Biomedical subjects

B Ho

Publications and source records attributed to B Ho.

At least 55 records · Page 3Linked to original sources

PubMed1997

An HPLC/RIA method for dynorphin A1-13 and its main metabolites in human blood.

A selective HPLC/RIA procedure for the determination of dynorphin A1-13 (Dyn A1-13) and its major metabolites in human blood was developed. In order to block peptidase activity, blood samples were transferred into an aliquot of a blocking solution (5% aqueous ZnSO4 solution-acetonitrile-methanol; 5:3:2, v/v/v). After solid phase extraction, reconstituted aliquots were injected into an isocratic reversed phase HPLC system to separate Dyn A1-13 from its main metabolites (Dyn A2-13, Dyn A1-12 and Dyn A2-12). The isolated and concentrated HPLC-fractions were assayed by RIA using a commercially available antiserum. Intra-day variabilities for quality controls (0.07, 0.25, and 1 ng ml-1) of Dyn A1-13, A2-13, A1-12, A2-12 were between 9 and 41%. Accuracy was between 86 and 132%. Inter-day variability for single quality controls analyzed on five days for Dyn A1-13, A2-13, A1-12, A2-12 was between 4 and 49% for 0.07, 0.25 and 1 ng ml-1 samples, respectively. Accuracy was between 72 and 129%. Five different batches of control blood showed blood levels no different from zero. Considering the complexity of the assay, the method is selective, accurate and reproducible with a limit of detection of 0.07 ng ml-1 for Dyn A1-13, Dyn A2-13, Dyn A1-12 and 0.21 ng ml-1 for Dyn A2-12. The assay was applied to the determination of Dyn A1-13 and its metabolites in blood samples of 2 subjects receiving i.v. infusions of 250 micrograms or 1000 micrograms kg-1 Dyn A1-13 over 10 min.

Acetonitriles↗

PubMed1996

Neuroblastoma cell culture assay shows that Carcinoscorpius rotundicauda haemolymph neutralizes tetrodotoxin.

Veratridine facilitates the influx of Na+ into Neuro-2A cells and this process is exacerbated by the presence of a Na+/K(+)-ATPase inhibitor, ouabain, leading to a reduction in cell viability. However, tetrodotoxin neutralizes the cytotoxic effects of veratridine, hence sustaining the viability of Neuro-2A cells. This neutralizing ability was negated when TTX was first reacted with cell-free haemolymph of Carcinoscorpius rotundicauda, resulting in reduced cell viability. These results therefore indicate a bona fide effect of the cell-free haemolymph against tetrodotoxin as demonstrated by in vitro cell culture technique.

Animals↗

PubMed1996

Anticonvulsant activity of novel derivatives of 2- and 3-piperidinecarboxylic acid in mice and rats.

The relative ability of derivatives of 2-piperidinecarboxylic acid (2-PC; pipecolic acid) and 3-piperidinecarboxylic acid (3-PC; nipecotic acid) to block maximal electroshock (MES)-induced seizures, elevate the threshold for electroshock-induced seizures and be neurotoxic in mice was investigated. Protective index (PI) values, based on the MES test and rotorod performance, ranged from 1.3 to 4.5 for 2-PC benzylamides and from < 1 to > 7.2 for 3-PC derivatives. PI values based on elevation of threshold for electroshock-induced seizures and rotorod performance ranged from > 1.6 to > 20 for both types of derivatives. Since preliminary data indicated that benzylamide derivatives of 2-PC displace [3H]1-[1-(2-thienyl)-cyclohexyl]piperidine (TCP) binding to the phencyclidine (PCP) site of the N-methyl-D-aspartate (NMDA) receptor in the micromolar range and such low affinity uncompetitive antagonists of the NMDA receptor-associated ionophore have been shown to be effective anticonvulsants with low neurological toxicity, the 2-PC derivatives were evaluated in rat brain homogenates for binding affinity to the PCP site. Although all compounds inhibited [3H]TCP binding, a clear correlation between pharmacological activity and binding affinity was not apparent. Select compounds demonstrated minimal ability to protect against pentylenetetrazol-, 4-aminopyridine- and NMDA-induced seizures in mice. Corneal and amygdala kindled rats exhibited different sensitivities to both valproic acid and the nonsubstituted 2-PC benzylamide, suggesting a difference in these two models. Enantiomers of the alpha-methyl substituted benzylamide of 2-PC showed some ability to reduce seizure severity in amygdala kindled rats.

4-Aminopyridine↗

PubMed1996

Binding of human plasminogen and lactoferrin by Helicobacter pylori coccoid forms.

The interactions between Helicobacter pylori spiral and coccoid forms, extracellular matrix (ECM) and plasma proteins were studied in an 125I-labelled protein assay. The range of binding of collagen V, plasminogen, human lactoferrin (HLf) and vitronectin to coccoid forms of H. pylori NCTC 11637 was 26-48%. In contrast, binding of radiolabelled fibronectin and collagen types I and III was low (3-8%). The coccoid forms of 14 strains of H. pylori showed significant HLf binding (median 26%). With plasminogen, no significant difference was found between binding to the coccoid (median = 13%) and spiral (median = 12%) forms, of 13 of the 14 strains of H. pylori tested; the exception was strain NCTC 11637. 125I-plasminogen showed a dose-dependent binding to both the coccoid and spiral forms. Plasminogen binding to both forms was specific; the binding was inhibited by non-labelled plasminogen, plasmin, lysine, EACA (epsilon-aminocaproic acid) but not by fetuin or various carbohydrates. Similarly, HLf binding was found to be specific and was inhibited by non-labelled HLf and BLf. The coccoid forms showed either similar or enhanced ECM binding capabilities compared with the spiral forms. As the binding of ECM proteins may be an important mechanism of tissue adhesion for various pathogenic bacteria, the coccoid differentiated form of H. pylori can be considered as an infective form in the pathogenesis of helicobacter infection and type B gastritis.

Collagen↗

PubMed1996

Genome macrorestriction analysis of sequential Pseudomonas aeruginosa isolates from bronchiectasis patients without cystic fibrosis.

The respiratory tracts of bronchiectasis patients may be persistently colonized with Pseudomonas aeruginosa, despite intensive chemotherapy. The organism may undergo phenotypic changes in these patients, providing misleading typing results by conventional methods. We prospectively studied eight bronchiectasis patients without cystic fibrosis over a period of 1 year. A high microbial load of P. aeruginosa was found in 70% of sputum samples collected. Of these, 55 sequential P. aeruginosa isolates were characterized by a genotyping method, pulsed-field gel electrophoresis, to overcome the problem of differentiating the P. aeruginosa strains during chemotherapy. Genome macrorestriction fingerprinting patterns were analyzed after digestion with XbaI restriction endonuclease. Of the eight patients, six harbored a single dominant strain of P. aeruginosa, with an intrapatient macrorestriction similarity pattern range of 96 to 100%. The other two patients were infected with mixed bacterial isolates including P. aeruginosa. However, diversity was observed in the P. aeruginosa isolates from all eight patients, with a relatedness of only 55 to 65%. The study further strengthens the fact that pulsed-field gel electrophoresis can be used efficiently and effectively to differentiate P. aeruginosa strains in bronchiectasis patients without cystic fibrosis.

Adult↗

PubMed1996

Is the coccoid form of Helicobacter pylori viable?

Helicobacter pylori was grown on solid medium for up to 5 weeks. Morphological conversion from spiral to coccoid forms began after 7 days incubation under microaerophilic conditions. Similar to the exponential cultures, the ageing bacteria produced alkaline phosphatase, acid phosphatase, leucine arylamidase and naphthol-AS-beta 1-phosphohydrolase, although there was a reduction in the levels of the latter two enzymes. Unlike the other enzymes, urease was not detected in 5-week-old cultures. By using primers based on urease subunit C and 26 kD protein genes for polymerase chain reaction (PCR) these two important gene fragments remained conserved despite the morphological conversion from spiral to coccoid forms. Furthermore, PCR-based random amplified polymorphic DNA (RAPD) fingerprinting showed similar DNA banding patterns from bacteria of various ages, demonstrating the conservation of the DNA composition despite morphological changes. This study shows that the aging coccoid form of H. pylori, although reportedly non-culturable in vitro, remains genetically unchanged indicating that it is likely to be viable.

Acid Phosphatase↗

PubMed1995

Data clustering and other archive retrieval strategies for teleradiology and picture archiving and communication systems.

A key advantage in the conversion from film-based to digital radiology is the possibility of a long-term on line electronic archival of patient studies. The popular approach based on optical disk jukeboxes for the long-term archive and magnetic disk storage for data caching is not economically attractive because of the cost of both the jukebox and the medium. Strategies for extending the archival system design with a tape jukebox have been studied. The proposed strategy calls for the use of high-ratio lossy compression together with low-cost tape storage to make long-term on line archiving more affordable. An intelligent prefetching algorithm based on hospital information system and radiologic information system triggers, which in turn are augmented by manual case preparation, can effectively overcome the longer latency of ad hoc retrievals. This longer latency is caused by both system-level bottlenecks and the sequential access constraint of the tape drive. Strategies for image clustering and tape allocation by patient classification also enhance retrieval efficiency. This archival design using image compression, prefetching, and clustering could be implemented in many of the existing teleradiology and picture archiving and communication systems.

Algorithms↗

PubMed1995

Molecular cloning and sequence analysis of factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda.

Two forms of Factor C cDNAs: CrFC21 (3448 bp) and CrFC26 (4182 bp) have been cloned into lambda gt22. CrFC26 includes 568 nucleotides of 5' untranslated region (5' UTR) containing seven ATGs before the real initiation site, an open reading frame (ORF) of 3249 nucleotides, a stop codon, and 365 nucleotides of 3' untranslated sequence. There are four polyadenylation signals and six potential glycosylation sites. The ORF codes for a signal peptide of 24 amino acids and a Factor C zymogen of 1059 residues. The CrFC21 lacks most of the 5' UTR, and has some base changes in its ORF. The predicted secondary mRNA structures of the 5' end of CrFC26 showed numerous stem-and-loop structures, thus obscuring its real start codon. In contrast, CrFC21 has a well-exposed AUG start site, and expresses Factor C in transcription-translation reactions in vitro. There is a typical serine protease catalytic triad of Asp-His-Ser, which is structurally like prothrombin, but catalytically more similar to trypsin. Although an overall homology of 97.7% was observed in comparison with the Tachypleus tridentatus Factor C (TtFC) cDNA, there were notable differences in the restriction sites and subtle base substitutions in the CrFC cDNA. The high degree of homology between Factor C from T. tridentatus and C. rotundicauda substantiates, at the molecular level, the proximity of these two species in the course of evolution. This finding contravenes the apparent disparities with respect to their morphology, ecological habitat, and taxonomical classification.

Amino Acid Sequence↗

PubMed1995

Expression of Carcinoscorpius rotundicauda factor C cDNA.

The cDNA encoding Factor C (FC) from the Singaporean horseshoe crab Carcinoscorpius rotundicauda has been studied for in vitro coupled transcription-translation (TnT) under the T7 promoter. Two species of full length cDNA, CrFC26 and CrFC21 which differ in length and nucleotide sequence at their 5' untranslated regions (UTR) were used in this study. Wild type CrFC26 with a long 5' UTR containing multiple "false" ATGs failed to generate a translated product. With a more accessible ATG codon in CrFC21, the recombinant construct gave a high yield of FC when transcribed and translated in vitro. CrFC26 deletion mutants which lack the entire 5' UTR and portions of the putative leader peptide were translatable, albeit at lower efficiency as compared to CrFC21. In vitro and in vivo expression of truncated portions of the CrFC21-T7 gene 10 fusions have been compared. In vitro reactions yielded single gene products from each of the expression constructs whereas E. coli produced three major immunoreactive bands of FC.

Animals↗

PubMed1995

The adhesion of Helicobacter pylori extract to four mammalian cell lines.

OBJECTIVE: To study the adherence of acid-glycine extract (AGE) from Helicobacter pylori to four mammalian cell lines: KATO III, CCL17, CCL156 and Neuro-2A. DESIGN: In vitro assays to assess H. pylori adherence were based on the principle of the affinity of the bacterial antigens to mammalian cells in culture. Protein extracts from mammalian cells were either coated onto 96-well microtitre plates or electrophoretically resolved and blotted onto immobilon-P membranes. When the adhesive proteins derived from the AGE of H. pylori adhered to the mammalian cell proteins, these adhesive antigens were detected by rabbit anti-H. pylori AGE antibodies. METHODS: The adhesive proteins in H. pylori NCTC11637 AGE were detected by enzyme-linked immunosorbent assay (ELISA) and Western blot under non-denaturing conditions. RESULTS: Adhesive proteins constitute 5% H. pylori AGE proteins. The adhesive proteins adhered more readily to mammalian cell extracts than to bovine serum albumin. Modified Western blot analysis showed their affinity to a number of mammalian proteins ranging in molecular weight from 40 to 900 kDa, and in particular to the 140-230 kDa proteins. CONCLUSIONS: The two in vitro adherence assays used effectively demonstrated the adherence of H. pylori to different mammalian cell lines. The 5% adhesive proteins from H. pylori may play an essential role in infection and colonization by H. pylori. Furthermore, adhesive proteins have similar adherence properties to mammalian cell components and may play an important role in the pathogenesis of H. pylori infection.

Adhesins, Bacterial↗

PubMed1995

The pathogenic role of the coccoid form of Helicobacter pylori.

Adherence of the coccoid forms of Helicobacter pylori to the gastric carcinoma cell line (KATO III) was examined by transmission electron microscopy. Specialized attachment sites such as the 'adhesion pedestal', 'cup-like indentation' and 'abutting adhesion' were seen in the interaction between coccoids and epithelial cells. These adherence patterns were similar to those observed with spiral forms in gastric biopsy specimens in vivo, suggesting a possible pathogenic role for the coccoids of H. pylori. With antigens prepared from both the coccoid and spiral forms, IgG antibodies reactive to H. pylori were detected using ELISA. Patients with gastroduodenal disease accounted for 74% (37/50) ELISA positives. Of the 50 healthy blood donors, 32 and 28% were seroreactive to coccoid and spiral antigens, respectively. These sera were further characterized by Western blot where immunoreactive protein bands of 128, 116, 110, 95, 91, 66, 60, 54, 50 and 33 kD were conserved in both the coccoid and spiral forms. These findings suggest that the coccoids could be a differentiated infective form of H. pylori, and that they could evoke an immune response from the host after invading the cells via specialized attachment sites.

Antigens, Bacterial↗

PubMed1994

A tetrodotoxin neutralizing system in the haemolymph of the horseshoe crab, Carcinoscorpius rotundicauda.

The horseshoe crab, Carcinoscorpius rotundicauda, when injected intracardially with tetrodotoxin (TTX) at a dosage of 10 x LD50 of mice showed no mortality. The resulting haemolymph extracted 24 hr later from these crabs caused no fatalities when injected into mice. TLC analysis revealed that cell free haemolymph (CFH) had converted a single yellow fluorescent spot of TTX into two blue fluorescent components when the reaction was carried out at 25 degrees C. However, no fluorescence was observed when TTX was reacted with CFH at 37 degrees C. There was also an increase in the intensity of protein bands on SDS-PAGE gel corresponding to 26,000 and 28,000 mol. wts. This may be the result of the TTX-neutralization reaction in CFH. The pretreatment of TTX with CFH reduced the toxicity of TTX in mice by two-fold. The anti-TTX activity is temperature and time dependent as well as heat stable. Protein denaturation of the haemolymph suggests that proteinaceous factor(s) may also play a role in TTX-neutralizing activity of CFH. It is therefore possible that the detoxification system is attributed to concerted actions of both proteinaceous and non-proteinaceous factors in the haemolymph.

Animals↗

PubMed1993

Two forms of factor C from the amoebocytes of Carcinoscorpius rotundicauda: purification and characterisation.

The two apparent form of the endotoxin-sensitive Factor C which were found to exist in the amoebocytes of horseshoe crabs have been separately purified to homogeneity from the lysate of the South-East Asian species, Carcinoscorpius rotundicauda. Both forms are serine proteinase zymogens having an apparent molecular mass of 132 kDa. By reducing SDS-PAGE, one was shown to consist of a single polypeptide while the other has a heavy chain (80 kDa) and a light chain (52 kDa) bridged by disulfide linkage(s). Both zymogen forms have endotoxin (lipopolysaccharide) receptors to which endotoxin binds to activate their catalytic sites. However, single-chain Factor C appears to have higher-affinity endotoxin-binding sites which are competitively but reversibly occupied by DMSO when the latter was added during its purification. Another salient difference between the two forms of Factor C is exhibited in their manner of activation by endotoxin. While double-chain Factor C appears similar to that of Tachypleus tridentatus, single-chain Factor C did not undergo any proteolytic cleavage upon activation. This conformational transition of zymogen activation suggests an alternative reversible pathway of endotoxin activation for the single-chain Factor C.

Animals↗

PubMed1993

A computer program in QuickBasic for the selection of tests for the identification of Helicobacter pylori.

Reliable microbiological tests are needed for the identification of bacteria. A program has been written in QuickBasic to identify such tests by using a formula that is based on Gyllenberg's Sum of C(i) and Gyllenberg's Rank R(i). A total of 139 papers on a newly isolated bacterium, Helicobacter pylori, was used as data source for the coding of test results into an input file. The program outputs a list that aids in the determination of suitable tests for the identification of H. pylori. These tests chosen by the formula were found to be correctly identified as supported by later publications on the bacterium.

Decision Support Techniques↗

PubMed1993

Slow and incomplete inactivations of voltage-gated channels dominate encoding in synthetic neurons.

Electrically excitable channels were expressed in Chinese hamster ovary cells using a vaccinia virus vector system. In cells expressing rat brain IIA Na+ channels only, brief pulses (< 1 ms) of depolarizing current resulted in action potentials with a prolonged (0.5-3 s) depolarizing plateau; this plateau was caused by slow and incomplete Na+ channel inactivation. In cells expressing both Na+ and Drosophila Shaker H4 transient K+ channels, there were neuron-like action potentials. In cells with appropriate Na+/K+ current ratios, maintaining stimulation produced repetitive firing over a 10-fold range of frequencies but eventually led to "lock-up" of the potential at a positive value after several seconds of stimulation. The latter effect was due primarily to slow inactivation of the K+ currents. Numerical simulations of modified Hodgkin-Huxley equations describing these currents, using parameters from voltage-clamp kinetics studied in the same cells, accounted for most features of the voltage trajectories. The present study shows that insights into the mechanisms for generating action potentials and trains of action potentials in real excitable cells can be obtained from the analysis of synthetic excitable cells that express a controlled repertoire of ion channels.

Action Potentials↗

PubMed1993

A simple and efficient continuous culture system for Helicobacter pylori.

Helicobacter pylori has been successfully maintained in a continuous culture system. The culture was grown in 0.6 litre of brain heart infusion broth with supplements in a 1 litre fermenter with a flow rate of 25 ml/h, an 80% dilution rate and a specific growth rate of 0.06/h. Unlike the typical growth curve which occurred in batch culture, the growth pattern in continuous culture showed a short lag phase following which growth was maintained in exponential phase with > or = 10-fold increase in viability and the pH was stabilised at 6.95 +/- 0.05. The urease activity remained constant in relation to viability while the urea was completely hydrolysed. The system provides cells with growth characteristics similar to the fresh isolate which are highly adaptable to the in vitro environment. This study shows that continuous culture is a simple and efficient growth system for the cultivation of a 'synchronous' spiral form of H. pylori.

Bacteriological Techniques↗

PubMed1993

Morphological changes of Carcinoscorpius rotundicauda amoebocyte and Escherichia coli during their interaction.

Interaction between Carcinoscorpius rotundicauda amoebocytes and Escherichia coli or Staphylococcus aureus has shown that whilst E. coli was observed to lyse, S. aureus remained intact. The heavily granulated amoebocytes were activated by the lipopolysaccharide of E. coli to undergo dramatic morphological changes with time, leading to degranulation which caused lysis of the E. coli. Degranulation of the amoebocytes involved maturation of the larger electron dense granules (0.7-2.0 micron) which were apparently mobilised towards the cell membrane whilst undergoing a loss in electron density. Some of these larger granules were expelled at 15-30 min while other intracellular granules underwent further degranulation, where parallel filaments appearing like microtubules were evident, giving the granules a striated and less dense form. By 60 min, there was complete depolymerisation of the microtubular filaments giving rise to undefined, fused and homogeneously packed striations and particulate material in the cytoplasm; the smaller electron dense granules (0.3-0.7 micron) remained unchanged. This asynchronous degranulation and preferential expulsion of granules from only some amoebocytes while maintaining others intact, implies that the horseshoe crab has safeguards in its defence mechanism, hence ensuring its continued survival as a 'living fossil'.

Animals↗

PubMed1993

Electrophoretic analysis of endotoxin-activated gelation reaction of Carcinoscorpius rotundicauda amoebocyte lysate.

Electrophoretic analysis of a 60 min reaction between E. coli endotoxin and the amoebocyte lysate showed that the coagulation reaction was complete by 15 min, with the conversion of coagulogen (21 kDa) to coagulin (17 kDa). Coincident with this observation was the maximal activities at 15 min, of Factor C and proclotting enzyme. On agitation of the coagulin gel clots, bioactive endotoxin was recovered. Densitometric scan of the electrophoretically-resolved proteins showed that the sum of coagulogen and coagulin remained almost constant at various time intervals of the coagulation reaction. Electrophoresis serves as a convincing and visually discernible method of studying the kinetics of coagulation, and defining the onset and completion of gelation. Furthermore, it is a useful means of examining the integrity of fresh lysate preparations based on the presence or absence of the 17 kDa coagulin band.

Animals↗