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Biomedical subjects

Y Shi

Publications and source records attributed to Y Shi.

At least 289 records · Page 16Linked to original sources
PubMed1999

Role of ERAB/L-3-hydroxyacyl-coenzyme A dehydrogenase type II activity in Abeta-induced cytotoxicity.

Endoplasmic reticulum-associated amyloid beta-peptide (Abeta)-binding protein (ERAB)/L-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer's disease (AD)-affected brain, binds Abeta, and contributes to Abeta-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a Km of approximately 68 microM and a Vmax of approximately 430 micromol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Abeta-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant beta-amyloid precursor protein (betaAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and betaAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2-C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17beta-estradiol. Addition of micromolar levels of synthetic Abeta(1-40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (Ki approximately 1.6 microM), as well as oxidation of 17beta-estradiol (Ki approximately 3.2 microM) and (-)-2-octanol (Ki approximately 2.6 microM). Because micromolar levels of Abeta were required to inhibit ERAB/HADH II activity, whereas Abeta binding to ERAB/HADH II occurred at much lower concentrations (Km approximately 40-70 nM), the latter more closely simulating Abeta levels within cells, Abeta perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and betaAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or betaAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and betaAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Abeta-rich environment.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1999

p53 overexpression in synovial tissue from patients with early and longstanding rheumatoid arthritis compared with patients with reactive arthritis and osteoarthritis.

OBJECTIVE: The p53 tumor suppressor gene is overexpressed in synovial tissue (ST) from patients with longstanding rheumatoid arthritis (RA), and may contain somatic mutations. The aim of this study was to determine p53 expression in ST from RA patients in different stages of the disease, compared with disease controls. METHODS: ST biopsy specimens were obtained from the knee joints of 31 RA patients in varying disease phases, 8 patients with reactive arthritis (ReA), 10 patients with inflammatory osteoarthritis (OA), and 6 control patients (4 with meniscus pathology, 2 with vascular insufficiency). ST was also obtained from the clinically uninvolved knee joints of 9 RA patients. Expression of p53 was determined by immunohistology with DO1 monoclonal antibody (mAb) in all patients and by Western blot analysis with DO7 mAb in a subgroup of the patients. RESULTS: The p53 protein was detected by immunohistology in 10 of the 13 patients with early RA (duration <6 months) and in 12 of the 14 patients with longstanding RA (duration >5 years). The p53 protein was also demonstrated in clinically uninvolved knee joints. Western blots revealed immunoreactive p53 in ST extracts from all RA patients. Expression of p53 was about twice as high in ST from patients with longstanding RA as in early RA samples, but the difference did not reach statistical significance. Small amounts of p53 were also detected in ST from ReA and OA patients, although the expression in RA synovium was significantly higher. Immunohistologic analysis of normal ST gave negative results for p53. CONCLUSION: This study shows that p53 overexpression is specific for RA, compared with OA and ReA. This phenomenon is probably secondary to increased production of wild-type p53 protein in response to DNA damage and secondary to somatic mutations caused by the genotoxic local environment in inflamed ST. Of interest, p53 overexpression can also be found in the earliest stages of RA and in clinically uninvolved joints.

Aged↗
PubMed1999

Dominant-negative p53 mutations in rheumatoid arthritis.

OBJECTIVE: Studies were performed to determine if p53 mutations identified in rheumatoid arthritis (RA) synovial tissue are dominant negative. METHODS: Site-directed mutagenesis was used to produce 2 RA-derived mutants: asparagine-->serine at codon 239 (N239S) and arginine-->stop at codon 213 R213*). HS68 dermal fibroblasts were transfected with either empty vector, wild-type p53 cDNA (wt), or the N239S or R213* mutant p53 cDNA clones. Interleukin-6 (IL-6) and bax gene expression were determined by Northern blot analysis. Bax transcription was determined using a bax promoter/reporter gene construct (bax-luc). RESULTS: Transfection of HS68 cells with wt increased bax mRNA levels. This process was blocked by cotransfection with either mutant. The mutant p53 genes also increased IL-6 gene expression. Low levels of bax promoter activity were detected in HS68 cells co-transfected with bax-luc and empty vector, N239S, or R213*, indicating that the RA mutants lacked transcriptional activity. Transfection with wt and bax-luc led to a 10-fold increase in luciferase expression. When the wt gene was cotransfected with either of the mutants, there was a dose-dependent inhibition of bax promoter activity. CONCLUSION: These data indicate that at least 2 of the p53 mutants identified in RA joint samples are dominant negative and suppress endogenous wild-type p53 function.

Arthritis, Rheumatoid↗
PubMed1999

Determination of selenium in Canadian food items by cyclic instrumental neutron activation analysis.

Instrumental neutron activation analysis (INAA) and pseudocyclic INAA (PCINAA) have been used to determine the selenium content of a variety of Canadian food items. Use of the 162-keV gamma ray of short-lived 77mSe in INAA allowed relatively simple and rapid determinations and was suitable for many of the foods. PCINAA was found to give lower detection limits and was used for the low-selenium food samples. Both internal and external quality assessments were used to evaluate and assure the accuracy and precision of the methods developed.

Food Analysis↗
PubMed1999

An autoinhibitory peptide from the erythrocyte Ca-ATPase aggregates and inhibits both muscle Ca-ATPase isoforms.

We have studied the effects of C28R2, a basic peptide derived from the autoinhibitory domain of the plasma membrane Ca-ATPase, on enzyme activity, oligomeric state, and E1-E2 conformational equilibrium of the Ca-ATPase from skeletal and cardiac sarcoplasmic reticulum (SR). Time-resolved phosphorescence anisotropy (TPA) was used to determine changes in the distribution of Ca-ATPase among its different oligomeric species in SR. C28R2, at a concentration of 1-10 microM, inhibits the Ca-ATPase activity of both skeletal and cardiac SR (CSR). In skeletal SR, this inhibition by C28R2 is much greater at low (0.15 microM) than at high (10 microM) Ca2+, whereas in CSR the inhibition is the same at low and high Ca2+. The effects of the peptide on the rotational mobility of the Ca-ATPase correlated well with function, indicating that C28R2-induced protein aggregation and Ca-ATPase inhibition are much more Ca-dependent in skeletal than in CSR. In CSR at low Ca2+, phospholamban (PLB) antibody (functionally equivalent to PLB phosphorylation) increased the inhibitory effect of C28R2 slightly. Fluorescence of fluorescein 5-isothiocyanate-labeled SR suggests that C28R2 stabilizes the E1 conformation of the Ca-ATPase in skeletal SR, whereas in CSR it stabilizes E2. After the addition of PLB antibody, C28R2 still stabilizes the E2 conformational state of CSR. Therefore, we conclude that C28R2 affects Ca-ATPase activity, conformation, and self-association differently in cardiac and skeletal SR and that PLB is probably not responsible for the differences.

Animals↗
PubMed1999

FHIT gene abnormalities in both benign and malignant thyroid tumours.

FHIT, a candidate tumour suppressor gene, has recently been identified at chromosomal region 3p14.2, and deletions of the gene have been reported in many types of human cancers. Loss of heterozygosity (LOH) at this region has also been found frequently in follicular thyroid carcinoma (FTC). To investigate the potential role of FHIT in thyroid tumorigenesis, we examined 57 thyroid tumour specimens (eight benign adenomas, 40 papillary, four follicular and five anaplastic carcinomas), and two thyroid carcinoma cell lines (NPA, SW579) for genetic alterations by using reverse transcription-polymerase chain reaction (RT-PCR), PCR product sequencing, single-strand conformation polymorphism (SSCP) and Southern blot analysis. Two cervical carcinoma cell lines (C-33A, HeLa) were included as positive controls. We detected truncated FHIT transcripts in three of eight (38%) benign adenomas, nine of 40 (23%) papillary, and two of five (40%) anaplastic carcinomas, and in three cell lines (SW579, C-33A, HeLa). Most of the truncated transcripts lacked exons 4 or 5 to 7 or 8 of the gene and were presumably non-functional as the translation start site is located in exon 5. SSCP analysis of the coding exons failed to detect any point mutations among the samples without abnormal FHIT transcripts. Southern blot analysis demonstrated either loss or reduced intensity of major Bam HI restriction fragments in the three cell lines found to have abnormal FHIT transcripts, indicating, respectively, either intragenic homozygous or heterozygous deletions of the FHIT gene. Intragenic homozygous deletions were also found in two papillary thyroid carcinoma specimens: one was missing a 13 kb Bam HI fragment which contains exon 4, the other had deletions of 15.5, 13 and 4.2 kb fragments which contain exons 2 and 9, 4, and 5, respectively. The absence of a defective FHIT gene in FTC indicates that an additional tumour suppressor gene may reside in this region and be involved in the development of FTC. Given that defective FHIT genes were found in both benign and malignant thyroid tumours, the inactivation of this putative tumour suppressor gene is likely to be an early event in the pathogenesis of some forms of thyroid neoplasms.

Acid Anhydride Hydrolases↗
PubMed1999

Transforming growth factor-alpha antisense vectors can inhibit glioma cell growth.

The effects of transforming growth factor-alpha (TGF-alpha) on cell growth were studied in human glioma U251 cells transfected with antisense TGF-alpha vectors (pcDNAI.neo). Several antisense clones showed a marked decrease in growth rate in serum-free medium but not in medium containing 10% FBS, compared with those of parental cells and clones from sense or vector transfectants. Antisense clones also produced fewer and smaller colonies in anchorage-independent growth assays. Moreover, there was a reduction in TGF-alpha expression in these antisense clones at both the protein and mRNA levels, as determined by enzyme linked immuno-sorbent assay and reverse transcriptase polymerase chain reaction analysis. A U251 clone transfected by TGF-alpha antisense in a different vector (pMT/Ep) also showed a marked suppression in cell growth and TGF-alpha mRNA level. Finally, transfected clones with either vector system, showed decreased tumorigenicity in nude mice. In summary, a strong correlation between the inhibition of glioma cell growth and TGF-alpha expression was obtained from two different plasmid vectors, indicating that the expression of TGF-alpha could be specifically and effectively down-regulated by TGF-alpha antisense vector, which in turn led to growth inhibition. These studies suggests that TGF-alpha plays an essential role in controlling human glioma cell proliferation and may serve as a potential target for treatment of malignant glioma.

Animals↗
PubMed1999

Tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA is elevated in advanced stages of thyroid carcinoma.

Tumour cell invasion and metastasis is a multistep process that involves the degradation of extracellular matrix proteins by matrix metalloproteinases (MMPs). Tissue inhibitors of metalloproteinases (TIMPs) act as negative regulators of MMPs and thus prevent tumour cell invasion and metastasis by preserving extracellular matrix (ECM) integrity. In the present study we examined the expression of one member of TIMPs, TIMP-1, in 39 thyroid tumour specimens and two thyroid carcinoma cell lines (NPA and SW579). We also investigated the effect of high TIMP-1 expression on the invasive potential of NPA cells. Northern blot analysis showed that TIMP-1 mRNA levels correlated directly with tumour aggressiveness: the highest number of TIMP-1 transcripts was found in stages III and IV vs benign goitre (P < 0.0001). However, TIMP-1 expression was not increased in NPA and SW579 cells, both of which are derived from poorly differentiated thyroid tumours. Immunohistochemical study showed strong TIMP-1 staining in the stroma cells of advanced stages of carcinomas. Overexpression of TIMP-1 by gene transfer resulted in a significant suppression of the malignant phenotype of NPA cells as judged by an in vitro tumour invasion assay. These results suggest that high levels of TIMP-1 transcripts in advanced stages of thyroid carcinoma likely come from stroma rather than thyroid cancer cells, and TIMP-1 may function as a thyroid tumour invasion/metastasis suppressor.

Adult↗
PubMed1999

The yins and yangs of ceramide.

Since their discovery over 100 years ago, sphingolipids have caught the eyes and the imagination of scientists. Modern science has made many new insights on the cell biology and day-to-day functions of many integral sphingolipids, especially those of ceramide. Ceramide is recognized as a vital second messenger in the signal transduction process mediated by receptors of many cytokines and growth factors. A great part of our current understanding of ceramide has been achieved from apoptosis-related studies, however recent data in the fields of immunology, endocrinology and neurobiology, also suggest a fundamental involvement of ceramide in the onset of diseases. Therefore, understanding the biology of ceramide could be a key to unraveling many biological mechanisms and provide information for the treatment of some common diseases.

Animals↗
PubMed1999

Intra-articular IL-4 gene therapy in arthritis: anti-inflammatory effect and enhanced th2activity.

Gene therapy has been explored as a potential method for treating chronic inflammatory diseases such as rheumatoid arthritis. To determine the efficacy of intra-articular IL-4 gene therapy in an animal model of arthritis using a retroviral vector, a retrovirus encoding rat IL-4 (DA-IL-4) was engineered, purified and concentrated to high titer (>/=109 CFU/ml). Infectivity and expression levels were demonstrated in vitro using cultured fibroblast-like synoviocytes. Efficacy was evaluated in the rat adjuvant arthritis model. DA-IL-4 or DA-beta-gal retrovirus was injected into the intra-articular joint space of the right ankle on day 12 after immunization. Three days after joint injection, the injected paw contained increased levels of IL-4 compared with control or with the contralateral uninjected paw, demonstrating successful transgene expression. Surprisingly, 8 days after treatment IL-4 levels continued to increase in the injected and contralateral paw compared with DA-beta-gal-treated animals. Serum IL-4 levels were also elevated in DA-IL-4-treated rats. RT-PCR studies demonstrated that the transgene was expressed in the injected ankle but not in the contralateral joint. IL-4 gene therapy resulted in a significant reduction in paw swelling and decreased radiographic evidence of bone destruction. This is the first demonstration of successful intra-articular retroviral gene treatment using a therapeutic gene. In addition to its anti-inflammatory effect, this study supports the potential application of intra-articular gene therapy as a method for enhancing systemic Th2 function.

Animals↗
PubMed1999

Furazolidone-containing short-term triple therapies are effective in the treatment of Helicobacter pylori infection.

BACKGROUND: A furazolidone-containing therapeutic regimen for Helicobacter pylori infection has attracted special interest in the face of a rising world-wide metronidazole resistant H. pylori, and the expense of currently used antimicrobial regimens. AIM: To evaluate the efficacy of furazolidone-containing regimens in eradicating H. pylori. METHODS: One-hundred and forty H. pylori positive patients with endoscopically confirmed duodenal ulcer or functional dyspepsia received one of four different regimens to eradicate H. pylori. In the first trial, the patients were randomly assigned to receive a 1-week course of furazolidone 100 mg b.d. and clarithromycin 250 mg b.d., with either tripotassium dicitrato bismuthate (TDB) 240 mg b.d. (FCB group) or lansoprazole 30 mg daily (FCL group). In the second trial, the patients were randomly assigned to receive a 1-week course of clarithromycin 250 mg b.d. and omeprazole 20 mg daily, with either furazolidone 100 mg b.d. (FCO group) or metronidazole 400 mg b.d. (MCO group). Endoscopy was repeated 4 weeks following completion of therapy with re-assessment of H. pylori status on gastric biopsies by histology and culture. RESULTS: Four patients (1 in FCB, 1 in FCO and 2 in MCO groups) dropped out because they refused a follow-up endoscopy. Eradication rates of H. pylori on an intention-to-treat basis in the FCB, FCL, FCO and MCO groups were 91% (32/35, 95% CI: 82-99%), 91% (32/35, CI: 82-99%), 86% (30/35, CI: 74-97%) and 74% (26/35, CI: 60-89%) (all P > 0.05), respectively. Mild side-effects occurred in 15% of the 140 patients. In MCO group, the eradication rate in the patients infected with metronidazole-sensitive isolates of H. pylori was 86%, but dropped to 67% in those with metronidazole-resistance strains (P = 0.198). CONCLUSION: One-week regimens containing furazolidone and clarithromycin in combination with TDB or a proton pump inhibitor fulfil the criteria for successful H. pylori therapy.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1999

Factors influencing the levels of fatty acid synthase complex activity in fowl.

There is a notable discrepancy between the FAS (fatty acid synthase) activity of four types of fowl (egg chicken, meat chicken, egg duck, and meat duck) with distinctively different body fat levels. There is a 14.8 fold difference per unit body weight between the maximum and minimum FAS activities. The three major factors affecting this discrepancy are liver weight per unit body weight, which is 2.3 times greater in meat ducks than in egg chickens, the amount of FAS protein per gram of liver, which is 1.85 times greater in meat ducks than in egg chickens, and the FAS specific activity in meat ducks, which is 3.5 times greater in meat ducks than in egg chickens. Within the same species of egg chickens, the abdomen fat per kg of body weight at 470 days after egg production is 66 times greater than 90 days before egg production and the liver FAS activity is increased 9.6 fold. The 9.6 fold FAS activity increase resulted from an increase in the specific activity, since the liver weight per kilogram of body weight remained constant at approx. 20 grams and the FAS weight per gram of liver also remained constant at approx. 4.5 mg. This shows that the control of the basic FAS activity level which is closely related to the level of body fat does not mainly arise from genetic control. For the same kind of fowl, the control of the basic FAS activity level occurs after gene expression. It is suggested that control may be imposed in the folding phase when new peptides give rise to functional proteins.

Adipose Tissue↗
PubMed1999

Measles virus infection in rhesus macaques: altered immune responses and comparison of the virulence of six different virus strains.

Measles remains a major cause of childhood mortality, with questions about virus virulence and pathogenesis still requiring answers. Rhesus macaques were infected with 5 different culture-adapted strains of measles virus, including 2 from patients with progressive vaccine-induced disease, and a sixth nonculture-adapted strain, Bilthoven. All caused infection detectable by reverse transcriptase-polymerase chain reaction and induction of antibody. Chicago-1 and Bilthoven induced viremias detectable by leukocyte cocultivation. Bilthoven induced Koplik's spots, conjunctivitis, and rash. Lymphopenia and depressed interleukin (IL)-2 production were followed by monocytosis and eosinophilia. All monkeys, including 41 involved in a primate facility outbreak, showed suppressed responses to phytohemagglutinin. As the rash resolved production of IL-2, IL-1beta, tumor necrosis factor-alpha, IL-6, and IL-5 mRNA increased. Monkeys are useful for studies of measles immunopathogenesis, but virus strains must be carefully chosen. Increased virulence of vaccine strains isolated from immunocompromised infants with fatal infections was not evident.

Animals↗
PubMed1999

Effects of salicylic acid derivatives on red blood cell membranes.

Salicylamide, sodium salicylate and acetylsalicylic acid are salicylic acid derivates. They differ in their substitution on the benzene ring and may have different effects on membranes. Red blood cells were used as a prototypical cellular system regarding drug mediated plasma bilayer effects. Established photometric methods sensing tiny changes of red blood cell morphology at rest (red blood cell shape) and at very low shear forces (red blood cell stiffness, red blood cell relaxation time) were applied. The derivative induced effects were detected in a time- and dose-dependent manner. Salicylamide induced a most pronounced echinocytic shape at 5 mM. The shape effect was smaller above as well as below 5 mM. Sodium salicylate induced echinocytes with increasing concentrations showing a saturation above 10 mM. In contrast, the shape was not affected by acetylsalicylic acid. All shape changes occurred within 2 min, and were reversible. The above tendencies were in parallel to a slight red blood cell stiffening. The relaxation time continuously increased with increasing concentrations in both salicylamide and sodium salicylate, with salicylamide always acting stronger. Acetylsalicylic acid again showed no effect. We hypothesize that the observed effects of sodium salicylate and salicylamide are due to their phenolic character mediating a molecular hydrophobicity. According to the bilayer couple hypothesis this would lead to an insertion into the red blood cells outer plasma bilayer leaflet. The extension induced here would cause a positive membrane bending leading to echinocytic shapes and the observed loss of red blood cell fluidity. In contrast, the hydrophilic aspirin would penetrate and thus not affect the red blood cell plasma membrane.

Aspirin↗
PubMed1999

Molecular analysis of microbial community structures in pristine and contaminated aquifers: field and laboratory microcosm experiments.

This study used phylogenetic probes in hybridization analysis to (i) determine in situ microbial community structures in regions of a shallow sand aquifer that were oxygen depleted and fuel contaminated (FC) or aerobic and noncontaminated (NC) and (ii) examine alterations in microbial community structures resulting from exposure to toluene and/or electron acceptor supplementation (nitrate). The latter objective was addressed by using the NC and FC aquifer materials for anaerobic microcosm studies in which phylogenetic probe analysis was complemented by microbial activity assays. Domain probe analysis of the aquifer samples showed that the communities were predominantly Bacteria; Eucarya and Archaea were not detectable. At the phylum and subclass levels, the FC and NC aquifer material had similar relative abundance distributions of 43 to 65% beta- and gamma-Proteobacteria (B+G), 31 to 35% alpha-Proteobacteria (ALF), 15 to 18% sulfate-reducing bacteria, and 5 to 10% high G+C gram positive bacteria. Compared to that of the NC region, the community structure of the FC material differed mainly in an increased abundance of B+G relative to that of ALF. The microcosm communities were like those of the field samples in that they were predominantly Bacteria (83 to 101%) and lacked detectable Archaea but differed in that a small fraction (2 to 8%) of Eucarya was detected regardless of the treatment applied. The latter result was hypothesized to reflect enrichment of anaerobic protozoa. Addition of nitrate and/or toluene stimulated microbial activity in the microcosms, but only supplementation of toluene alone significantly altered community structures. For the NC material, the dominant subclass shifted from B+G to ALF, while in the FC microcosms 55 to 65% of the Bacteria community was no longer identifiable by the phylum or subclass probes used. The latter result suggested that toluene exposure fostered the proliferation of phylotype(s) that were otherwise minor constituents of the FC aquifer community. These studies demonstrated that alterations in aquifer microbial communities resulting from specific anthropogenic perturbances can be inferred from microcosm studies integrating chemical and phylogenetic probe analysis and in the case of hydrocarbon contamination may facilitate the identification of organisms important for in situ biodegradation processes. Further work integrating and coordinating microcosm and field experiments is needed to explore how differences in scale, substrate complexity, and other hydrogeological conditions may affect patterns observed in these systems.

Bacteria, Anaerobic↗
PubMed1999

Targeted disruption of mouse Yin Yang 1 transcription factor results in peri-implantation lethality.

Yin Yang 1 (YY1) is a zinc finger-containing transcription factor and a target of viral oncoproteins. To determine the biological role of YY1 in mammalian development, we generated mice deficient for YY1 by gene targeting. Homozygosity for the mutated YY1 allele results in embryonic lethality in the mouse. YY1 mutants undergo implantation and induce uterine decidualization but rapidly degenerate around the time of implantation. A subset of YY1 heterozygote embryos are developmentally retarded and exhibit neurulation defects, suggesting that YY1 may have additional roles during later stages of mouse embryogenesis. Our studies demonstrate an essential function for YY1 in the development of the mouse embryo.

Animals↗