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Biomedical subjects

Y Zhao

Publications and source records attributed to Y Zhao.

At least 775 records · Page 43Linked to original sources
PubMed1996

Regulation of tumor necrosis factor-and Fas-mediated apoptotic cell death by a novel cDNA TR2L.

A novel cDNA, TR2L, isolated from murine NIH 3T3 fibroblasts, was found to modulate tumor necrosis factor (TNF)-mediated apoptosis in murine L929 fibrosarcoma cells. The full-length cDNA (853 bp) encodes a predicted coding region of 56 amino acids (6.3 kD), with 53.6% identity to the C-terminus of rat transcriptional activator FE65. When expressed stably in L929 cells, TR2L protein inhibited TNF cytotoxic response. In contrast, TR2L enhanced anti-Fas antibodies/actinomycin D (ActD)-mediated L929 apoptosis. Alteration of TR2L function occurred by tagging this protein with a 6xHis fragment to the N-terminus (designated 6xH-TR2L). L929 cells which stably expressed 6xH-TR2L acquired a significantly enhanced TNF apoptotic response and increased genomic DNA fragmentation compared to control cells. Enhanced cell death also occurred in these 6xH-TR2L-expressing cells under serum starvation conditions. In contrast, the anti-Fas/ActD-mediated apoptosis was blocked by the 6xH-TR2L protein. Functional role of TR2L protein in regulation of cancer cell susceptibility to TNF-and Fas ligand-mediated apoptosis is suggested.

3T3 Cells↗
PubMed1996

Glial cell line-derived neurotrophic factor signals through the RET receptor and activates mitogen-activated protein kinase.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta family of growth factors, was first identified by its ability to promote the survival of midbrain dopaminergic neurons in culture. We demonstrate that GDNF treatment of several neuroblastoma cell lines leads to dose-dependent tyrosine phosphorylation of the RET receptor and that other transforming growth factor-beta family members are not able to activate the RET receptor. GDNF treatment of neuroblastoma cells also results in increased transcription of an Elk luciferase reporter gene, suggesting that GDNF activates the mitogen-activated protein kinase signal transduction pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

The gene encoding the catalytic subunit C alpha of cAMP-dependent protein kinase (locus PRKACA) localizes to human chromosome region 19p13.1.

We have determined the chromosomal localization of the gene for the catalytic subunit C alpha of cAMP-dependent protein kinase (locus PRKACA) to human chromosome 19 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. In addition, PCR analysis of a chromosome 19 mapping panel revealed the presence of a human C alpha-specific amplification product only in cell lines containing the region 19p13.1 to 19q12. Finally, two-color fluorescence in situ hybridization to metaphase chromosomes using the human C alpha cDNA and human chromosome 19 inter-Alu-PCR product as probes localized the human C alpha gene to chromosome region 19p13.1.

Animals↗
PubMed1996

Reactivity of alcohols toward the phosphoenzyme intermediate in the protein-tyrosine phosphatase-catalyzed reaction: probing the transition state of the dephosphorylation step.

In solution phosphate monoesters hydrolyze via a highly dissociative mechanism involving a "loose" or "exploded" metaphosphate-like transition state where bond formation to the incoming nucleophile is minimal and bond breaking between phosphorus and the leaving group is substantial. To better understand how protein-tyrosine phosphatase (PTPase) effects catalysis, it is important to determine the nature of the enzymic transition state. PTPases catalyze the hydrolysis of phosphate monoesters by a two-step mechanism that proceeds through a phosphoenzyme intermediate (E-P). Extensive heavy atom kinetic isotope effect and leaving group dependency studies have provided insights into the nature of the transition state for the first step (E-P formation) of the PTPase reaction. In this paper we have probed the transition state for the low M(r) PTPase-catalyzed dephosphorylation step by studying the effect of changing the alcohol basicity on its reactivity toward E-P. The Brønsted beta nu value for the reactions of alcohols and E-P is determined to be 0.14, which indicates that the enzymic transition state is highly dissociative and similar to that in uncatalyzed solution reactions. We show that the conserved hydroxyl group in the PTPase signature motif is primarily involved in the E-P dephosphorylation step. We further demonstrate that elimination of the hydroxyl group renders the transition state for E-P dephosphorylation less dissociative, suggesting that the main function of the hydroxyl group in the PTPase active site is to promote the E-P going through a dissociative pathway and to stabilize the dissociative transition state.

Alcohols↗
PubMed1996

Narrowing the position of the Werner syndrome locus by homozygosity analysis-extension of homozygosity analysis.

Werner syndrome (WS) is an autosomal recessive disorder characterized by the premature occurrence of many age-related features. Previously, the WS gene (WRN) was mapped between D8S131 and D8S87, in an 8.3-cM interval. In this study, regions of homozygosity in 36 WS patients from inbred families were searched for by genotyping for 35 dinucleotide repeat polymorphic markers to narrow down the WRN critical region. The region most consistently homozygous in these patients was between the D8S1219/D8S1220 cluster and D8S278, within a 4.4-cM interval. For 16 markers mapped in this interval, 24 WS patients (22 Japanese patients and 2 Caucasian patients) in whom consanguinity failed to be proved were also genotyped, under the assumption that some of these patients might still be from consanguineous marriages. The data were analyzed by Fisher's exact test with a 2 x 2 contingency table for the 22 Japanese patients, excluding the 2 Caucasian patients. The frequencies of homozygosity in the 22 patients at 10 of 16 markers tested were significantly higher than those detected in the general population. Analysis of homozygosity patterns indicated that the region most consistently homozygous was between D8S1445 and D8S278. Thus the WRN locus is most likely between the two markers D8S1445 and D8S278, in a 1.6-cM interval.

Asian People↗
PubMed1996

Molecular cloning of delta 9 fatty acid desaturase from the protozoan Tetrahymena thermophila and its mRNA expression during thermal membrane adaptation.

In response to a decrease in its growth temperature, the protozoan Tetrahymena is known to increase the level of unsaturated fatty acids in its membrane phospholipids so as to maintain the correct physical state (fluidity) of the membranes. In this organism, synthesis of unsaturated fatty acids is initiated by delta 9 acyl-CoA desaturase. Our previous studies have shown that, during cold adaptation, the activity of microsomal palmitoyl- and stearoyl-CoA desaturase increases, reaching a maximal level at 2 h after a temperature down-shift to 15 degrees C. Two hypotheses have been proposed to explain this increase in desaturase activity: (1) self-regulation via a direct effect of reduced membrane fluidity, and (2) induction of desaturase mRNA. However, the precise mechanism is not clearly understood. In order to obtain further insight into the mechanism of regulation of the desaturase, we have isolated a gene that encodes delta 9 fatty acid desaturase from T. thermophila and examined its expression during cold adaptation. The nucleotide sequence indicates that the 1.4 kbp gene encodes a polypeptide of 292 amino acid residues which shows marked sequence similarity to delta 9 acyl-CoA desaturases from other sources, e.g. rat, mouse, Amblyomma americanum and Saccharomyces cerevisiae. This protein has three histidine-cluster motifs (one HXXXXH and two HXXHH), and two hydrophobic regions which are conserved among delta 9 acyl-CoA desaturases. The level of desaturase mRNA was sensitive to decreasing the temperature of the culture media, and was close to maximal immediately after the temperature was shifted down from 35 degrees C to 15 degrees C (0.8 degrees C/min). Thereafter, the amount of mRNA gradually decreased with time, but remained above the control level for at least 5 h. Furthermore, during the course of the cooling process to 15 degrees C, the increased expression of desaturase mRNA became evident at 27 degrees C. Nuclear run-on analysis and actinomycin D chase experiments revealed that the elevation of the mRNA level was due to increases in both transcription and mRNA stability. These results suggest that the enhanced desaturase activity is controlled, at least in part, at the transcriptional level.

Adaptation, Physiological↗
PubMed1996

Crystal structure implies that cyclophilin predominantly catalyzes the trans to cis isomerization.

The crystal structure of human recombinant cyclophilin A complexed with a substrate of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF) has been determined and refined to an R-factor of 0.189 at 2.4 A resolution. The structure revealed only the cis form of the substrate bound to cyclophilin A in a stoichiometry of 1:1. This binding ratio is different from the structure of cyclophilin A complexed with the tetrapeptide N-acetyl-Ala-Ala-Pro-Ala-amidomethylcourmarin. Model docking revealed that the trans form of AAPF does not fit into the active site. The observation that only the trans cis form of AAPF binds to cyclophilin A implies that cyclophilin A predominantly catalyzes the trans to cis isomerization of a peptidylprolyl amide bond. On the basis of the structure, it is proposed that Arg55 hydrogen-bonds to the nitrogen to deconjugate the resonance of the prolyl amide bond and thus facilitates the cis-trans rotation.

Amino Acid Isomerases↗
PubMed1996

Mechanistic implication of crystal structures of the cyclophilin-dipeptide complexes.

The structures of cyclophilin A complexed with dipeptides of Ser-Pro, His-Pro, and Gly-Pro have been determined and refined at high resolution. Comparison of these structures revealed that the dipeptide complexes have the same molecular conformation and the same binding of the dipeptides. The side chains of the N-terminal amino acid of the above dipeptides do not strongly interact with cyclophilin, implying their minor contribution to the cis-trans isomerization and thus accounting for the broad catalytic specificity of the enzyme. The binding of the dipeptides is similar to that of the common substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in terms of the N-terminal hydrogen bonding and the hydrophobic interaction of the proline side chain. However, substantial difference between these structures are observed in (1) hydrogen bonding between the carboxyl terminus of the peptides and Arg55 and between Arg55 and Gln63, (2) the side chain conformation of Arg55, and (3) water binding at the active site. These differences imply either that dipeptides are not substrates but competitive inhibitors of peptidyl-prolyl cis-trans isomerases or that dipeptides are subject to different catalytic mechanisms from tetrapeptides.

Amino Acid Isomerases↗
PubMed1996

Serum albumin is a significant intermediate in cholesterol transfer between cells and lipoproteins.

The function of albumin in the movement of cholesterol into and out of non-cholesterol-loaded fibroblasts has been investigated. Cholesterol efflux from cholesterol labeled normal human skin fibroblasts to fatty acid-free human serum albumin (HSA) is biphasic with a rapid first phase that plateaus at about 15 min followed by a nearly linear phase up to 90 min, the longest incubation in this study. Saturation of efflux is observed at about 10 mg of albumin/mL. Efflux is specific to albumin since other molecules, such as ovalbumin or gelatin, do not induce efflux. The ability of HSA to induce cellular cholesterol efflux is low compared to reconstituted discoidal lipoprotein A-I (LpA-I). HSA at 2 mg/mL produces a rate of cholesterol efflux similar to that of LpA-I at 45 micrograms of protein/mL; however, these concentrations are within the physiological range for both HSA and apolipoprotein A-I (apoA-I). The efflux to the medium containing both LpA-I and HSA is greater than that to each of them alone but does not show complete additivity, indicating a competition between HSA and LpA-I. The HSA-mediated cholesterol movement is bidirectional as demonstrated by the transfer of cholesterol from HSA-(3H)- cholesterol complexes to fibroblasts; moreover, the HSA-mediated transfer is much faster than that from cholesterol-containing LpA-I (0.8 versus 0.2 pmol (micrograms of cell protein)-1 (90 min)-1. However, the presence of either low-density lipoprotein (LDL) or LpA-I in the incubation medium significantly inhibits the transfer of cholesterol from HSA-(3H)-cholesterol complexes to fibroblasts, thus allowing the bidirectional transfer of cholesterol between HSA and cells to possibly operate as a net efflux. In conclusion, albumin plays a significant role in cholesterol transfer between cells and lipoproteins.

Apolipoprotein A-I↗
PubMed1996

Mapping protein-protein interactions by affinity-directed mass spectrometry.

A precise and rapid method for identifying sites of interaction between proteins was demonstrated; the basis of the method is direct mass spectrometric readout from the complex to determine the specific components of the proteins that interact--a method termed affinity-directed mass spectrometry. The strategy was used to define the region of interaction of a protein growth factor with a monoclonal antibody. A combination of proteolytic digestion and affinity-directed mass spectrometry was used to rapidly determine the approximate location of a continuous binding epitope within the growth factor. The precise boundaries of the binding epitope were determined by affinity-directed mass spectrometric analysis of sets of synthetic peptide ladders that span the approximate binding region. In addition to the mapping of such linear epitopes, affinity-directed mass spectrometry can be applied to the mapping of other types of molecule-molecule contacts, including ligand-receptor and protein-oligonucleotide interactions.

Amino Acid Sequence↗
PubMed1996

Expression of transcripts of interleukin-6 and related cytokines by human breast tumors, breast cancer cells, and adipose stromal cells.

The expression of transcripts of cytokines of the interleukin-6 (IL-6) family has been examined in human breast tumors, breast cancer cell lines, and adipose stromal cells, by means of reverse transcription polymerase chain reaction amplification. Of the six breast tumor samples examined, all expressed transcripts encoding IL-6 and Leukemia Inhibitory Factor (LIF). Four of the samples also expressed transcripts for oncostatin M (OSM) and IL-11, and three expressed the IL-6 receptor. Adipose stromal cells expressed IL-6, IL-11 and LIF, but not the IL-6 receptor, consistent with previous conclusions that IL-6 activity in these cells required addition of IL-6 soluble receptor. In the case of T47D cells, expression of IL-11 protein was confirmed by immunotitration. Moreover, in these cells, expression of IL-11 transcripts was induced 3-fold by addition of estradiol to the culture medium. These results add credence to our previous proposal that breast cancer development is regulated in part by local autocrine and paracrine mechanisms via epithelial/mesenchymal interactions, in which estrogen produced by stromal cells surrounding the tumor acts to stimulate the production of growth factors and cytokines by the tumor cells. Some of these may act to stimulate further the growth and development of the tumor, while these or other factors may act on the surrounding mesenchymal cells in a paracrine fashion to stimulate aromatase expression in the presence of glucocorticoids. Thus, a positive feedback loop is established which leads to the development and growth of the tumor.

Adipose Tissue↗
PubMed1996

Catabolism of isobutyrate by colonocytes.

Isolated colonocytes have more capacity for the oxidation of isobutyrate and alpha-ketoisovalerate than isolated enterocytes. Both enterocytes and colonocytes express high levels of 3-hydroxyisobutyryl-CoA hydrolase, an enzyme activity important in maintaining low intracellular concentrations of methacrylyl-CoA, a common, potentially toxic intermediate in the catabolic pathways of these compounds. In spite of comparable 3-hydroxyisobutyryl-CoA hydrolase activities in both cell types, and much greater amounts of 3-hydroxyisobutyrate dehydrogenase in colonocytes than in enterocytes, only the colonocytes produced 3-hydroxyisobutyrate as an endproduct of alpha-ketoisovalerate and isobutyrate catabolism. Butyrate very effectively inhibits isobutyrate catabolism by colonocytes, most likely by competitively inhibiting activation of isobutyrate to its CoA ester. Oleate also inhibits isobutyrate catabolism, but at a site more distal than butyrate. Starvation of rats for 72 h decreased the capacity of colonocytes for butyrate but not isobutyrate catabolism. We conclude that isobutyrate could function as a carbon source for energy and anapleurosis in colonocytes under conditions of defective butyrate oxidation or low butyrate availability.

Animals↗
PubMed1996

Requirement of transforming growth factor-beta (TGF-beta) type II receptor for TGF-beta-induced proliferation and growth inhibition.

Growth regulation of fibroblasts is important for lung development and repair of lung injury. In this study, we investigated the role of transforming growth factor-beta (TGF-beta) type II receptor in the TGF-beta-dependent proliferative response of lung fibroblasts. TGF-beta stimulated the proliferation of adult lung fibroblasts at a low concentration (1 ng/ml), but inhibited the growth of fetal lung fibroblasts in a dose-dependent fashion (0.1-10 ng/ml). Cross-linking and Northern analysis demonstrated that the two lung fibroblast cell lines expressed the TGF-beta type I receptor (T beta RI) and type II receptor (T beta RII). We overexpressed in lung fibroblasts a truncated derivative of T beta RII that lacked the cytoplasmic serine/threonine kinase domain (T beta RII delta K). T beta RII delta K was a dominant-negative inhibitor of TGF-beta signal transduction blocking not only TGF-beta-induced mitogenic action upon adult lung fibroblasts but also TGF-beta-induced growth inhibition of fetal lung fibroblasts. The results indicate that the type II receptor is indispensable for mediating both the mitogenic and antiproliferative effects of TGF-beta upon lung fibroblasts.

Animals↗
PubMed1996

Polarization of counterions in polyelectrolytes.

A theory of the polarization of counterions bound to a polyion, such as a DNA, in low and high electric field strengths is developed using statistical mechanics of inhomogeneous systems. For low fields, one finds that the polarizability p is (Zq)2rho0betaL3/(12[l + Lrho0sigma(L, b, xi, Z, I, rho0)]), where sigma = integral1 0(lambda' - lambda0) ¿dc(lambda - lambda')/dlambda¿lambda = lambda0 dlambda']), Z and L are the valence and the length of the polyion, respectively, q is the proton charge, beta = 1/kBT, T is the temperature, kB is the Boltzmann constant, I is the ionic strength, lambda = x/L and lambda0 = x0/L are scaled distances, x0 is a reference point such that the inhomogeneous counterion density at x0 is equal to rho0--the uniform density in the absence of an electric field E--and c(x) is the direct correlation function of the homogeneous counterion-polyion phase, which includes attractive and repulsive interactions. If Lsigma(L, ...) is much less than one, then the polarizability is proportional to L3. If the term Lsigma(L, ...) is much larger than one, the polarizability scales as L2. The induced dipole moment saturates and its value is the same as that of Mandel-Manning theories. The onset of the saturation, however, depends critically on the direct correlation function and hence polyelectrolyte effects. In the formalism, the polarization of the counterions is the equilibrium response to an electric field provided E is less than Esaturated. A dynamical scheme that incorporates the fact that in high fields the bound counterions conduct is discussed.

Chemical Phenomena↗
PubMed1996

Thrombin-induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein in bovine pulmonary artery endothelial cells.

Myristoylated alanine-rich C kinase substrates (MARCKS) is a prominent protein kinase C (PKC) substrate that is targeted to the plasma membrane by an aminoterminal myristoyl group. In its nonphosphorylated form, MARCKS cross-links Factin and binds calmodulin (CaM) reciprocally. However, upon phosphorylation by PKC, MARCKS release the actin or CaM MARCKS may therefore act as a CaM sink in resting cells and regulate CaM availability during cell activation. We have demonstrated previously that thrombin-induced myosin light chain (MLC) phosphorylation and increased monolayer permeability in bovine pulmonary artery endothelial cells (BPAEC) require both PKC-and CaM-dependent pathways. We therefore decided to investigate the phosphorylation of MARCKS in BPAEC to ascertain whether this occurs in a temporally relevant manner to participate in the thrombin-induced events. MARCKS is phosphorylated in response to thrombin with a time course similar to that seen with MLC. As expected, MARCKS is also phosphorylated by phorbol 12-myristate 13 acetate (PMA), a PKC activator, but with a slower onset and more prolonged duration. Bradykinin also enhances MARCKS phosphorylation in BPAEC, but histamine does not. MARCKS is distributed evently between the membrane and cytosol in BPAEC, and neither thrombin nor PMA caused significant translocation of the protein. Specific PKC inhibitors attenuated MARCKS phosphorylation by either thrombin or PMA. Since thrombin-induced MLC phosphorylation is also attenuated by these inhibitors, MARCKS may be involved in MLC kinase activation and subsequent BPAEC contraction. W7, a CaM antagonist, enhances the phosphorylation of MARCKS. This was expected since CaM binding to MARCKS has been shown to decrease MARCKS phosphorylation by PKC. On the other hand, tyrosine kinase inhibitors, genistein and tyrphostin, attenuate MARCKS phosphorylation but have no effect on MLC phosphorylation, suggesting that MARCKS may be phosphorylated by kinases other than PKC. Phosphorylation of MARCKS outside the PKC phosphorylation domain would not be expected to induce the release of CaM. These data provide support for the hypothesis that MARCKS may serve as a regulator of CaM availability in BPAEC.

Animals↗
PubMed1996

Mg2+ antagonism on Ni(2+)-induced changes in microtubule assembly and cellular thiol homeostasis.

As an essential metal for cell metabolism, Mg2+ is known to exert antagonism on Ni2+ genotoxic and other effects. This study examined the influence of Mg2+ on Ni(2+)-induced changes in microtubule (MT) assembly in vitro, cytoplasmic MT organization, cellular glutathione (GSH), and cytoskeletal and cytosolic protein sulfhydryls (PSH). As determined by a turbidity assay at 27 degrees C, Ni2+ enhanced the in vitro MT assembly in a Pipes buffer by shortening the initial lag (nucleation phase) and increasing the rate of polymerization with a higher final plateau. However, presence of 1 mM exogenous MgCl2 abolished the Ni2+ enhancing effect. Exposure of 3T3 cells to 2 mM NiCl2 for 20 hr resulted in perinuclear bundling of MTs and decreases in cytoskeletal and cytosolic PSH and cellular GSH levels. However, coincubation of cells with MgCl2 (1.25-20 mM) added to the culture medium markedly diminished the Ni2+ injury to MT organization. Under these conditions the Ni2+ interference with PSH was blocked such that both the cytoskeletal and cytosolic PSH levels returned to the range of control cells without metal treatment. Treatment of cells with Mg2+ (1.25-5 mM) for 20 hr slightly increased, while with higher Mg2+ doses (> 10 mM) decreased, cellular GSH content. Importantly, in Ni(2+)-treated cultures, addition of Mg2+ (1.25-10 mM) elevated GSH levels to > or = 200% of that in cells treated with Ni2+ alone. Furthermore, these Ni2+ and Mg2+ (1.25-10 mM) treated cells actually maintained GSH levels which were essentially unchanged from the basal level of control cells with no metal treatment. Although Mg2+ replacement of Ni2+ bound to MT proteins could be an important mechanism, cellular GSH may also be a critical factor in Mg2+ antagonism on Ni(2+)-enhanced MT assembly in view of the essential role of tubulin PSH in modulating MT assembly and, in turn, the GSH modulation of PSH.

3T3 Cells↗