repression of the dramatic activation of this pathogen-responsive gene by LPS is probably effected via competition between the dorsal-like motifs over the proximal LPS-responsive kappaB unit, or through inhibition from the upstream repressive element(s), which accounts for the gene expression pattern observed in vivo. Our findings demonstrate that blood coagulation and innate immune response are integrated at the transcriptional level in this ancient organism, and that this LPS-responsive serine protease is controlled by an evolutionarily conserved NFkappaB pathway.

Amino Acid Motifs↗

PubMed2003

Sequence and structural diversity in endotoxin-binding dodecapeptides.

For the study of sequence or structure requirement of short peptides for endotoxin binding, and to search for potential endotoxin antagonists, biopanning was carried out on a phage-displayed random dodecapeptide library against immobilized lipopolysaccharide (LPS) or lipid A (LA), the core toxic portion of LPS. Specific binding of selected phage-displayed peptides to LPS/LA was confirmed by surface plasmon resonance (SPR) analysis. These peptides are rich in basic and hydrophobic amino acids, especially histidine, proline and tryptophan, highlighting apparent amphiphilicity and bacterial membrane activity. These dodecapeptide sequences have no predictable secondary structure in solution, indicating the importance of a random structure before their interaction with LPS/LA. Sequence alignment reveals various potential secondary structures with these selected peptides, which contain specific signature motifs such as b(p)hb(p)hb(p), bbbb, hhhh (b-basic, p-polar, h-hydrophobic residue), capable of binding LPS/LA. However, none of these peptides exhibit a significant calculated structural amphiphilicity while assuming a secondary structure. This study suggests that for these short dodecapeptides to bind LPS/LA, the potential for their structural adaptation is more important than an amphipathic structure.

Amino Acid Sequence↗

PubMed2003

Surface localized Heat Shock Protein 20 (HslV) of Helicobacter pylori.

BACKGROUND: Heat Shock Protein (HSP) has been regarded as a pathogenic factor in Helicobacter pylori infection. Heat Shock Protein 20 (HSP20) of H. pylori is identified as Hs1V based on open reading frame predication of genome sequences. It is a homologue of HslV of E. coli, a peptidase involved in protein degradation. METHODS: The HSP20 gene was cloned and inserted into pET16b fused with His-tag. Recombinant HSP20 protein (rHSP20) was expressed and purified by nickel column. Rabbit anti-rHSP20 was purified by Protein A affinity chromatography and used as a probe to localize HSP20 in H. pylori by immuno-gold labeling and Western blotting. rHSP20 was also used as antigen to test for antibody against HSP20 in patients with H. pylori infection by enzyme-linked immunosorbant assay. RESULTS: Immuno-gold labeled transmission electron microscopy shows that HSP20 is located on the cell surface of H. pylori. Western blotting of 2-D gel shows that HSP20 has a pI of approximately 5.5 and a molecular weight of approximately 18 kDa. The ELISA result shows that there is no significant difference in antibody titre against rHSP20 in all sera tested. CONCLUSION: The presence of IgG to rHSP20 may imply an earlier exposure of the patients and normal subjects to H. pylori. However, the mechanism has not been established. HSP20 has been shown to localize on the surface of H. pylori. Surface localization of H. pylori HSP20 may provide the path to a better understanding of the role and function of HSP20 in bacteria-host interaction.

Amino Acid Sequence↗

PubMed2003

Tandem repeats of Sushi3 peptide with enhanced LPS-binding and -neutralizing activities.

Endotoxin, also known as lipopolysaccharide (LPS), is the major mediator of septic shock due to Gram-negative bacterial infection. Chemically synthesized S3 peptide, derived from Sushi3 domain of Factor C, which is the endotoxin-sensitive serine protease of the limulus coagulation cascade, was previously shown to bind and neutralize LPS activity. For large-scale production of this peptide and to mimick other pathogen-recognizing molecules, tandem multimers of the S3 gene were constructed and expressed in Escherichia coli. The recombinant tetramer of S3 (rS3-4mer) was purified by anion exchange and digested into monomers (rS3-1mer). Both the rS3-4mer and rS3-1mer were functionally analyzed for their ability to bind LPS by an ELISA-based method and surface plasmon resonance. The LAL inhibition and TNFalpha-release test showed that rS3-1 mer can neutralize the LPS activity as effectively as the synthetic S3 peptide, while rS3-4mer displays an enhanced inhibitory effect on LPS-induced activities. Both recombinant peptides exhibited low cytotoxicity and no haemolytic activity on human cells. This evidence suggests that the recombinant sushi peptides have potential use for the detection, removal of endotoxin and/or anti-endotoxin strategies.

Arthropod Proteins↗

PubMed2003

Immune responses to differentiated forms of Helicobacter pylori in children with epigastric pain.

Helicobacter pylori infection affects human populations of all ages. This gastric bacterium exists in spiral form and the reported viable but nonculturable coccoid form. The present study aims to examine the probable role of the coccoid form in H. pylori infection by comparing the seroprevalences of the spiral and the coccoid forms in children with epigastric pain. Four hundred eighty-nine children (mean age, 8.5 years) with epigastric pain formed the basis of this study. Five hundred ninety-nine schoolchildren of comparable ages and with no record of dyspepsia served as controls. The seroprevalence of antigens prepared from both morphological forms was examined by enzyme-linked immunosorbent assay. The results showed that 65 (13.3%) and 273 (55.8%) of 489 symptomatic children were seropositive for antigens of the H. pylori spiral and coccoid forms, respectively. In contrast, only 7.0% of the control group had elevated levels of immunoglobulin G antibodies against the spiral form, while 26.5% were positive for antibodies against the coccoid form. There were no significant differences between genders or among ethnic groups. The study showed a rise in seroprevalence corresponding with age: 7.1% for those < or =5 years to 21.4% for those > or =11 years. The seroprevalence of antigens of the H. pylori spiral and coccoid forms in children with epigastric pain was twofold higher than that in the control subjects. Interestingly, there was a fourfold increase in seropositivity for coccoid-form antigen compared to that for the spiral-form antigen among the symptomatic pediatric patients as well as the control group, indicating a possible infective role of the coccoid form of H. pylori in the pediatric patients with epigastric pain.

Abdominal Pain↗

PubMed2003

Metronidazole-resistant Helicobacter pylori is more prevalent in patients with nonulcer dyspepsia than in peptic ulcer patients in a multiethnic Asian population.

The trend of increasing prevalence of antibiotic resistance among Helicobacter pylori strains has been suggested as a cause of the failure of treatment of H. pylori infections. In this study, 120 of 211 antral biopsy specimens from patients with dyspeptic symptoms were found to harbor H. pylori. The isolates from the 120 specimens were tested by the agar dilution method, and 38 (31.7%) were found to be metronidazole resistant. Among the 211 subjects, 81 of 115 (70.4%) patients with peptic ulcer (PU) were infected with H. pylori, whereas 39 of 96 (40.6%) patients with nonulcer dyspepsia (NUD) were infected with H. pylori. Interestingly, significantly more NUD patients than PU patients harbored metronidazole-resistant H. pylori (22 of 39 [56.4%] and 16 of 81 [19.8%], respectively; P < 0.001). A similar pattern was also observed among NUD patients of different ethnicities but not between male and female patients (23 of 78 [29.5%] and 15 of 42 [35.7%], respectively; P = 0.54). In the posttreatment follow-up, five of six patients who had positive urea breath test results, indicating treatment failure, were NUD patients. Of these, four harbored metronidazole-resistant H. pylori strains. This further illustrates the relevance of metronidazole-resistant H. pylori in NUD patients. The significantly higher percentage of metronidazole-resistant H. pylori isolates in NUD patients may be attributed to the protection offered by the mucus layer of the nonulcerated stomach to the bacteria that reside below it, resulting in organism exposure to sublethal concentrations of metronidazole and leading to the induction of metronidazole resistance. The results demonstrate that the H. pylori isolates colonizing NUD patients are more likely to be resistant to metronidazole. It will therefore be useful to reevaluate the use of metronidazole in the treatment of NUD patients infected with H. pylori.

Asian People↗

PubMed2003

Identification of H. pylori strain specific DNA sequences between two clinical isolates from NUD and gastric ulcer by SSH.

AIM: The genomes of Helicobacter pylori (H. pylori) from different individuals are different. This project was to identify the strain specific DNA sequences between two clinical H. pylori isolates by suppression subtractive hybridization (SSH). METHODS: Two clinical H. pylori isolates, one from gastric ulcer (GU, tester) and the other from non-ulcer dyspepsia (NUD, driver), were cultured and the genomic DNA was prepared and submitted to Alu I digestion. Then two different adaptors were ligated respectively to the 5'-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNA. The un-hybridized tester DNA sequences were amplified by two sequential PCR and cloned into pGEM-T-Easy Vector. The tester strain specific inserts were screened and disease related DNA sequences were identified by dot blotting. RESULTS: Among the 240 colonies randomly chosen, 50 contained the tester strain specific DNA sequences. Twenty three inserts were sequenced and the sizes ranged from 261 bp to 1 036 bp. Fifteen inserts belonged to the H.pylori plasmid pHPO100 that is about 3.5 kb and codes a replication protein A. Other inserts had patches of homologous to the genes of H.pylori in GenBank. Various patterns of dot blots were given and no GU strain unique DNA sequences were found when 4 inserts were used as probes to screen the genomic DNA from 27 clinical isolates, 8 from GU, 12 from duodenum ulcer (DU), 4 from GU-DU, 2 from NUD and 1 from gastric cancer (GC). But a 670 bp DNA fragment (GU198) that was a bit homologous to the 3'-end of the gene of thymidylate kinase was positive in 7 GU strains (7/8), 3 GU-DU strains (3/4) and 3 DU strains (3/12). A 384 bp fragment (GU79) of the replication gene A (repA) was positive only in 4 H.pylori isolates, 2 from GU and 2 from GU-DU. CONCLUSION: Differences exist in the genes of different H.pylori isolates. SSH is very effective to screen H.pylori strain specific DNA sequences between two clinical isolates, and some of these sequences may have clinical significance.

DNA, Bacterial↗

PubMed2003

Stability of randomly amplified polymorphic DNA fingerprinting in genotyping clinical isolates of Helicobacter pylori.

AIM: H pylori genomes are highly diversified. This project was designed to genotype H pylori isolates by the polymerase chain reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) fingerprinting technique and to verify its stability by Southern blotting and DNA sequencing. METHODS: Clinical isolates of H pylori were cultured from gastric antra and cardia of 73 individuals, and genomic DNA was prepared for each isolate. RAPD was carried out under optimized conditions. 23S rDNA was regarded as an internal control, and a 361 bp rDNA fragment (RDF) was used as a probe to screen the RAPD products by Southern blotting. Ten RDFs from different clinical isolates and the flanking regions (both upstream and downstream) of four RDFs were amplified and sequenced. RESULTS: H pylori isolates from different individuals had different RAPD profiles, but the profiles for isolates cultured from different gastric sites of a given individual were identical in all but one case. Isolates from 27 individuals were RDF positive by Southern blotting. Sequences of the RDFs and their flanking regions were almost the same between the RDF positive and negative isolates as determined by Southern blotting. There was no binding site for random PCR primer inside the sequences. CONCLUSION: RAPD is very useful in genotyping H pylori grossly on a large scale. However, it seems unstable in amplification of low yield fragments, especially those that do not appear as visible bands on the agarose gel stained with EB, since the primer is partially matched to the template.

Blotting, Southern↗

PubMed2002

Modular arrangement and secretion of a multidomain serine protease. Evidence for involvement of proline-rich region and N-glycans in the secretion pathway.

The Limulus Factor C (FC), a multidomain glycoprotein that binds bacterial endotoxin with high affinity, belongs to the serine protease family of the complement and blood coagulation cascade. Here, we provide compelling evidence for the importance of modular arrangement and relevance of the proline-rich region (PRR) and N-glycosylation to the secretion and function of FC. We propose that PRR could be a universal conformational domain that regulates protein folding and targeting. FCs lacking PRR preceding the serine protease domain, were localized intracellularly. Misfolded conformers of the intracellular FCs were more susceptible to trypsin digestion. Glycosylation inhibition studies indicate that the presence but not the exact structure of the N-glycans affects the secretion of FC, although the complexity of glycosylation may influence its endotoxin-induced proteolytic cleavage with resultant enzymatic activity. Disruption of specific N-glycan sites at positions 740, 767, and 912, downstream of the PRR, at or near the serine protease domain, blocks its secretion. Co-expressed molecular chaperones like canine calnexin associates with glycosylated FCs to increase its solubility and secretion level but did not alter their expression profiles. Our results clearly demonstrate that the folding and secretion of a multidomain serine protease like FC are determined by its modular domain arrangement and site-specific N-glycans. The secreted FCs containing the N-terminal portion of FC are able to detect lipopolysaccharide with high sensitivity. We also identified the lectin-like and sushi 4 domains to contribute to the binding of lipopolysaccharide.

Amino Acid Sequence↗

PubMed2002

Engineering a novel secretion signal for cross-host recombinant protein expression.

Protein secretion is conferred by a hydrophobic secretion signal usually located at the N-terminal of the polypeptide. We report here, the identification of a novel secretion signal (SS) that is capable of directing the secretion of recombinant proteins from both prokaryotes and eukaryotes. Secretion of fusion reporter proteins was demonstrated in Escherichia coli, Saccharomyces cerevisiae and six different eukaryotic cells. Estrogen-inducibility and secretion of fusion reporter protein was demonstrated in six common eukaryotic cell lines. The rate of protein secretion is rapid and its expression profile closely reflects its intracellular concentration of mRNA. In bacteria and yeast, protein secretion directed by SS is dependent on the growth culture condition and rate of induction. This secretion signal allows a flexible strategy for the production and secretion of recombinant proteins in numerous hosts, and to conveniently and rapidly study protein expression.

Arabinose↗

PubMed2002

Rational design of green fluorescent protein mutants as biosensor for bacterial endotoxin.

Enhanced green fluorescent protein (EGFP) was selected as a signalling scaffold protein for design of a fluorescent biosensor for bacterial endotoxin [or lipopolysaccharide (LPS)]. Virtual mutagenesis was utilized to model EGFP variants containing binding sites for LPS and lipid A (LA), the bioactive component of LPS. Cationic amphipathic sequences of five alternating basic and hydrophobic residues were introduced to beta-sheets located on the surface of EGFP barrel, in the vicinity of the chromophore. Computational methods were employed to predict binding affinity of Escherichia coli LA, to the models of virtual EGFP mutants. DNA mutant constructs of five predicted best binding EGFP variants were expressed in COS-1 cells. The EGFP-mutant proteins exhibited differential expression and variable degrees of fluorescence yield at 508 nm. The EGFP mutants showed a range of LA binding affinities that corresponded to the computational predictions. LPS/LA binding to the mutants caused concentration-dependent fluorescence quenching. The EGFP mutant, G10 bearing LPS/LA amphipathic binding motif in the vicinity of the chromophore (YLSTQ(200-204)-->KLKTK) captured LA with a dissociation constant of 8.5 microm. G10 yielded the highest attenuation of fluorescence intensity in the presence of LPS/LA and demonstrated capability in fluorescence-mediated quantitative detection of LPS in endotoxin-contaminated samples. Thus, the EGFP mutant can form the basis of a novel fluorescent biosensor for bacterial endotoxin.

Biosensing Techniques↗

PubMed2002

A novel fluorescent protein-based biosensor for gram-negative bacteria.

Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFP(i)) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host. The EGFP(i) proteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide from the E. coli lysates, the binding affinities of the EGFP(i) G10 and G12 to lipid A were established. The K(D) values of 7.16 x 10(-7) M for G10 and 8.15 x 10(-8) M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFP(i) biosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFP(i) specifically tagged gram-negative bacteria like E. coli and Pseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment. This dual function of the EGFP(i) in detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.

Biosensing Techniques↗

PubMed2001

Involvement of the heparan sulphate-binding proteins of Helicobacter pylori in its adherence to HeLa S3 and Kato III cell lines.

To determine whether Helicobacter pylori heparan sulphate-binding proteins (HSBPs) are involved in the adherence of H. pylori to HeLa and Kato III cells, monolayers were pre-incubated with various preparations and concentrations of H. pylori HSBPs at 37 degrees C, washed and then challenged with bacteria. HSBPs did not prevent but enhanced H. pylori adherence. However, challenging cultured cells with H. pylori previously incubated with rabbit anti-HSBP IgG resulted in significant inhibition of bacterial adherence. These data demonstrate that the extracellular HSBP plays an important role in promoting H. pylori attachment to Kato III and HeLa S3 cells, that adhesion of H. pylori to Kato III and HeLa S3 cells is promoted by the presence of the 71.5-kDa extracellular HSBP and that rabbit polyclonal antibodies against this HSBP can inhibit adhesion of H. pylori to the cultured cell lines and detach cell-bound H. pylori.

Animals↗