Biomedical subjects

E C Chan

Publications and source records attributed to E C Chan.

At least 73 records · Page 4Linked to original sources

PubMed1996

Vacuolating toxin from Helicobacter pylori activates cellular signaling and pepsinogen secretion in human gastric adenocarcinoma cells.

We investigated cellular signaling and pepsinogen secretion in the human gastric adenocarcinoma cell line AGS which was pretreated with the purified vacuolating cytotoxin from Helicobacter pylori. Results indicated that vacuolating toxin increased the levels of inositol phosphates, cytosolic free calcium concentration, adenosine 3',5'-cyclic monophosphate, and phosphorylation of 31 kDa and 22 kDa proteins in the host cells. Moreover, pepsinogen secretion from AGS cells was stimulated with increasing concentrations of cytotoxin. We conclude that besides the H. pylori cytotoxin-induced cellular vacuoles, cytotoxin-stimulated signaling mediators and pepsinogen release are important factors involved in the etiology of chronic active gastritis and peptic ulcer disease.

Adenocarcinoma↗

PubMed1996

Helicobacter pylori induces gene expression in human gastric cells identified by mRNA differential display.

Changes in gene expression patterns in gastric cells infected with Helicobacter pylori were characterized by means of mRNA differential display. Total RNA preparations were extracted from the H. pylori infected gastric cells and paired non-infected cells, and were probed with candidate clones identified after screening up to 6,000 mRNA species. Among them, four clones, 04G-1, 04G-2, 01G-1, and Cppa-2 show significant expression in the infected cells by Northern blot analysis, and they are 199 bp, 196 bp, 228 bp, and 276 bp in length, respectively. Database search revealed that nucleotide sequences of these clones share very low identity with any known sequence. These results indicate that H. pylori can significantly affect gene expression in gastric cells. Furthermore mRNA differential display can be used in pathogenesis studies to identify new genes in gastric cells in response to insults such as H. pylori.

Adenocarcinoma↗

PubMed1996

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Amino Acid Sequence↗

PubMed1996

An extracellular enzyme with hyaluronidase and chondroitinase activities from some oral anaerobic spirochaetes.

Treponema denticola, Treponema vincentii and Treponema socranskii produce an enzyme that hydrolyses hyaluronic acid (HA) and chondroitin sulphate (CS). The secreted enzyme is specifically inhibited by gold sodium thiomalate and anti-bee-venom antibodies. The use of saturated substrate (HA or CS) transblots allowed the visualization of active enzyme directly from culture supernatants and is a useful tool in clarification of complex polysaccharide-degrading enzyme specificities. The affinity-purified extracellular enzyme of T. denticola contains a single molecular species with a molecular mass of 59 kDa. Since it hydrolyses both HA and CS, it can more appropriately be termed a hyaluronoglucosaminidase (HGase). The HGase has been localized at the cell surface by electron microscopy and may play an active role in the degradation of connective tissue ground substance in the initiation and progression of periodontal disease.

Antibodies, Blocking↗

PubMed1996

Characterization of a 4.2-kb plasmid isolated from periodontopathic spirochetes.

Oral anaerobic treponemes are assoicated with active periodontal disease and may comprise up to 57% of the microbiota in periodontal pockets. Four treponeme strains (designated U2a, U2b, U9b, and U9c) isolated from clincial cases were found to harbor a new 4.2-kb plasmid when plasmid DNA was extracted and purified employing the Qiagen Plasmid Kit. This plasmid differs from the smaller plasmids (2.0-, 2.6-, and 2.7-Kb) reported previously by others in Treponema denticola. The newly discovered 4.2-kb plasmid was found to be the same in all four treponeme strains by restriction endonuclease analysis. It is a circular plasmid since restriction with PstI, Pvu II, Sma I, Xma I, Ava 1 or Bam HI produced a single band of the same size. Bacterial strain U2b was shown to be Treponema socranskii and U9c to be T. denticola. The plasmid is designated "pTS1". The presence of the same plasmid in different species of the treponemes isolated from the same patient suggests the possibility of a naturally occurring genetic transfer system within the oral spirochetes or their ability to take up and maintain mobilizable plasmids.

DNA, Bacterial↗

PubMed1996

Determination of optimal liquid medium for enzyme expression by Helicobacter pylori.

AIMS: To determine the best medium for the growth and enzyme expression of Helicobacter pylori. METHODS: Twelve H pylori isolates from histologically confirmed infected patients were cultured on Brucella broth (BB), Brucella broth minus bisulphite (BLBB), and brain-heart infusion broth (BHIB), all supplemented with 5% (v/v) fetal calf serum. Growth rates and enzyme expressions of all H pylori isolates cultivated in these media were evaluated. RESULTS: Although both BLBB and BHIB supported good growth of H pylori under microaerophilic conditions, the total protein content of H pylori detected was much higher with BHIB cultivation. Measurement of the specific activities of urease, phospholipase C, and sphingomyelinase for 12 H pylori isolates cultivated in these media, showed that BHIB supported the highest expression of these enzymes. Although BLBB supported better growth of H pylori than BB, it did not increase enzyme expression. CONCLUSIONS: Cultivation of H pylori in BHIB is recommended for studies on the physiology, metabolism, and enzyme expression of the organism.

Animals↗

PubMed1996

A novel fluorochrome for the microscopic observation of microbial morphology in wet mounts.

A family of novel nontoxic fluorochromes, including trans-4-(p-N,N-dimethylaminostyryl)-N-butoxycarbonyl-methylpyridin ium bromide and other dimethylaminostyrylpyridinium salts, has been used for the examination of microorganisms in the living state. Equipment required is similar to that for immunofluorescence, but the technique does not necessitate the utilization of specific antibodies. Procaryotic vegetative cells and endospores were readily visualized by their fluorescence with no observable quenching. Other bacteria observed included capsule-forming species, acid-fast mycobacteria, and spirochetes. In addition, the technique is also applicable to the examination of eucaryotic microorganisms. The technique as described is simple and convenient, and serves as an alternate means for the microscopic study of viable microorganisms.

Bacteria↗

PubMed1996

Iron acquisition by oral hemolytic spirochetes: isolation of a hemin-binding protein and identification of iron reductase activity.

Oral anaerobic spirochetes (OAS) have been implicated in the etiology of periodontal disease. To adapt to the environment of the subgingiva, OAS must be able to acquire iron from limited sources. OAS have previously been shown not to produce siderophores but are beta-hemolytic and can bind hemin via a proteinaceous 47-kDa outer membrane sheath (OMS) receptor. Present studies show that [3H]hemin is not transported into the cytoplasm, that hemin and ferric ammonium citrate, as the sole iron sources, can support the growth of OAS and that protoporphyrin IX and Congo red are inhibitory, thereby implying an important in vivo role for hemin as an iron source. Treponema denticola ATCC 35405 produces an iron reductase. The iron reductase can reduce the central ferric iron moiety of hemin. The 47-kDa OMS hemin-binding protein has been purified to apparent homogeneity by methanol-chloroform extraction of cellular lipoproteins and the use of a hemin-agarose bead affinity column. A model of iron acquisition by OAS is presented.

Bacterial Outer Membrane Proteins↗

PubMed1995

Distribution and characterization of plasmids in oral anaerobic spirochetes.

Fifteen oral spirochete strains belonging to the species Treponema denticola, Treponema vencentii and Treponema socranskii as well as 9 fresh clinical isolates were screened for the presence of extrachromosomal plasmid DNA by a modified alkaline lysis procedure. A 2.6-kb plasmid was detected in both T. denticola ATCC 33520 and T. denticola e'. The 2.6-kb plasmid from T. denticola e' was shown to be similar to pTD1, previously reported by Ivic et al. in T. denticola ATCC 33520 on the basis of molecular weight, restriction endonuclease profile and DNA:DNA hybridization. T. denticola ATCC 33520 and T. denticola e' share 65% DNA homology and belong to different serological groups. This dissimilarity has been reconfirmed by specific immunofluorescence using polyclonal and monoclonal antibodies. A plasmid-free T. denticola ATCC 33520 was identified. Comparative studies have shown no antigenic, morphological, or genetic differences between the plasmid-bearing and the plasmid-free strain. In addition, screening of fresh clinical isolates of spirochetes revealed the presence of a 4.2-kb plasmid in 4 of these strains.

Bacterial Typing Techniques↗

PubMed1995

Evidence for two distinct locomotory phenotypes of Treponema denticola ATCC 35405.

Using a two-layer system, a bottom layer containing treponema cells suspended in NOS (New Oral Spirochete)-Noble agar medium or NOS-Bacto agar medium and overlaid with cell-free NOS-agarose medium resulted in the spirochete cells migrating into the top layer. However, if the positions of the medium layers were reversed with the cells inoculated into the bottom layer containing NOS-agarose, there was no migration into the upper layer. This suggests migration of the spirochetes away from Bacto and Noble agars. Using a 3-layer system in which cells were inoculated into a middle layer consisting of NOS-agarose medium and sandwiched between cell-free NOS-agarose medium layers, cells remained within the middle layer. If the cells were inoculated into a middle layer consisting of NOS-Bacto agar medium while the upper and lower layers remained unchanged, cells migrated into both upper and lower layers. If cells that had migrated into the upper layer were transferred into a middle layer, they virtually all migrated into the upper layer repeatedly. Cells that had migrated into the lower layer and transferred to the middle layer migrated repeatedly into the lower layer. These results suggest the possible existence of two distinct locomotory phenotypes within this strain of treponeme.

Agar↗

PubMed1995

Contaminated toothbrushes and their disinfection.

Twenty toothbrushes used by healthy subjects were screened for the presence of microorganisms. Microbes were dislodged from the brushes by vortexing, and an average of 4 x 10(3) CFU/mL were recovered from the suspending fluid. Bristles removed from the vortexed brushes still yielded confluent bacterial growth on brain-heart infusion agar medium. Virkon (one per cent), Listerine, Cepacol, Scope, and Plax were tested for their bactericidal effects on microorganisms sedimented from the suspending fluid, on toothbrush bristles and proxabrushes, and on various test species including Candida albicans, Mycobacterium smegmatis, M. bovis, and Streptococcus mitis. Virkon and Listerine killed all the test species and virtually all the microorganisms on the toothbrush bristles and proxabrushes. Six volunteers tested the efficacy of a Listerine soaking regime to prevent the bacterial contamination of toothbrushes. Soaking the toothbrush head (bristles) in Listerine for 20 minutes after brushing was sufficient to eliminate bacterial contamination.

Bacteria↗

PubMed1994

Enumeration of viable oral spirochetes from periodontal pockets.

We recently developed a successful method for quantifying oral anaerobic spirochetes in pure culture by a viable count. New oral spirochete medium was used with low temperature-gelling agarose in polystyrene tissue-culture flasks. We have extended the use of this method to determine the viable count of spirochetes from periodontal pockets. Sixteen subgingival plaque samples were obtained by insertion of sterile paper points into deep periodontal pockets. The points were placed into reduced transport medium at chairside, vortexed in the microbiology laboratory and aliquots of the medium inoculated into molten new oral spirochete-agarose medium (37 degrees C) containing rifampin (20 micrograms/ml) in a flask. Subsequent dilutions were made from this initial flask to other flasks containing selective medium in sequence. All flasks were incubated anaerobically. Most other subgingival bacteria were selectively inhibited by rifampin. Spirochete colonies were typically spherical and were either dense or cottony. Their identities were checked by darkfield examination. Counts of colony-forming units of cultivable spirochetes ranged from 12.5% to 28.2% of the total cultivable anaerobic flora by the method described.

Colony Count, Microbial↗

PubMed1993

Disruption of targeted gene in bacterial chromosome by using a temperature-sensitive plasmid.

The temperature-sensitive plasmid, pSAK3, was used in tryptophan-producing strains for tryptophanase gene disruption to block the degradation of tryptophan. Plasmid pSAK3, which consisted of the pSC103 plasmid containing a tetracycline resistant gene and a disrupted tryptophanase gene inserted by a kanamycin resistant gene, was integrated into the homologous site on the chromosome by gene recombination. Through raising the temperature of cultivation to 42 degrees C and double antibiotics screening process, the strains with disrupted tryptophanase genes could be easily selected and resulted in the generation of stable mutants that no longer exist activity of tryptophanase. Once the strain with a defected tryptophanase gene was obtained, the infection of phage P1 was applied to specifically disrupt the tryptophanase gene of any other strains. This approach also can be applied to disrupt the other targeted genes as an alternative to the conventional mutagenesis.

Base Sequence↗

PubMed1993

Treponema denticola (ex Brumpt 1925) sp. nov., nom. rev., and identification of new spirochete isolates from periodontal pockets.

Standard growth and isolation methods were used to obtain five new treponema strains in pure culture from deep periodontal pockets. The strains were identified to the species level by various methods, including agglutination, immunofluorescence, a dot blot immunoassay, electron microscopy, gas-liquid chromatography of metabolic volatile fatty acids, and DNA hybridization. Two isolates were strains of Treponema socranskii; the other three were strains of "Treponema denticola," a species described in 1925 by Brumpt. Because no type strain was designated for this species previously, the name was not included on the Approved Lists of Bacterial Names and has no current nomenclatural standing. We propose that Treponema denticola (ex Brumpt) sp. nov., nom. rev. is a valid and distinct species of the genus Treponema and designate strain ATCC 35405 as the type strain and strains ATCC 33520 and ATCC 35404 as reference strains. T. denticola appears to be the species that is most frequently isolated from periodontal pockets. Unless new isolation and cultivation techniques are introduced, it appears that present technology can yield only isolates belonging to the currently described oral anaerobic spirochete species and that there is little chance of isolating the larger treponemes.

Agglutination Tests↗

PubMed1993

A successful method for quantifying viable oral anaerobic spirochetes.

Spirochetes are markedly prevalent in periodontal disease but are not included as predominant cultivable organisms because of the inability to quantify them by viable count. A successful method was developed for enumerating viable oral spirochetes as colony-forming units (CFU) in an agarose-based medium. Treponema denticola, Treponema vincentii and Treponema socranskii in log-phase growth in new oral spirochete (NOS) broth were used for evaluation of the method. Critical components of the method include enzyme-free low temperature-gelling (37 degrees C) agarose in NOS medium in small tissue-culture flasks into which the spirochetes were seeded and diluted. The flasks were anaerobically incubated in a glove-box. Reliable, consistent and reproducible viable counts of pure spirochete cultures were obtained. The injurious effects of spirochete temperature-sensitivity were averted by using molten agarose at 37 degrees C. Distinctive colony morphologies of spirochete species could be compared from pure cultures. Addition of rifampin into the medium showed no decrease in spirochete CFU count. The method as described allows for selection of mutants and detection of biochemical activity and is potentially useful for enumeration of spirochetes from periodontal pockets as members of the predominant cultivable flora.

Agar↗

PubMed1993

Viscosity-dependent locomotion of oral spirochetes.

Spirochetes are thought to remain motile in environments (such as intercellular spaces) that immobilize extracellularly flagellated eubacteria. This attribute suggests that the viscosity of the milieu is of importance to locomotion. We sought to determine the interdependence of oral spirochete locomotion with media viscosity. Video time-lapse microscopy using darkfield optics was used. The motility of the spirochetes in media of different viscosities (various concentrations of Noble agar) was measured. Treponema denticola exhibited the fastest speed (18.7 +/- 4.4 microns/min) at a viscosity of 30 mPa.s. The highest speeds for Treponema vincentii and Treponema socranskii were 41.9 +/- 14.9 and 33.4 +/- 13.2 microns/min, respectively, at 88 mPa.s. These data show that optimal migration of spirochetes is viscosity-dependent. The results support the hypothesis that such viscosity-dependent locomotion could be a virulence factor that enables oral spirochetes to initiate and sustain periodontal disease.

Cell Movement↗

PubMed1993

Binding of hemin and congo red by oral hemolytic spirochetes.

Colony-forming units or cells in suspension of oral anaerobic spirochetes (Treponema denticola, Treponema vincentii and Treponema socranskii) bind hemin and Congo red. Hemin or Congo red binds to a hydrophobic polypeptide receptor that is located in the outer membrane of the bacterial cells and it has a relative molecular mass of 47 kDa. These oral spirochetes also lyse sheep erythrocytes to produce beta-hemolytic zones around colony-forming units. The oral spirochetes may acquire iron for growth when they lyse erythrocytes and bind heme from which they may sequester and transport iron into the cells.

Bacterial Outer Membrane Proteins↗

PubMed1993

Corticotropin-releasing hormone in baboon pregnancy.

During human pregnancy, plasma CRH immunoreactivity (CRH-IR) rises progressively, peaking during labor and falling after delivery. Among animal species, only higher primates have elevated CRH-IR during pregnancy. This study examines whether changes in plasma CRH-IR in the baboon (Papio hamadryas) are similar to those in the human. CRH-IR was determined by RIA in 16 baboons at different stages of gestation (44 samples) and in 3 males. Assays were performed on Vycor extracts of plasma and CRH-IR diluted in parallel to synthetic human (h) CRH-41 standard. Reverse phase high pressure liquid chromatography and size-exclusion chromatography with Sephadex G-50 showed that baboon CRH-IR eluted in a position similar to that of hCRH-41. Regression analysis revealed a cubic association between plasma CRH-IR and gestational age, with peak concentrations occurring at 60 days gestation (term = 182 days). Although greatly elevated concentrations persisted throughout pregnancy, concentrations in the first half (1-91 days) were significantly higher (mean +/- SEM, 1.9 +/- 0.3 nM/L; n = 27) than in the second half (92-182 days; 1.0 +/- 0.2 nM/L; n = 11; P < 0.003 by t test). CRH-IR fell to low levels by day 1 postpartum. The concentration of total cortisol in nonpregnant animals was 1370.9 +/- 134.9 nM/L (n = 5), which was similar to pregnancy levels (1346.3 +/- 356.1 nM/L; n = 28); there was no gestational age-related pattern evident. Plasma corticosteroid-binding globulin was estimated by RIA, and plasma free cortisol was calculated to be 73 +/- 14 nM/L in pregnant animals and showed no gestational age-related changes. The mean progesterone concentration in the pregnant baboon was 12.5 +/- 2.2 nM/L (7-169 days; n = 27). There was no significant change in progesterone levels during the period of gestation studied; however, they were higher than nonpregnant levels. Baboon and human plasma (0.1 mL each) were incubated with [125I]Tyr-hCRH in Tris-HCl buffer (pH 7.5) and chromatographed with Sephadex G-75, using the same buffer. The radioactivity of fractions was determined, and no CRH-binding protein was identified in baboon plasma. This study indicates that gestational changes in CRH-IR in the baboon are different from those observed in humans. There is a dissociation between maternal plasma CRH and cortisol. The apparent lack of bioactivity of baboon plasma CRH is not due to a circulating binding protein, which is absent in this species.

Animals↗