Biomedical subjects

Bow Ho

Publications and source records attributed to Bow Ho.

At least 19 recordsLinked to original sources

PubMed2006

Alterations in rdxA and frxA genes and their upstream regions in metronidazole-resistant Helicobacter pylori isolates.

Metronidazole resistance among Helicobacter pylori strains has been related to alterations in gene products having metronidazole nitroreductase activities. RdxA and FrxA proteins are the two major contributing factors. In this investigation, the rdxA and frxA genes and their upstream regions were analyzed in 19 H. pylori isolates, 8 of which were metronidazole-sensitive (MIC < or = 8 microg/mL) and 11 of which were metronidazole-resistant (MIC > or = 8 microg/mL), as determined by the E-test. Among the metronidazole-resistant isolates, three contained both RdxA and FrxA proteins with premature truncation caused by gene nonsense mutations or frameshift mutations, while three contained only stop mutations in FrxA and two only in RdxA. Substitutions of amino acids occurred in other RdxA (5/6) and FrxA (4/5) proteins from metronidazole resistant isolates as compared with those from metronidazole-sensitive ones. All metronidazole-resistant isolates had alterations in RdxA and/or FrxA proteins. Moreover, the upstream regions (-1 to -35) of rdxA and frxA genes in some metronidazole-resistant isolates varied by nucleotide insertion and/or deletion or substitution. The patterns of variation in both genes and their upstream regions were highly diversified. Alterations in rdxA and frxA genes and their upstream regions may be involved in the development of metronidazole resistance in H. pylori.

Amino Acid Sequence↗

PubMed2006

An evolutionarily conserved 16-kDa thioredoxin-related protein is an antioxidant which regulates the NF-kappaB signaling pathway.

Thioredoxin (TRX) is generally a 12-kDa protein-disulfide reductase. Here, we report the discovery of a 16-kDa thioredoxin-related protein designated Cr-TRP16, from a "living fossil," the horseshoe crab (Carcinoscorpius rotundicauda). Cr-TRP16 contains an atypical WCPPC catalytic motif and possesses the classical thiodisulfide reductase activity, as indicated by the insulin reduction assay. Furthermore, Cr-TRP16 can function as an antioxidant and protect against DNA nicking by reactive oxygen species. Overexpression of Cr-TRP16 regulated the transcription of NF-kappaB-dependent genes probably by enhancing NF-kappaB DNA-binding activity, suggesting possible roles for Cr-TRP16 in modulating the NF-kappaB signaling pathway. In vivo, the antioxidant pyrrolidine dithiocarbamate suppressed the expression of NF-kappaB-regulated genes such as IkappaB and inducible nitric oxide synthase. This further supports the notion that oxidative stress is also a regulatory factor of the NF-kappaB signaling pathway, a phenomenon which has been entrenched for several hundred million years. Furthermore, we demonstrated that the 16-kDa thioredoxins are evolutionarily conserved from Caenorhabditis elegans to human. Interestingly, thioredoxin-like 6, a human homologue of Cr-TRP16, could also enhance NF-kappaB DNA-binding activity, suggesting that the regulatory role of the 16-kDa thioredoxins on NF-kappaB is well conserved through evolution.

Amino Acid Sequence↗

PubMed2006

Diversity in lectins enables immune recognition and differentiation of wide spectrum of pathogens.

Carbohydrate-binding lectins play essential roles as pattern recognition receptors in innate immunity in both vertebrates and invertebrates. The carcinolectins 5 (CL5a and CL5b, the CL5 isoforms of horseshoe crab, Carcinoscorpius rotundicauda, with apparent sizes of 36 and 40 kDa, respectively) are prominent plasma lectins that bind all representative microbes and pathogen-associated molecular pattern molecules. Different cDNA isoforms of both CL5a and CL5b were isolated, leading to our speculation on their functional divergence. Characterization of CL5 isoforms bound to microbial cell surfaces demonstrates the diversity of these lectins. The resolution patterns of the isoforms that associate with fungus differ from those that associate with bacteria, suggesting the unique roles these lectins play in the recognition and differentiation of microbes. We postulate that different populations of plasma lectins act in collaboration in frontline innate immune defense against disparate pathogens. The functional diversity of lectins in invertebrates appears to evolutionarily compensate for the lack of acquired immunity.

Amino Acid Sequence↗

PubMed2006

Molecular mechanisms that govern the specificity of Sushi peptides for Gram-negative bacterial membrane lipids.

Factor C-derived Sushi peptides (S1 and S3) have been shown to bind lipopolysaccharide (LPS) and inhibit the growth of Gram-negative bacteria but do not affect mammalian cells. On the premise that the composition of membrane phospholipids differs between the microbial and human cells, we studied the modes of interaction between S1 and S3 and the bacterial membrane phospholipids, POPG, in comparison to that with the mammalian cell membrane phospholipids, POPC and POPE. S1 exhibits specificity against POPG, suggesting its preference for bacterial anionic phospholipids, regardless of whether the phospholipids form vesicles in a solution or a monolayer on a solid surface. The specificity of the Sushi peptides for POPG is a consequence of the electrostatic and hydrophobic forces. The unsaturated nature of POPG confers fluidity to the lipid layer, and being in the proximity of LPS in the microenvironmental milieu, POPG probably enhances the insertion of the peptide-LPS complex into the bacterial inner membrane. Furthermore, during its interaction with POPG, the S1 peptide underwent a transition from random to alpha-helical coil, while S3 became a mixture of beta-sheet and alpha-helical structures. This differential structural change in the peptides could be responsible for their different modes of disruption of POPG vesicles. Conceivably, the selectivity for POPG spares the mammalian membranes from undesirable effects of antimicrobial peptides, which could be helpful in designing and developing a new generation of antibiotics and in offering some clues about the specific function of Factor C, a LPS biosensor.

Anions↗

PubMed2006

Iron-withholding strategy in innate immunity.

The knowledge of how organisms fight infections has largely been built upon the ability of host innate immune molecules to recognize microbial determinants. Although of overwhelming importance, pathogen recognition is but only one of the facets of innate immunity. A primitive yet effective antimicrobial mechanism which operates by depriving microbial organisms of their nutrients has been brought into the forefront of innate immunity once again. Such a tactic is commonly referred to as the iron-withholding strategy of innate immunity. In this review, we introduce various vertebrate iron-binding proteins and their invertebrate homologues, so as to impress upon readers an obscured arm of innate immune defense. An excellent comprehension of the mechanics of innate immunity paves the way for the possibility that novel antimicrobial therapeutics may emerge one day to overcome the prevalent antibiotic resistance in bacteria.

Animals↗

PubMed2006

Evidence for the ancient origin of the NF-kappaB/IkappaB cascade: its archaic role in pathogen infection and immunity.

The evolutionary conservation of the NF-kappaB transcription factors, from Drosophila to humans, underscores its pivotal role in immune response. Unexpectedly, the canonical NF-kappaB signaling pathway is not functional in the immune system of Caenorhabditis elegans. Therefore, the ancient origin of the NF-kappaB signaling pathway is still unknown. Here, we report the discovery and characterization of a primitive and functional NF-kappaB/IkappaB pathway in the immune defense of a "living fossil," the horseshoe crab, Carcinoscorpius rotundicauda. The ancient NF-kappaB/IkappaB homologues, CrNFkappaB, CrRelish, and CrIkappaB, share numerous signature motifs with their vertebrate orthologues. CrNFkappaB recognizes both horseshoe crab and mammalian kappaB response elements. CrIkappaB interacts with CrNFkappaB and inhibits its nuclear translocation and DNA-binding activity. The activation of the CrNFkappaB is autoregulated by a feedback mechanism mediated by CrIkappaB, the natural inhibitor of CrNFkappaB. We further show that Gram-negative bacteria infection causes rapid degradation of CrIkappaB and nuclear translocation of CrNFkappaB. Infection also leads to an increase in the kappaB-binding activity and up-regulation of immune-related gene expression, like inducible nitric oxide synthase and Factor C, an LPS-activated serine protease. Altogether, our study shows that, although absent in C. elegans, the NF-kappaB/IkappaB signaling cascade remains well conserved from horseshoe crab to humans, playing an archaic but fundamental role in regulating the expression of critical immune defense molecules.

Animals↗

PubMed2006

The molecular mechanism of interaction between sushi peptide and Pseudomonas endotoxin.

Septic shock is caused by Gram-negative bacterial infection. Lipopolysaccharide (LPS) is the bioactive molecule present on the outer membrane of the Gram-negative bacteria. It is generally thought that LPS interacts with sensors on the host cell membrane to activate the intracellular signaling pathway resulting in the overproduction of cytokines such as TNF-alpha. This causes inflammation and ultimately, septic shock. Lipid A is the pharmacophore of the LPS molecule. Thus, developing bio-molecules which are capable of binding LPS at high affinity, especially to the lipid A moiety is an efficient way to neutralize the LPS toxicity. Factor C, a serine protease in the horseshoe crab ameobocytes, is sensitive to trace levels of LPS. We have derived Sushi peptides from the LPS-binding domains of Factor C. Our earlier study showed that the Sushi peptides inhibit LPS-induced septic shock in mice. Here, we demonstrate that the molecular interaction between LPS and Sushi 1 peptide is supported by the hydrophobic interaction between the lipid tail of LPS and Sushi 1 peptide. Furthermore, in the presence of LPS, the peptide transitions from a random structure into an alpha-helical conformation and it disrupts LPS aggregates, hence, neutralizing the LPS toxicity.

Animals↗

PubMed2005

Validation of four Helicobacter pylori rapid blood tests in a multi-ethnic Asian population.

AIM: To validate the accuracy of four rapid blood tests in the diagnosis of Helicobacter pylori. METHODS: Consecutive dyspeptic patients scheduled for endoscopy at the National University Hospital, Singapore, were interviewed and had blood drawn for serology. The first 109 patients were tested with BM-test (BM), Pyloriset Screen (PS) and QuickVue (QV), and the next 99 subjects were tested with PS and Unigold (UG). Endoscopies were performed blinded to rapid blood test results and biopsies were taken for culture and rapid urease test. Urea breath tests were performed after endoscopies. The rapid blood test results were compared with four reference tests (rapid urease test, culture, serology, and breath test). RESULTS: The study population composed of 208 patients (mean age 43.1 years; range 18-73 years; 119 males; 174 Chinese). The number of evaluable patients for BM, QV, UG and PS were 102, 102, 95, and 197, respectively. The sensitivity and specificity, respectively were: PS 80.2%, 95.8%; UG 55.9%, 100%; QV 43.3%, 100%; BM 67.2%, 97.1%. CONCLUSION: The rapid blood test kits showed high specificity and positive predictive value (97-100%), while sensitivity and negative predictive value ranged widely (43%-80% and 47%-73%, respectively). Among test kits, PS showed the best sensitivity (80%), best negative predictive value (73%) and best negative likelihood ratio (0.207). PS had a specificity of 96%, positive predictive value of 97% and positive likelihood ratio of 19.1.

Adolescent↗

PubMed2005

Investigation of a novel artificial antimicrobial peptide by fluorescence correlation spectroscopy: an amphipathic cationic pattern is sufficient for selective binding to bacterial type membranes and antimicrobial activity.

Fluorescence Correlation Spectroscopy (FCS) is used to study the interaction of a recently designed antimicrobial peptide, called V4, with LPS and lipids of varying head and tail groups. V4 is designed based on a known amphipathic cationic pattern BHPHB (B: basic; H: hydrophobic; P: polar residue, respectively) and shows a good combination of high antimicrobial activity, low cytotoxic activity and low hemolytic activity. It is shown that V4 has high binding affinity for LPS, which is the major component of the outer membrane of Gram-negative bacteria, and shows selectivity for negatively charged lipids in contrast to zwitterionic lipids at a low peptide/lipid ratio. At high peptide/lipid ratio, V4 can permeabilize vesicles composed of negatively charged lipids and eventually cause vesicle fusion. The identification of the amphipathic cationic pattern as the mediator of selectivity and antimicrobial activity could be a first step in the rational design of better antimicrobial peptides.

Anti-Infective Agents↗

PubMed2005

The antimicrobial properties of C-reactive protein (CRP).

C-reactive protein, CRP, is a predominant pattern-recognition receptor (PRR) in the plasma of the horseshoe crab, which recognizes lipopolysaccharide (LPS). Native CRP2 has previously been shown to exhibit agglutination activity against the polysialic capsule of Escherichia coli K1 but its role in bacterial clearance is not well characterized. In this work, the antimicrobial activity of a recombinant CRP2 isoform (rCRP2) was tested against E. coli, Pseudomonas aeruginosa and Staphylococcus aureus. rCRP2 agglutinates bacteria and exhibits bactericidal activity against Gram-negative bacteria. In addition, the antimicrobial activity of rCRP2 is calcium-independent. GST pulldown experiments suggest that in the naïve physiological state, CRP2 interacts with hemocyanin, native CRPs, a 35-kDa plasma lectin and an as yet unidentified 40-kDa protein. This interaction was enhanced upon Pseudomonas infection. We propose that rCRP2 is a PRR with potent antimicrobial activity and its interacting partners contribute to effective bacterial clearance.

Agglutination Tests↗

PubMed2005

The response of ferritin to LPS and acute phase of Pseudomonas infection.

Plasma ferritin is an important extracellular iron storage molecule, whose concentration increases drastically in cancer and infection. During infection, the pathogen usurps host iron for its survival and pathogenicity; hence, maintenance of the plasma ferritin level during infection is a crucial host defence mechanism. In this study, the horseshoe crab plasma ferritin complex was purified, characterized, and its involvement in innate immune defence was investigated. The plasma ferritin appears as a 21-kDa subunit on SDS-PAGE. Full-length ferritin-H cDNAs (CrFer-H1 and CrFer-H2) were cloned. Analysis of the 5' UTR indicates the existence of a functional iron-response element, suggesting that both the CrFer-H genes may be post-transcriptionally regulated. Northern analysis shows that the CrFer-H is ubiquitously expressed. Within 3 h of lipopolysaccharide challenge, the gene is up-regulated by > 12-fold. In contrast, iron-loading did not result in any significant change. When challenged with Pseudomonas aeruginosa, the plasma ferritin disappeared between 6-48 h and re-appeared thereafter, suggesting that during infection, ferritin may be concealed intracellularly as it withholds iron from the invading pathogen. Taken together, these results provide insights into the importance of plasma ferritin as an evolutionarily conserved molecule for the iron-withholding strategy of innate immunity.

Acute Disease↗

PubMed2004

The ancient origin of the complement system.

The complement system has been thought to originate exclusively in the deuterostomes. Here, we show that the central complement components already existed in the primitive protostome lineage. A functional homolog of vertebrate complement 3, CrC3, has been isolated from a 'living fossil', the horseshoe crab (Carcinoscorpius rotundicauda). CrC3 resembles human C3 and shows closest homology to C3 sequences of lower deuterostomes. CrC3 and plasma lectins bind a wide range of microbes, forming the frontline innate immune defense system. Additionally, we identified CrC2/Bf, a homolog of vertebrate C2 and Bf that participates in C3 activation, and a C3 receptor-like sequence. Furthermore, complement-mediated phagocytosis of bacteria by the hemocytes of horseshoe crab was also observed. Thus, a primitive yet complex opsonic complement defense system is revealed in the horseshoe crab, a protostome species. Our findings demonstrate an ancient origin of the critical complement components and the opsonic defense mechanism in the Precambrian ancestor of bilateral animals.

Amino Acid Sequence↗

PubMed2004

Neither gastric topological distribution nor principle virulence genes of Helicobacter pylori contributes to clinical outcomes.

AIM: Studies on Helicobacter pylori (H pylori) and gastroduodenal diseases have focused mainly on the distal sites of the stomach, but relationship with the gastric cardia is lacking. The aim of this study is to determine if the gastric topology and genotypic distribution of H pylori were associated with different upper gastrointestinal pathologies in a multiethnic Asian population. METHODS: Gastric biopsies from the cardia, body/corpus and antrum were endoscoped from a total of 155 patients with dyspepsia and/or reflux symptoms, with informed consent. H pylori isolates obtained were tested for the presence of 26 kDa, ureC, cagA, vacA, iceA1, iceA2 and babA2 genes using PCR while DNA fingerprints were generated using random amplification polymorphic DNA (RAPD). RESULTS: H pylori was present in 51/155 (33%) of patients studied. Of these, 16, 15 and 20 were isolated from patients with peptic ulcer diseases, gastroesophageal reflux diseases and non-ulcer dyspepsia, respectively. Of the H pylori positive patients, 75% (38/51) had H pylori in all three gastric sites. The prevalence of various genes in the H pylori isolates was shown to be similar irrespective of their colonization sites as well as among the same site of different patients. The RAPD profiles of H pylori isolates from different gastric sites were highly similar among intra-patients but varied greatly between different patients. CONCLUSION: Topographic colonization of H pylori and the virulence genes harboured by these isolates have no direct bearing to the clinical state of the patients. In multi-ethnic Singapore, the stomach of each patient is colonized by a predominant strain of H pylori, irrespective of the clinical diagnosis.

Adult↗

PubMed2004

Prominent role of gamma-glutamyl-transpeptidase on the growth of Helicobacter pylori.

AIM: gamma-glutamyl transpeptidase (GGT) has been reported as a virulence and colonizing factor of Helicobacter pylori (H pylori). This study examined the effect of GGT on the growth of H pylori. METHODS: Standard H pylori strain NCTC 11637 and 4 clinical isolates with different levels of GGT activity as measured by an enzymatic assay were used in this study. Growth inhibition and stimulation studies were carried out by culturing H pylori in brain heart infusion broth supplemented with specific GGT inhibitor (L-serine sodium borate complex, SBC) or enhancer (glutathione together with glycyl-glycine), respectively. The growth profiles of H pylori were determined based on viable bacterial count at time interval. RESULTS: Growth was more profuse for H pylori isolates with higher GGT activity than those present with lower GGT activity. However, in the presence of SBC, growth of H pylori was retarded in a dose dependent manner (P = 0.034). In contrast, higher growth rate was observed when GGT activity was enhanced in the presence of glutathione and glycyl-glycine. CONCLUSION: Higher GGT activity provides an advantage to the growth of H pylori in vitro. Inhibition of GGT activity by SBC resulted in growth retardation. The study shows that GGT plays an important role on the growth of H pylori.

Borates↗

PubMed2004

Perturbation of Lipopolysaccharide (LPS) Micelles by Sushi 3 (S3) antimicrobial peptide. The importance of an intermolecular disulfide bond in S3 dimer for binding, disruption, and neutralization of LPS.

S3 peptide, derived from the Sushi 3 domain of Factor C, which is the lipopolysaccharide (LPS)-sensitive serine protease of the horseshoe crab coagulation cascade, was shown previously to harbor antimicrobial activity against Gram-negative bacteria. However, the mechanism of action remains poorly understood at the molecular level. Here we demonstrate that the intermolecular disulfide bonding of S3 resulting in S3 dimers is indispensable for its interaction with LPS. The binding properties of the S3 monomer and dimer to LPS were analyzed by several approaches including enzyme-linked immunosorbent assay (ELISA)-based assay, surface plasmon resonance, and fluorescence correlation spectroscopy (FCS). It is evident that the S3 dimer exhibits stronger binding to LPS, demonstrating 50% LPS-neutralizing capability at a concentration of 1 mum. Circular dichroism spectrometry revealed that the S3 peptide undergoes conformational change in the presence of a disulfide bridge, transitioning from a random coil to beta-sheet structure. Using a fluorescence correlation spectroscopy monitoring system, we describe a novel approach for examining the mechanism of peptide interaction with LPS in the native environment. The strategy shows that intermolecular disulfide bonding of S3 into dimers plays a critical role in its propensity to disrupt LPS micelles and consequently neutralize LPS activity. S3 dimers display detergent-like properties in disrupting LPS micelles. Considering intermolecular disulfide bonds as an important parameter in the structure-activity relationship, this insight provides clues for the future design of improved LPS-binding and -neutralizing peptides.

Dimerization↗

PubMed2004

Effect of biopsies on sensitivity and specificity of ultra-rapid urease test for detection of Helicobacter pylori infection: a prospective evaluation.

AIM: To prospectively assess the sensitivity, specificity and time to positivity of theUltra-rapid urease test (URUT) for Helicobacter pylori (H pylori), and compare the results of one with those of two biopsies. METHODS: Five antral biopsies were taken in consecutive patients undergoing upper endoscopy: one and two biopsies for URUT, and one each for H pylori culture and histology. URUT was read at 1, 5, 10, 20 and 30 min, 1, 2, 3 and 24 h after biopsy insertion into the reagent. A positive histology and/or culture was used as positive reference "gold standards". RESULTS: URUT was more sensitive for detecting H pylori with two biopsies rather than one, at all time points up to 120 min. The sensitivity improved from 3.6% to 82.1% for one biopsy and 10.7% to 85.7% for two biopsies from 1 to 120 min. The sensitivity reached 96.4% at 24 h for both, but the specificity reduced from 100% to 96% and 92% for one and two biopsies, respectively. CONCLUSION: Development of a positive URUT result is hastened by doubling the number of gastric biopsies. We recommend taking two instead of one biopsy to achieve an earlier positive URUT result so that H pylori eradication therapy can be initiated before patient is discharged from the endoscopy suite.

Adult↗

PubMed2004

C-reactive protein: a predominant LPS-binding acute phase protein responsive to Pseudomonas infection.

As a structural component of the outer membrane of Gram-negative bacteria, endotoxin, also known as lipopolysaccharide (LPS) exhibits strong immunostimulatory properties, rendering it a pivotal role in the pathogenesis of Gram-negative septicaemia. Our attempt to identify LPS-binding proteins from the hemolymph of the horseshoe crab led to the isolation and identification of Creactive protein (CRP) as the predominant LPS-recognition protein during Pseudomonas infection. CRP is an evolutionarily ancient member of a superfamily of 'pentraxins'. It is a major protein in acute phase of infection in humans. Our investigation of CRP response to Pseudomonas aeruginosa unveiled a robust innate immune system in the horseshoe crab, which displays rapid suppression of a dosage of 10(6) CFU of bacteria in the first hour of infection and effected complete clearance of the pathogen by 3 days. Such a high dose would have been lethal to mice. Full-length CRP cDNA was cloned. Analysis of the untranslated regions suggests their crucial role in post-transcriptional regulation of CRP transcript levels. Northern blot analysis demonstrated an acute up-regulation of CRP by about 60-fold in 6-48 h of Pseudomonas infection. Taken together, our results provide new insights into the importance of CRP as a conserved molecule for pathogen recognition.

Amino Acid Sequence↗

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↗