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Biomedical subjects

L Yan

Publications and source records attributed to L Yan.

At least 235 records · Page 13Linked to original sources
PubMed1995

Long-term supplementation of canthaxanthin does not inhibit gastric epithelial cell proliferation in Helicobacter mustelae-infected ferrets.

The effect of canthaxanthin (CX), a nonprovitamin A carotenoid, on gastric epithelial proliferation was studied in ferrets colonized with Helicobacter mustelae, which causes a chronic gastritis and an increased gastric epithelial cell proliferation. Seven spayed female ferrets were dosed by gavage with CX beadlets (50 mg/kg body wt, 5 d/wk) over 2 y, whereas seven control animals were given placebo beadlets. At the end of the 2-y-period, ferrets were killed, and gastric tissues were obtained from the antrum and body regions. A cell-proliferating biomarker, proliferating cell nuclear antigen (PCNA), was assayed by immunohistochemistry. The PCNA positivity was measured by a computer-based image analysis system. Serum concentrations of carotenoids, retinoids and tocopherols were analyzed by HPLC. Serum antioxidant status was measured by the oxygen radical absorbance capacity (ORAC) assay. The PCNA positivity in both antrum and body regions was not significantly different between CX-fed ferrets and controls. Serum CX concentrations were significantly higher in CX-fed ferrets vs. controls (P < 0.001), whereas levels of other carotenoids, retinoids and tocopherols were not significantly different. The ORAC values were not significant different between groups. This study does not suggest inhibitory effects of CX on gastric epithelial cell proliferation in H. mustelae infected ferrets.

Animals↗
PubMed1995

In-vitro hepatotoxic factor in Helicobacter hepaticus, H. pylori and other Helicobacter species.

Several inbred strains of mice in closed breeding colonies were found to have spiral-shaped bacteria associated with active, chronic hepatitis. A new species of Helicobacter, H. hepaticus, was isolated from the infected livers of some strains of mice. Other strains of mice were colonised with H. hepaticus in the caecum and colon, but not the liver. Filtersterilised supernatant fluid from five strains of H. hepaticus was tested in a mouse liver cell line (ATCC no. CCL 9.1) for cytotoxic activity. All strains produced a toxic factor causing morphological changes in the cells at dilutions up to 1 in 1000. Toxicity was observed after exposure to the supernatant fluid for 48-72 h. Other Helicobacter spp. that also produced the cytopathic effect (CPE) in the liver cell line were H. felis, H. acinonyx, H. pylori and one strain of H. mustelae. "Helicobacter rappini" and H. muridarum did not cause CPE in the liver cells. The soluble factor was stable at 4 degrees C for up to 3 months. It was also stable at 56 degrees C for 30 min, but was inactivated by boiling for 15 min. It was inactivated by incubation with trypsin. A partially purified preparation of the cytotoxin had a mol. wt of c. 100,000 and did not have urease activity. The cytotoxin produced by H. hepaticus did not cause vacuole formation in HeLa cells.

Animals↗
PubMed1995

Evaluation of antibiotic therapies for eradication of Helicobacter hepaticus.

The newly recognized murine pathogen Helicobacter hepaticus is known to colonize the ceca and colons of several strains of mice from a variety of commercial suppliers. Additionally, the organism persistently infects mice, causes a chronic hepatitis, and is linked to hepatic tumors in the A/JCr inbred mouse strain. For this reason, eradication of the organism from infected mouse colonies is desirable. Treatment modalities for eradication of H. hepaticus from the gastrointestinal system consisted of oral administration of various antibiotic combinations previously evaluated for eradication of experimental H. felis gastric infection in mice. A/JCr mice (8 to 10 weeks old) naturally infected with H. hepaticus were divided into six treatment groups of 10 animals each. Animals received monotherapy of amoxicillin, metronidazole, or tetracycline or triple therapy of amoxicillin-metronidazole-bismuth (AMB) or tetracycline-metronidazole-bismuth (TMB). All medications were administered by oral gavage three times daily for 2 weeks. One month after the final treatment, mice were euthanatized and livers, ceca, and colons were cultured for H. hepaticus. All untreated control animals had H. hepaticus isolated from the cecum and/or colon. H. hepaticus was not recovered from the livers, ceca, or colons of the AMB or TMB treatment groups. All animals receiving the various antibiotic monotherapies had H. hepaticus isolated from the cecum and colon. We conclude that at the doses and the route evaluated, AMB and TMB triple therapies are effective for eradication of H. hepaticus in 8- to 10-week old A/JCr mice.

Amoxicillin↗
PubMed1995

Helicobacter pylori-induced gastritis in the domestic cat.

Helicobacter pylori has been cultured from the inflamed gastric mucosae of naturally infected cats; the lesions in H. pylori-infected cat stomachs mimic many of the features seen in H. pylori-infected human stomachs. To determine whether H. pylori-negative specific-pathogen-free cats with normal gastric mucosae were susceptible to colonization by this bacterium and whether gastritis developed after infections, four H. pylori-negative cats treated with cimetidine were orally dosed three times with 3 ml (1.5 x 10(8) CFU/ml) of H. pylori every 4 days. All four cats became persistently colonized as determined by gastric cultures and PCRs from serial gastric biopsy samples and necropsy samples at 7 months postinfection. H. pylori was not isolated from the two control cats, nor were their gastric tissues positive by PCR; one of the two cats had a few focal lymphocytic aggregates in the body submucosa, whereas the second cat had a normal gastric mucosa. All four H. pylori-infected cats had multifocal gastritis consisting of lymphoid aggregates plus multiple large lymphoid nodules, which were most noticeable in the antral mucosa. In addition, one H. pylori-infected cat had a moderate diffuse infiltration of polymorphonuclear leukocytes in the subglandular region of the antrum. H. pylori-like organisms were focally distributed in glandular crypts of the antrum. Two of the H. pylori-infected cats had significant (eightfold) increases over baseline in levels of immunoglobulin G H. pylori serum antibody. The H. pylori isolates from the four experimentally infected cats had restriction fragment length polymorphism patterns specific for the flaA gene that were identical to those of the inoculating strain. H. pylori readily colonizes the cat stomach and produces persistent gastritis.

Animals↗
PubMed1995

Inability of an isogenic urease-negative mutant stain of Helicobacter mustelae to colonize the ferret stomach.

Eight ferrets specific-pathogen-free for Helicobacter mustelae were given, per dose, approximately 3.0 x 10(7) CFU of either the wild-type parent strain of H. mustelae (NCTC 12032) (two ferrets) the isogenic urease-negative mutant strain of H. mustelae (10::Tn3Km) (four ferrets), or sterile culture broth (two ferrets). Infection status was monitored by endoscopic gastric biopsy for urease activity, histopathology, and culture and by serology at 3, 6, 10, and 21 weeks. All ferrets were necropsied at 25 weeks. Both negative control ferrets remained uninfected, both ferrets receiving the H. mustelae wild-type parent strain became infected after two doses of the organism, and all four ferrets given two doses of the isogenic urease-negative mutant strain of H. mustelae remained uninfected throughout the 6-month study. Histopathology correlated with infection status. H. mustelae-infected ferrets exhibited diffuse mononuclear inflammation in the subglandular region and the lamina propria of the gastric mucosa, while uninfected ferrets showed no or minimal inflammation. These results suggest that urease activity is essential for colonization of the ferret stomach by H. mustelae.

Animals↗
PubMed1995

Identification of widespread Helicobacter hepaticus infection in feces in commercial mouse colonies by culture and PCR assay.

The identification of a new murine pathogen, Helicobacter hepaticus, and its association with chronic active hepatitis and liver tumors prompted an evaluation of the prevalence of H. hepaticus in commercially available mice. Of the 28 different strains or stocks, totaling 160 mice from four major commercial vendors, cultured for H. hepaticus, 100% of mice from two outbred strains from one vendor were infected with H. hepaticus, whereas 9 of 13 inbred mouse strains from another vendor were infected. This high prevalence of H. hepaticus established a need for a rapid and reproducible, noninvasive assay for the screening of colony-maintained mice being used for biomedical research. The culturing of fecal material by using 0.45-microns-pore- size filtration for H. hepaticus consistently yielded reproducible results but required extended periods of time. (1 to 3 weeks) to obtain a definitive answer. Although it is rapid, the use of a direct PCR-based detection assay with fecal specimens is restricted by inhibitory agents. to circumvent these inhibitory agents and to augment our H. hepaticus culture technique, we have developed a novel PCR system in which the bacteria are isolated from fecal material in the presence of polyvinylpyropyrollidone and lysed by treatment with Chelex 100. The PCR is performed with Tth polymerase supplemented with a polymerase enhancer. By this PCR method, 24 H. hepaticus culture-positive and 30 H. hepaticus culture-negative fecal samples were correctly identified. Moreover, two samples which were PCR positive and culture negative initially were positive by both methods upon retesting of fresh material. Southern blot hybridizations and sequencing of PCR products showed them to be H. hepaticus specific. A comparison of results obtained under identical conditions indicated a 100-fold increase in sensitivity with Tth polymerase over Taq polymerase. This PCR method can be used as a noninvasive means of rapidly screening large numbers of colony mice for H. hepaticus.

Animals↗
PubMed1995

Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting.

The gastric pathogen Helicobacter pylori establishes long-term chronic infections that can lead to gastritis, peptic ulcers, and cancer. The species is so diverse that distinctly different strains are generally recovered from each patient. To better understand the dynamics of long-term carriage, we characterized H. pylori isolates from initial and follow-up biopsy specimens from a patient population at high risk of H. pylori infection and gastric cancer. Eighty-five isolates were obtained from 23 patients and were analyzed by genomic restriction enzyme analysis, arbitrarily primed PCR fingerprinting, (random amplified polymorphic DNA analysis), and/or restriction of specific PCR-amplified genes (restriction fragment length polymorphism analysis). A single strain was found in sequential biopsy specimens from 12 of 15 patients (80%) receiving sucralfate. In the remaining three patients treated with sucralfate, two strains were identified in two patients and three strains were identified in the third patient. In contrast, a single strain was found in sequential biopsy specimens from only three of eight patients (37%) receiving bismuth, metronidazole, and nitrofurantoin. Two strains were identified in five other patients receiving bismuth-antibiotic (63%). Immunoglobulin G antibodies to H. pylori were present in the sera of all patients. Thus, H. pylori colonization can persist for long periods (up to at least 4 years), despite high titers of immunoglobulin G antibodies in serum. Resistance to metronidazole was noted in some strains before and/or after treatment, but all strains remained susceptible to amoxicillin, tetracycline, and nitrofurantoin. We conclude that H. pylori genotypes, as measured by several sensitive DNA fingerprinting methods, can remain stable for years in vivo, despite the acquisition or loss of drug resistance, circulating antibody, or exposure to antibiotics or sucralfate.

Anti-Bacterial Agents↗
PubMed1995

A 25.7 x 10(3) M(r) hydra metalloproteinase (HMP1), a member of the astacin family, localizes to the extracellular matrix of Hydra vulgaris in a head-specific manner and has a developmental function.

Hydra extracellular matrix (ECM) is composed of a number of components seen in vertebrate ECM such as laminin, type IV collagen, fibronectin, and heparan sulfate proteoglycan. A number of functional studies have shown that hydra ECM plays an important role in pattern formation and morphogenesis of this simple metazoan. The present study was designed to identify matrix degrading proteinases in hydra and determine their potential function in hydra morphogenesis. Using SDS-PAGE gelatin-zymography, five gelatinolytic bands were identified with relative molecular masses of 67 x 10(3), 51-58 x 10(3) (a triplet) and 25-29 x 10(3), respectively. Inhibition studies indicated that all of these gelatinases were metalloproteinases. Gelatin-zymography indicated that there was a differential distribution of these gelatinases along the longitudinal axis of hydra, with the 67 x 10(3) M(r) gelatinase being concentrated in the body column, while the 51-58 x 10(3) M(r) gelatinase triplet and the 25-29 x 10(3) M(r) gelatinase concentrated in the head region. Purification procedures were successfully developed for the 25-29 x 10(3) M(r) metalloproteinase which has been termed hydra metalloproteinase 1 (HMP1) and which appeared as a single band with a SDS-PAGE mobility of 25.7 x 10(3) M(r). The N-terminal sequence of purified HMP1 indicated that it has structural homology with metalloproteinases that belong to the astacin family. Subsequent cloning and sequencing of cDNA clones confirmed the identification of HMP1 as an astacin-like metalloproteinase. Immunocytochemical studies with antibodies generated against the purified enzyme and to a synthetic peptide indicated that HMP1 was localized to the ECM of tentacles. Functional studies were performed in which purified HMP1, anti-HMP1 IgG, or suspected substrates of HMP1 (e.g. growth factors such as TGF-beta 1) were introduced into the interepithelial compartment of hydra using a 'DMSO loading' procedure. These studies indicated that HMP1 has a functional role during a number of developmental processes such as head regeneration and cell differentiation/transdifferentiation of tentacle battery cells.

Amino Acid Sequence↗
PubMed1994

Determination of GDP-Fuc:Gal beta 1-4GlcNAc-R (Fuc to GlcNAc) alpha 1,3 fucosyltransferase activity by a solid-phase method.

We report the development of a solid-phase assay for the activity of the enzyme GDPFuc:Gal beta 1-4GlcNAc-R (Fuc to GlcNAc) alpha 1,3 fucosyltransferase (alpha 1,3FT). This enzyme generates the blood group antigen Lewis x (Lex)Gal beta 1-4(Fuc alpha 1-3)GlcNAc-R from the acceptor Gal beta 1-4GlcNAc-R. In our method, the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (lacto-N-neotetraose, LNnT) from human milk was chemically conjugated to bovine serum albumin (BSA) to generate LNnT-BSA. As a source of alpha 1,3FT to develop the assay, we used extracts of COS7 cells created to stably express the human FucTIII and FucTIV genes, both of which have alpha 1,3FT activity. LNnT-BSA was immobilized in microtiter wells and incubated with GDPFuc and cell extracts. The Lex antigen generated by alpha 1,3FT was detected with a monoclonal IgM antibody (anti-CD15). Binding of this IgM-type antibody to product was detected by one of two methods. Method 1 was based on the binding of alkaline phosphatase-conjugated goat anti-mouse IgM. Method 2 was based on the binding of a streptavidin conjugate of the recombinant bioluminescent protein aequorin to biotinylated goat anti-mouse IgM. The alpha 1,3FT assay was linear with respect to time (0-3 h), extract added (0-40 micrograms), and was dependent on GDPFuc (20 microM optimal) and LNnT-BSA. Both methods 1 and 2 allowed measurement of alpha 1,3FT in extracts of the human cell line HL-60.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1994

Human polymorphonuclear leukocytes lack detectable nitric oxide synthase activity.

Nitric oxide regulates polymorphonuclear leukocyte (PMN) function, but whether or not human PMNs express nitric oxide synthase (NOS) activity is controversial. We studied NOS activity in human PMNs by using human aortic endothelial cells (HAECs) for comparison. The conversion of L-arginine to L-citrulline, a relatively specific measure of NOS activity, was easily measured and inducible in fractionated HAECs, and > 90% of all L-arginine conversion was blocked by the NOS inhibitor, N omega-amino-L-arginine (L-NAA). In fractionated PMNs, L-arginine conversion was low and was unaffected by L-NAA. In addition, NOS activity was not induced in PMNs by LPS, IL-1 beta, or IFN-gamma. In a whole-cell assay, total L-arginine conversion was much lower in human PMNs compared with HAECs (3.38 +/- 0.21 vs 157.5 +/- 10.28 pmol/h/10(6) cells, respectively; p < 0.01). This conversion in whole PMNs was not increased in vitro by LPS, IFN-gamma, IL-1 beta, or TNF-alpha nor decreased by W13, a calmodulin inhibitor. Furthermore, PMNs isolated from four volunteers before and after challenge with i.v. LPS (4 ng/kg) showed no increase in L-arginine to L-citrulline conversion. Nitrite and nitrate release from human PMNs was 35-fold lower than for HAECs and was not inhibited by L-NAA. These data suggest that human PMNs do not express NOS activity.

Amino Acid Oxidoreductases↗
PubMed1994

Nitric oxide regulates endotoxin-induced TNF-alpha production by human neutrophils.

We studied the effect of nitric oxide on LPS-induced TNF-alpha production by human neutrophils. Human neutrophils exposed to LPS and IFN-gamma did not show measurable increases in intracellular cyclic GMP (cGMP). However, cGMP increased upto 30-fold (p < 0.01) in neutrophils incubated with both sodium nitroprusside (SNP), an exogenous source of nitric oxide, and N-acetylcysteine (NAC), which increases the bioavailability of nitric oxide; this increase indicates that neutrophils contain a nitric oxide-sensitive guanylate cyclase. SNP, with or without NAC, did not increase TNF-alpha production in human neutrophils cultured in medium alone. However, LPS-dependent TNF-alpha production was increased by exposure to SNP (p < 0.05); this effect was further increased by the addition of NAC (p < 0.02). IFN-gamma greatly increased LPS-mediated TNF-alpha production by human neutrophils (p < 0.01), and SNP plus NAC was found to further augment this production (p < 0.01). The up-regulation of TNF-alpha production by nitric oxide was not associated with increased amounts of LPS-induced TNF-alpha mRNA, and was not reproduced by exposing neutrophils to cGMP analogues. These data suggest that nitric oxide released by endothelial and vascular smooth muscle cells may exert a paracrine effect on human neutrophils and augment the inflammatory response in sepsis by increasing the production of cytokines. Although the mechanism of this effect remains unknown, it does not seem to be dependent on cGMP or increased levels of TNF-alpha mRNA.

Acetylcysteine↗
PubMed1994

Isomorphous binding of mercury-substituted thiosaccharides to pertussis toxin crystals yields crystallographic phases.

An isomorphous derivative of pertussis toxin crystals was prepared using a 2-alpha-mercuric analog of N-acetyl neuraminic acid in a method analogous to the use of inhibitors labelled with heavy atoms to solve crystal structures of enzymes. This derivative exploits the specific binding between pertussis toxin and terminal sialic acid residues on receptor glycoproteins. Difference Patterson maps yielded heavy-atom sites which refined with good statistics, indicating that the protein probably does not undergo a conformational change on receptor binding. Mercuric analogs of other monosaccharides should be easily obtainable using the same synthetic strategy, suggesting a general method for derivatizing crystals of carbohydrate-binding proteins.

Journal Article↗
PubMed1994

Cloning and biological function of laminin in Hydra vulgaris.

The Cnidarian, hydra, lends itself to studies related to the role of extracellular matrix (ECM) components in development because of its high regenerative capacity and its simple structure, which is organized as an epithelial bilayer with an intervening ECM termed the mesoglea. Previous immunocytochemical and biochemical studies have established that hydra mesoglea contains many of the major matrix components (e.g., fibronectin, laminin, type IV collagen, and heparan sulfate proteoglycan) associated with the ECM of vertebrate and more complex invertebrate species. Additional studies have also established that ECM components have a critical role in hydra development as monitored during head regeneration and morphogenesis of hydra cell aggregates. In the present study a monoclonal antibody (mAb52) raised to isolated hydra mesoglea was used as a probe in additional functional studies and to screen a cDNA expression library made from poly(A)+ RNA isolated from Hydra vulgaris. Immunofluorescent analysis indicated that mAb52 was localized along the entire longitudinal axis of adult polyps in what is termed the subepithelial zones of hydra mesoglea. Cytochemical studies found these subepithelial zones to be rich in anionic sites. Previous studies have shown that mAb52 blocks hydra cell aggregate development and experiments in the current study have shown that mAb52 also blocks in vivo interstitial cell (I-cell) migration in hydra grafts. Sequence analysis of cDNA clones isolated using mAb52 indicated that the protein encoded by these clones had structural homology with mammalian and Drosophila laminin B1 chain and hybridized to a single 6.75-kb band on Northern blots of total hydra RNA. One interesting difference in hydra laminin B1 was the presence of a FTGTQ amino acid sequence in place of the vertebrate YIGSR cell binding domain. Under nonreducing conditions, polyclonal antibodies against FTGTQ bound to the same > 200-kDa band on Western blots of mesoglea as mAb52 and also immunolocalized to the subepithelial zones. Under reducing conditions, anti-FTGTQ antibodies bound to a single band with a mass of approximately 200 kDa. In addition, FTGTQ peptide inhibited adhesion of dissociated hydra cells to mesoglea and anti-FTGTQ antibodies inhibited hydra cell binding to substrates coated with mesoglea or FTGTQ peptide. Anti-FTGTQ antibodies also inhibited in vivo I-cell migration in hydra grafts. Given the early divergence of Cnidarians during evolution, these studies indicate the highly conserved nature of laminin and provide additional information regarding the critical role of ECM components during hydra development.

Amino Acid Sequence↗
PubMed1994

Protein synthesis is not required for the inhibitory effect of selenite on cell colony formation and RNA synthesis.

Selenite has been shown to undergo intracellular metabolism that results in its conversion to other low molecular weight Se-containing species and also to its incorporation into a selenocysteine residue in selenoprotein. In order to investigate whether the incorporation into protein is required for the cytotoxic effects of selenite, we have examined whether inhibition of protein synthesis prevents the inhibitory effect of selenite on the ability of cells to form colonies or to synthesize RNA. We have found that treatment of HeLa cells with cycloheximide inhibited protein synthesis by > 90% but had no effect on the inhibitory effect of selenite on cell colony formation or RNA synthesis. Since protein synthesis is not necessary for these cytotoxic effects of selenite they are unlikely to result from an increase in the synthesis of selenoproteins.

Clone Cells↗
PubMed1994

Mass spectrometric detection of preproenkephalin A-derived peptides in bovine pituitary.

A new analytical system, including chromatography separation, enzyme digestion, radioimmunoassay detection, and mass spectrometry characterization, has been designed to detect native preproenkephalin A-derived peptides in the bovine pituitary. The direct evidence of these intact peptides was provided by the mass spectrometric detection of the (M+H)+ ions of ME-Lys at m/z 702 and of ME-Arg at m/z 730, following trypsin digestion and the detection of ME-like immunoreactivity in HPLC fractions.

Amino Acid Sequence↗
PubMed1994

Localization of two gonadotropin receptors in the salmon gonad by in vitro ligand autoradiography.

Receptors for two salmon gonadotropins, GTH I and GTH II, were localized by use of in vitro ligand autoradiography of coho salmon gonads at various stages of sexual maturation. The results in both sexes revealed the presence of two types of GTH receptors: type I (GTH-RI), which interacts with both GTHs, and type II (GTH-RII), which interacts specifically with GTH II. GTH-RI was found at all stages of spermatogenesis examined and was localized on cells that were most likely Sertoli cells; however, it could not be determined whether GTH-RI was also localized on Leydig cells. In contrast, GTH-RII was found only in Leydig cells in the testis from a spermiating fish. In the vitellogenic ovary, GTH-RI was localized in the thecal layer and intensely on granulosa cells; in the preovulatory follicle, in contrast, GTH-RI was found in the thecal layer and in interstitial connective tissue, but not in the granulosa layer. Among all the stages of oogenesis examined, only granulosa cells of the preovulatory follicle exhibited GTH-RII. The appearance of GTH-RII coincides well with the increase in plasma levels of GTH II that occurs during final oocyte maturation and spermiation in coho salmon. The nature, distribution, and timing of appearance of these two receptors can explain, at least in part, the results of previous studies on steroidogenic activities of the two GTHs. The present study also suggests the functional homology of salmon GTH I and GTH II to mammalian FSH and LH, respectively, during gonadal development.

Animals↗