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Biomedical subjects

Y Shi

Publications and source records attributed to Y Shi.

At least 559 records · Page 31Linked to original sources
PubMed1994

Expression of recombinant human casein kinase II and recombinant heat shock protein 90 in Escherichia coli and characterization of their interactions.

To assess the interaction of human casein kinase II (CKII) with the heat shock protein 90 (HSP90) class of chaperone proteins, human CKII alpha and beta subunits and beta S2A mutant were expressed and purified separately or from a tandem coexpression construct in Escherichia coli. Recombinant human HSP90 beta and recombinant yeast HSP90 as His6 constructs were also expressed in and purified from E. coli. The rhCKII S2A mutant removed the regulatory beta subunit autophosphorylation site but had no effect on catalytic efficiency with peptide or protein substrates. As a CKII substrate, recombinant hHSP90 beta displayed a Km of 9.8 microM and a kcat of 4.1 min-1 and was phosphorylated to 1.5 mol/mol, whereas ryHSP90, lacking the known serine CKII sites of hHSP90, was phosphorylated at a 19-fold lower kcat/Km ratio to levels of 0.8 mol/mol. The endoplasmic reticulum HSP90 family member Grp94 was phosphorylated to 1.4 mol/mol but, in contrast, HSC70 and FKBP25 chaperones were phosphorylated to < 0.01 mol/mol. Neither phospho nor dephospho forms of hHSP90 showed significant activation of CKII toward the peptide substrate RRREEETEEE in contrast to a previous report that activation was observed at high molar ratios of chaperone to kinase.

Amino Acid Sequence↗
PubMed1994

Role of DNA fragmentation in T cell activation-induced apoptosis in vitro and in vivo.

Apoptotic cell death, characterized by DNA fragmentation and morphologic changes, has previously been shown to occur in immature thymocytes and some T cell hybridomas after activation. Like some other forms of apoptosis, DNA fragmentation during activation-induced cell death precedes the morphologic events. For apoptosis to proceed, activation of the cells must persist at least to the time of DNA fragmentation, before which the cells can remain viable if the activation signal is removed. Aurintricarboxylic acid (ATA) blocks activation-induced apoptotic cell death in a T cell hybridoma, and kinetic studies show that this inhibition occurs at or near the time of DNA fragmentation in the cells. Taken together with the ability of ATA to inhibit DNA fragmentation in isolated nuclei exposed to Ca2+ and Mg2+, these data strongly suggest that ATA prevents apoptosis via its ability to inhibit endogenous endonuclease activity, and, conversely, that this activity is required for this form of cell death. In vivo, ATA inhibits thymocyte depletion and DNA fragmentation induced by anti-CD3 Ab. Further, specific loss of V beta 8+ thymocytes after administration of staphylococcal enterotoxin B is blocked by administration of ATA. These observations support an essential role for DNA fragmentation as an irreversible step in activation-induced apoptosis in T cell hybridomas and during T cell development. This is contrasted with heat shock-induced cell death, in which inhibition of DNA fragmentation does not prevent loss of cell viability.

Animals↗
PubMed1994

Low rate of ret proto-oncogene activation (PTC/retTPC) in papillary thyroid carcinomas from Saudi Arabia.

BACKGROUND: The ret proto-oncogene activation (PTC/retTPC oncogene) in thyroid papillary carcinoma has been reported in different populations with different frequencies. Thyroid papillary carcinoma appears to behave more aggressively in the Persian Gulf region than elsewhere. In the current study, the frequency of PTC/retTPC oncogene in thyroid tumors from Saudi Arabia was investigated. METHODS: PTC/retTPC oncogene transcripts were analyzed by the polymerase chain reaction amplification of cDNA synthesized by treatment of total RNA with reverse transcriptase. Seven multinodular goiters, 1 follicular adenoma, 4 follicular carcinomas, 40 papillary carcinomas, and 5 anaplastic carcinomas were studied. RESULTS: Only one papillary carcinoma specimen was found to have PTC/retTPC oncogene transcripts. The breakpoint of the rearranged PTC/retTPC oncogene is identical to that previously described. The PTC/retTPC-positive sample was also examined for p53 tumor suppressor gene mutations in exons 5-8. One transitional point mutation was detected at codon 161 (GCC to ACC), changing Ala to Thr. CONCLUSIONS: This study questions the relevance of PTC/retTPC oncogene in the carcinogenesis of thyroid papillary carcinomas in the Saudi population. Genetic background among races may contribute to the different frequencies of PTC/retTPC oncogene in thyroid papillary carcinoma.

Carcinoma, Papillary↗
PubMed1994

Immune responses, and autoimmune outcome, during virus infection of the central nervous system.

A combined role of a virus infection of the central nervous system (CNS) and an autoimmune response to myelin basic protein (MBP), an autoantigen of the CNS, is suggested in the pathogenesis of multiple sclerosis (MS). SJL mice are highly susceptible while B6 mice are less susceptible to the induction of experimental autoimmune encephalomyelitis (EAE), the autoimmune model of MS. Peripheral inoculation of Semliki forest virus (SFV) into SJL and B6 mice resulted in: (1) Higher viral titers, more severe clinical disease, and hence a stronger nonspecific and SFV-specific lymphoproliferation, and production of IFN-gamma and TNF/LT was observed by splenocytes (SPL) of B6 than by those of SJL mice, on Day 7 postinfection. (2) Following viral clearance, however, proliferation to SFV, and to MBP, and the production of IFN-gamma and TNF/LT by SPL of SFV-infected SJL mice were significantly higher, while the production of TGF-beta was significantly lower than by those of B6 mice. In conclusion, the immune responses to SFV, and to MBP, which were triggered by SFV infection were significantly higher and more prolonged in the SPL of SJL mice, the EAE-susceptible mice, than by those of B6 mice after the infection was cleared.

Alphavirus Infections↗
PubMed1994

Value of antibodies to GAD65 combined with islet cell cytoplasmic antibodies for predicting IDDM in a childhood population.

The value of a test for islet cell cytoplasmic antibodies together with a test for GAD65 antibodies to predict the subsequent development of diabetes over a period of 11.5 years was assessed in an open childhood population comprising 2,805 individuals. A single serum sample was obtained from each individual between 1975 and 1977 and screened for islet cell cytoplasmic antibodies for which eight individuals were positive (0.29%). During the average follow-up period of 11.5 years, four of eight islet cell antibody positive and three islet cell antibody negative individuals developed clinical diabetes. Sera from all individuals, who were islet cell antibody positive and/or developed diabetes (total of 11) and from 100 randomly selected control subjects were analysed for GAD65 antibodies. Six of eight islet cell antibody positive individuals were GAD65 antibody positive including all four who subsequently developed IDDM. Furthermore, one of the three islet cell antibody negative individuals who developed IDDM was GAD65 antibody positive both in 1976 and in 1989. Thus, a positive test for GAD65 antibodies alone correctly predicted diabetes in five of seven children, who developed the disease. Only one of the children, who developed diabetes was positive for insulin autoantibodies and this individual was also positive for islet cell cytoplasmic antibodies and GAD65 antibodies. One of the 100 control subjects was positive for GAD65 antibodies (1%). The results suggest that a single GAD65 antibody test may have a higher sensitivity for predicting IDDM than a test for islet cell cytoplasmic antibodies, but that a combined positive test for both antibodies increases the specificity for predicting IDDM over a period of 11.5 years.

Adolescent↗
PubMed1994

DNA fragmentation is an early event in cytokine-induced islet beta-cell destruction.

The cytokines, interleukin 1, tumour necrosis factor, and interferon gamma are cytotoxic to islet beta cells, however, their mechanisms of beta-cell killing are not fully characterized. Since DNA damage is a mechanism of cytokine-induced cell death in some cell types, we sought evidence for cytotoxic effects of cytokines at a nuclear level in islet beta cells by measuring DNA fragmentation in rat islets and islet beta-cell lines. The individual cytokines, interleukin 1 (10 U/ml), tumour recrosis factor (10(3) U/ml) and interferon gamma (10(3) U/ml) inhibited insulin release from rat islets, but did not cause DNA fragmentation or destroy islet cells; by contrast, combination of the three cytokines induced DNA fragmentation and islet-cell death. Cytokine-induced DNA fragmentation preceded cell lysis in islet beta-cell lines (RINm5F, rat insulinoma cells; and NIT-1, NOD/Lt mouse transgenic beta cells), whereas in non-islet cell lines (GH-3, rat pituitary; and PC-12, rat adrenal) the cytokines induced cell lysis and no or late DNA fragmentation. Nicotinamide prevented both DNA fragmentation and destruction of RINm5F islet cells by the cytokines. These findings identify DNA as an early target of cytokine action in islet beta cells, and implicate DNA fragmentation as a mechanism of cytokine-induced beta-cell destruction.

Animals↗
PubMed1994

Basic fibroblast growth factor identified in chronically stimulated cardiomyoplasties.

In the presence of myocardial ischemia, chronic electrical stimulation of a latissimus dorsi (LD) cardiomyoplasty enhances extramyocardial collateral blood flow. We postulated that basic fibroblast growth factor (bFGF) may mediate extramyocardial collateral formation. To test this hypothesis, LDs from goats with cardiomyoplasties were probed for the presence of bFGF by Western blot analysis and immunohistochemistry. Three groups were studied: static LD cardiomyoplasty (group 1); LD cardiomyoplasty stimulated at a 2-Hz frequency for 6 weeks (group 2); and LD cardiomyoplasty electrically stimulated and given human recombinant bFGF (group 3). There was no evidence of bFGF in the left LDs of group 1 by Western blot. Basic fibroblast growth factor-like immunoreactive evidence was found in the left LDs of group 2 goats by both Western blot and immunohistochemistry. In the right LDs of group 2, bFGF-like material was found by immunohistochemistry but not by Western blot, which suggests that the tissue concentrations were low (near the limits of detection). The left LDs of group 3 were positive for bFGF by Western blot and immunohistochemistry. Group 3 right LDs were positive for bFGF by immunohistochemistry. Immunohistochemical findings in group 2 indicate that bFGF is present in goat skeletal muscle. Western blot data from groups 1 and 2 suggest that bFGF may be increased in chronically stimulated cardiomyoplasties. From findings in group 3, we conclude that exogenous bFGF does not downregulate, and may upregulate, endogenous production. These results support the possibility that skeletal muscle bFGF is an important factor in extramyocardial collateral formation.

Animals↗
PubMed1994

Fibroblast growth factors mobilize peritoneal macrophage intracellular calcium.

Macrophages have been implicated in the propagation of inflammatory disease. The evidence linking macrophages to inflammation stems from their elicited responses to various extracellular ligands eventually culminating in the elaboration of a variety of inflammatory mediators. As part of an investigation of fibroblast growth factors role in promoting inflammation, we examined one aspect of transmembrane signal transduction, intracellular calcium mobilization following culture of murine peritoneal macrophages with acidic and basic fibroblast growth factor. Peritoneal macrophages displayed a rapid rise in cytosolic calcium from a basal level of 147.6 +/- 25.4 nM to 261.9 +/- 49.9 nM at 3.5 minutes following culture with acidic fibroblast growth. A similar rise in calcium was noted with basic fibroblast growth factor. Titration revealed the maximal effective dose of aFGF and bFGF with respect to calcium response to be 10 ng/ml. Using blockers of both voltage and non-voltage gated channels, the FGF induced rise in cytosolic calcium was specifically abolished. Similarly, using specific 5-lipoxygenase (A69412) or cyclooxygenase (Indomethacin) blockers, the aFGF induced rise in maximal calcium response was reduced by 41% and 96% respectively. On the basis of these data, we speculate on some possible roles that FGF may play in the inflammatory response.

Animals↗
PubMed1994

Composition of peritoneal macrophage membranes in autoimmune MRL lpr/lpr mice.

Adult MRL lpr/lpr mice display phenotypic features that are consistent with both rheumatoid arthritis and systemic lupus erythematosus. Previous studies have reported that peritoneal macrophages harvested from this model have an increased propensity for both spontaneous and elicited release of prostaglandins and leukotrienes relative to immunologically normal control mice. To investigate whether one aspect of the differences in secretory potential between autoimmune and normal mice was at the level of increased substrate availability, gas chromatographic analysis of peritoneal macrophage membranes from autoimmune MRL lpr/lpr, young lpr, wild type +/+, and immunologically normal mice was done. The results demonstrate enrichment of arachidonate in adult lpr macrophage membranes in all major phospholipid classes relative to young lpr, +/+ and immunologically normal C3H/HeN mice. Similarly, there was an increased mole % of arachidonic acid in lpr mice relative to controls. Elevated membrane arachidonate may contribute to the increased propensity of autoimmune strains to participate in the inflammatory process.

Animals↗
PubMed1994

Differential nm23 gene expression at the fetal-maternal interface.

The product of the nm23 gene has been proposed as a candidate tumour metastasis suppressor protein. A strong association has been observed between reduced expression of the nm23 gene and acquisition of metastatic behaviour in some tumour cells, including breast cancer and melanoma, but not in others, such as neuroblastoma and colon, cervical and thyroid cancers. During the early gestation period both human and murine trophoblast cells exhibit in vitro invasive properties similar to those of neoplastic cells. Such invasive properties, however, disappear in the late stage of gestation. In the present study, we examined the abundance of nm23 mRNA from various fetal-maternal interface tissues (uterus, decidua, placenta and embryo) during early (day 8), mid (day 14) and late (day 18) stages of gestation in CD1 mice, in order to determine whether nm23 plays any anti-invasive and/or biological roles during gestation. nm23 was found to be expressed in all the tissues during the early and mid stages of gestation. The expression levels were, however, variable among different tissues and development stages. In the early stage, nm23 mRNA levels were the highest and similar among tissues from the uterus, decidua, placenta and embryo. In the mid stage, the mRNA levels were reduced significantly in the uterus, decidua and placenta, but not in the embryo. In the late stage, nm23 mRNA was further reduced to the extent that it could not be seen in the decidua, was barely seen in the uterus and was weakly present in the placenta. However, the mRNA level of the embryo in the late stage was still high and similar to the early stage. We also examined nm23 expression in trophoblast cells from normal human term placenta and a highly metastatic human choriocarcinoma cell line, JAR. nm23 expression was significantly higher in JAR than in normal placenta, indicating that nm23 does not appear to have an anti-metastatic function in this cell line. Several cytokines--interleukin 2 (IL-2), tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)--and prostaglandin E2 (PGE2) known to modulate tumour growth and metastasis were examined to determine whether they regulate nm23 expression in JAR in vitro. The B16F10 melanoma cell line was used as control. No effect was found in the JAR cell line, whereas TNF-alpha, IFN-gamma and PGE2 down-regulated nm23 expression in the B16F10 cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65.

The smaller isoform of the GABA synthesizing enzyme glutamic acid decarboxylase, GAD65, is synthesized as a soluble protein that undergoes post-translational modification(s) in the NH2-terminal region to become anchored to the membrane of small synaptic-like microvesicles in pancreatic beta cells, and synaptic vesicles in GABA-ergic neurons. A soluble hydrophilic form, a soluble hydrophobic form, and a hydrophobic firmly membrane-anchored form have been detected in beta cells. A reversible and hydroxylamine sensitive palmitoylation has been shown to distinguish the firmly membrane-anchored form from the soluble yet hydrophobic form, suggesting that palmitoylation of cysteines in the NH2-terminal region is involved in membrane anchoring. In this study we use site-directed mutagenesis to identify the first two cysteines in the NH2-terminal region, Cys 30 and Cys 45, as the sites of palmitoylation of the GAD65 molecule. Mutation of Cys 30 and Cys 45 to Ala results in a loss of palmitoylation but does not significantly alter membrane association of GAD65 in COS-7 cells. Deletion of the first 23 amino acids at the NH2 terminus of the GAD65 30/45A mutant also does not affect the hydrophobicity and membrane anchoring of the GAD65 protein. However, deletion of an additional eight amino acids at the NH2 terminus results in a protein which is hydrophilic and cytosolic. The results suggest that amino acids 24-31 are required for hydrophobic modification and/or targeting of GAD65 to membrane compartments, whereas palmitoylation of Cys 30 and Cys 45 may rather serve to orient or fold the protein at synaptic vesicle membranes.

Amino Acid Sequence↗
PubMed1994

A direct sensitivity matrix approach for fast reconstruction in electrical impedance tomography.

In electrical impedance imaging, several proposed reconstruction algorithms have employed the concept of a sensitivity matrix, which can be used to relate the magnitude of a boundary voltage change of a 2D object to the change in conductivity inside the object that has given rise to it. The search for an appropriate inversion of the sensitivity matrix is the key to these algorithms. In this work, a method called the direct sensitivity matrix (DSM) approach for fast image reconstruction is proposed. Both theoretical and experimental results showing the efficiency of this proposed method are also presented.

Algorithms↗
PubMed1994

Clinical observation of treatment of hypertension with calcium.

The effect of oral calcium supplementation (1000 mg/day) on hypertension was studied in 57 borderline and mild-to-moderate hypertensive patients in a randomized, double-blind, placebo-controlled study for 14 weeks. Twenty-five patients from the above groups (11 from the calcium-treated group and 14 from the placebo group) were studied in a crossover fashion for 14 more weeks. The high calcium intake lowered systolic blood pressure by 17 mm Hg (P < .01), and diastolic blood pressure by 11 mm Hg (P < .01). Fifty percent of the calcium-treated patients showed a significant antihypertensive effect and were termed calcium responders. In the crossover study, serum sodium was lower after taking calcium than after placebo intake (P < .05). Pretreatment plasma free calcium content of the calcium-responsive patients was significantly lower (P < .05) than in the calcium nonresponsive patients, and was highly significantly increased (P < .01) after administering calcium. The result showed that oral calcium supplementation can lower blood pressure in a significant fraction of essential hypertensive subjects, and that the free calcium level in plasma may help identify calcium-responsive individuals. While the mechanism by which increased calcium intake lowers blood pressure in hypertension is still undetermined, these data support an underlying relationship between hypertension and calcium and possibly sodium metabolism.

Administration, Oral↗
PubMed1994

Chemical and molecular classification of Saccharomonospora strains.

The results of our experiments showed that the 5'-terminal sequences of 23S rRNAs can be used to distinguish different genera of actinomycetes, including the genera Streptomyces, Micromonospora, Amycolatopsis, and Saccharomonospora. There are small differences (< 1%) among the sequences of some strains belonging to the genera Streptomyces (two strains) and Saccharomonospora (seven strains). On the basis of the results of morphological and biochemical analyses, strain 9022 belongs in the genus Saccharomonospora; however, there are distinct differences in the cell wall compositions and the 5' termini of the 23S rRNA sequences of this strain and members of the genus Saccharomonospora. Hence, strain 9022 cannot be classified in the genus Saccharomonospora.

Actinomycetales↗