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Biomedical subjects

Y Shi

Publications and source records attributed to Y Shi.

At least 523 records · Page 29Linked to original sources
PubMed1995

Direct association of Grb2 with the p85 subunit of phosphatidylinositol 3-kinase.

Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to play a key role in growth factor signaling pathways, although its signaling mechanism has not been fully elucidated. Using the yeast interaction trap system, we have identified Grb2 as a PI 3-kinase interacting protein. Our experiments demonstrate that p85, the regulatory subunit of PI 3-kinase, interacts with Grb2 in vivo, and this interaction is independent of growth factor stimulation. The direct association between Grb2 and p85 was reconstituted in vitro with glutathione S-transferase fusion proteins. Domain analyses and peptide competition indicate that the association is mediated by the SH3 domains of Grb2 and the proline-rich motifs of p85 and that only one SH3 domain is required for minimal binding. The interaction does not displace the catalytic subunit of PI 3-kinase but is exclusive of Sos. Signaling through PI 3-kinase, therefore, may involve the ubiquitous adapter Grb2, which serves as a convergence point for multiple pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Relief of YY1 transcriptional repression by adenovirus E1A is mediated by E1A-associated protein p300.

YY1 represses transcription when bound upstream of transcriptional initiation sites. This repression can be relieved by adenovirus E1A. Here, we present genetic evidence that the ability of E1A to relieve YY1 repression was impaired by mutations that affect E1A binding to its associated protein p300. This suggests that E1A may modulate the repressor activity of YY1 by binding to p300, which may be physically complexed with YY1. A YY1/p300 protein complex in vivo was demonstrated by several independent approaches, and the YY1-interacting domain was mapped to the carboxy-terminal region of p300, distinct from the E1A-binding site. Unlike E2F/RB, the YY1/p300 complex is not disrupted by E1A. Functional studies using recombinant p300 demonstrated unequivocally that p300 is capable of mediating E1A-induced transcriptional activation through YY1. Taken together, these results reveal, for the first time, a YY1/p300 complex that is targeted by E1A and demonstrate a function for p300 in mediating interactions between YY1 and E1A. Our data thus identify YY1 as a partner protein for p300 and uncover a molecular mechanism for the relief of YY1-mediated repression by E1A.

Adenoviridae Infections↗
PubMed1995

WT1-mediated transcriptional activation is inhibited by dominant negative mutant proteins.

The WT1 tumor suppressor gene encodes four isoforms of a zinc finger transcription factor with both activation and repression functions which are dependent upon promoter architecture. Using a simple HSV-tk promoter containing 5'-Egr-1/WT1-binding sites, we found that WT1 isoforms (A) and (B) strongly activated transcription. WT1(A) and (B) bound equally well to the Egr-1/WT1-binding site, but WT1(B), which contains a 17 amino acid insertion compared to WT1(A), was a consistently stronger activator of transcription than WT1(A). Transcriptional activation by wild-type WT1 was inhibited by coexpression of WT(PM) or WT(AR), genetically defined dominant negative alleles of WT1. In vitro, as well as in the yeast two-hybrid system, WT1 protein associated with itself and with dominant negative mutant proteins. The major domain required for self-association and inhibition of transcriptional activation mapped to the first 182 amino acids of WT1. Dominant negative WT1 alleles may play a role in tumorigenesis by associating with wild-type WT1 proteins and decreasing their transcriptional activity.

Animals↗
PubMed1995

Rapamycin enhances apoptosis and increases sensitivity to cisplatin in vitro.

Apoptosis can be regulated in a number of different systems by the actions of cytokines. Rapamycin has been shown to exert its effects on growth factor-induced cell proliferation, at least in part, by blocking the activation of the p70 S6 kinase and thus preventing the downstream signaling process, such as the activation of the members of the cdk family. To determine whether this pathway plays a role in the regulation of apoptosis, we assessed the effect of rapamycin on apoptosis induced by interleukin 2 deprivation in murine T-cell lines, by T-cell receptor ligation in a murine T-cell hybridoma, by enforced c-myc expression in murine fibroblasts, and by corticosteroids in murine T-lymphoma cell lines. Although rapamycin did not induce apoptosis on its own, rapamycin augmented apoptosis in each of the cell lines used as indicated by increased genomic DNA fragmentation, decreased cell viability, and characteristic apoptotic changes in morphology. These results suggest that a signal transduction pathway(s) inhibited by rapamycin plays an important role in the susceptibility of cells to apoptosis. Many chemotherapeutic agents kill cancer cells through the induction of apoptosis. Strikingly, rapamycin increased the ability of the alkylating agent, cisplatin, to induce apoptosis in the human promyelocytic leukemia cell line HL-60 and the human ovarian cancer cell line SKOV3. These data suggest that a signal transduction pathway, likely related to p70 S6 kinase, inhibited by rapamycin may be an important component of the pathway which prevents cell death in many cell lineages and also indicate that rapamycin has the potential to augment the efficacy of selected anticancer therapies.

Animals↗
PubMed1995

Specific DNA-RNA hybrid binding by zinc finger proteins.

Zinc finger proteins of the Cys2His2 type represent a large class of proteins that have been assumed to function by means of specific interactions with DNA. Experiments motivated by structural characteristics of zinc finger protein-DNA complexes revealed that certain zinc finger proteins bound DNA-RNA hybrids with affinities comparable to or greater than those for DNA duplexes. The interactions between the zinc finger proteins and the DNA-RNA hybrids were dependent on which strand was RNA and were sequence-specific. Thus, interactions with DNA-RNA hybrids should be considered with regard to the biological roles of zinc finger proteins.

Amino Acid Sequence↗
PubMed1995

ATP-sensitive binding of a 70-kDa cytosolic protein to the glucose transporter in rat adipocytes.

We have identified a 70-kDa cytosolic protein (GTBP70) in rat adipocytes that binds to glutathione S-transferase fusion proteins corresponding to the cytoplasmic domains of the facilitative glucose transporter isoforms Glut1, Glut2, and Glut4. GTBP70 did not bind to irrelevant fusion proteins, indicating that the binding is specific to the glucose transporter. GTBP70 binding to the glucose transporter showed little isoform specificity but was significantly subdomain-specific; it bound to the C-terminal domain and the central loop, but not to the N-terminal domain of Glut4. The GTBP70 binding to Glut4 was not affected by the presence of 2 mM EDTA, 2.4 mM Ca2+, or 150 mM K+. The binding was inhibited by ATP in a dose-dependent manner, with 50% inhibition at 10 mM ATP. This inhibition was specific to ATP, as ADP and AMP-PCP (adenosine 5'-(beta, gamma-methylenetriphosphate)) were without effect. GTBP70 did not react with antibodies against phosphotyrosine, phosphothreonine, or phosphoserine, suggesting that it is not a phosphoprotein. The binding of GTBP70 to Glut4 was not affected by the pretreatment of adipocytes with insulin. When these experiments were repeated using rat hepatocyte cytosols, no ATP-sensitive 70-kDa protein binding to the glucose transporter fusion proteins was evident, suggesting that either GTBP70 expression or its function is cell-specific. These findings strongly suggest the possibility that GTBP70 may play a key role in glucose transporter regulation in insulin target cells such as adipocytes.

Adenosine Triphosphate↗
PubMed1995

Functional interactions between YY1 and adenovirus E1A.

YY1 is a C2H2-type zinc finger transcription factor that is a member of the human GLl-Kruppel family of proteins. YY1 represses transcription when bound upstream of transcription initiation sites. The repression can be relieved by adenovirus E1A and activation of target genes occurs. We have mapped the repression domain of YY1 to the C-terminal region, overlapping its DNA binding domain. We have also identified an activation domain within the first 69 amino acids of YY1. The YY1 C-terminal region is involved in physical interactions with E1A and is functionally necessary for YY1 to respond to E1A. This suggests that relief of YY1 repression by E1A involves YY1-E1A physical interactions. Although not involved in interactions with E1A, the N-terminal activation domain is also necessary for YY1 to respond to E1A. Presumably, under repressing conditions, the activation domain is masked by the conformation of YY1, but is released upon binding of E1A and is required to subsequently activate transcription. Consistent with this hypothesis, an ATF-2-YY1 chimeric protein containing the activation domain of ATF-2 and the C-terminal two-thirds of YY1 is still a potent repressor. Unlike the mutant YY1 lacking its own N-terminal activation domain, the chimeric protein is fully responsive to E1A.

Activating Transcription Factor 2↗
PubMed1995

Active site mapping of Escherichia coli D-Ala-D-Ala ligase by structure-based mutagenesis.

Eleven Escherichia coli D-Ala-D-Ala ligase (DdlB) mutants, at K144, K215, and E270 in the ATP binding site, at E15, S150, H63, and R255 in the first D-Ala subsite, and at Y216, S281, L282, and D257 in the second D-Ala subsite, were constructed, purified, and examined for steady-state kinetic parameters, kcat and Kms for ATP, and both first (D-Ala1), and second (D-Ala2) D-alanines. Of these, E270Q, K215A, R255A, and D257N retained very low or no detectable activity consistent with X-ray structure based predictions for roles in Mg2+ coordination to beta, gamma-P of ATP (E270), coordination to transferring gamma-PO3 of ATP (K215), and coordination/orientation of nucleophilic COO- of D-Ala1 that attacks gamma-PO3 of ATP (R255, D257) and the side chain of R255, respectively. The substantial retention of activity in the Y216F mutant argues against the possibility that Y216 may be a catalytic base that deprotonates the alpha-NH3+ of D-Ala2 to attack the acyl phosphate form of D-Ala1. While all seven mutants that retain activities have a 300-2000 fold elevation in Km for D-Ala1 (1-2 microM in wild type), the S281A mutant has a 500-fold elevation in Km for D-Ala2, consistent with a proposed interaction with the COO- of D-Ala2. Similarly, the kinetics of inhibition by a slow-binding phosphinate inhibitor in the presence of ATP are most altered in the S281A mutant.

Adenosine Diphosphate↗
PubMed1995

A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli.

Examination of x-ray crystallographic structures shows the tertiary structure of D-alanine:D-alanine ligase (EC 6.3.2.4). a bacterial cell wall synthesizing enzyme, is similar to that of glutathione synthetase (EC 6.32.3) despite low sequence homology. Both Escherichia coli enzymes, which convert ATP to ADP during ligation to produce peptide products, are made of three domains, each folded around a 4-to 6-stranded beta-sheet core. Sandwiched between the beta-sheets of the C-terminal and central domains of each enzyme is a nonclassical ATP-binding site that contains a common set of spatially equivalent amino acids. In each enzyme, two loops are proposed to exhibit a required flexibility that allows entry of ATP and substrates, provides protection of the acylphosphate intermediate and tetrahedral adduct from hydrolysis during catalysis, and then permits release of products.

Adenosine Diphosphate↗
PubMed1995

Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis.

We investigated the role of gp39-CD40 interaction in the development of glomerulonephritis in lupus mice. In contrast to normal mice, lupus mice had much higher percentages of intensely gp39+ T cells in their spleens even at the preautoimmune age of 1 mo, and the further increase in gp39 expression by anti-CD3 Ab stimulation was markedly greater in lupus T cells. The pathogenic autoantibody-inducing ability of Th clones and splenic Th cells from lupus mice could be blocked in vitro by anti-gp39 Ab. Acceleration of lupus nephritis by the transfer of pathogenic autoantibody-inducing Th clones in vivo could also be completely blocked by anti-gp39 Ab. Surprisingly, a brief treatment of lupus mice with anti-gp39 Ab had a sustained beneficial effect on their spontaneous disease long after the Ab had been cleared from their systems. Only three injections of anti-gp39 Ab given to prenephritic lupus mice at 3 mo of age markedly delayed and reduced the incidence of lupus nephritis up to 12 mo of age by which time almost all the control mice had developed severe glomerulonephritis. Remarkably, pathogenic Th cells were left intact in these anti-gp39-treated mice but their B cells could not produce pathogenic autoantibodies even 9 mo after the therapy. Our studies suggest that blocking the interaction between gp39 on pathogenic Th cells and CD40 on lupus B cells at a crucial window of time delays the expansion autoimmune memory B cells resulting in long-term therapeutic benefits.

Animals↗
PubMed1995

Effects of cytokine-mediated modulation of nm23 expression on the invasion and metastatic behavior of B16F10 melanoma cells.

The molecular mechanisms of tumor invasion and metastasis are yet to be fully elucidated. A potential tumor-metastasis-suppressor gene nm23 has been described in certain rodent and human tumors. In the present study, we examined the potential anti-invasive and anti-metastatic effect of nm23 gene in B16F10 cells, a malignant murine melanoma cell line. Transfection of nm23 gene into B16F10 melanoma cells resulted in significant suppression of the invasiveness and metastatic ability of melanoma cells and significantly enhanced the survival of tumor-bearing mice. B16F10 melanoma cells transfected with nm23 produced significantly less soluble ICAM-I and were more susceptible to LAK-cell-mediated cytotoxicity. Co-culture of B16F10 melanoma cells with IL-2 had no effect on nm23 expression, whereas treatment with PGE2, TNF-alpha and IFN-gamma resulted in down-regulation of nm23 expression. Concomitantly, in vivo treatment with TNF-alpha or IFN-gamma in experimental mice increased pulmonary metastases and lowered the overall survival period, as compared with IL-2 treatment alone. These results provide evidence that nm23, in addition to its anti-metastatic function, could also be involved in modulating tumor-target-structure expression, in down-regulating invasive potential and in production of soluble intracellular adhesion molecules. The down-regulation of nm23 by TNF-alpha, IFN-gamma and particularly by PGE2 warrants re-examination of current immunotherapeutic protocols and of the role played by PGE2 in tumor progression.

Animals↗
PubMed1995

Selective inhibition of the slow K+ current at motor nerve ending by plasma from a myasthenia gravis patient.

The effect of plasma from a myasthenia gravis (MG) patient, containing anti-presynaptic membrane receptor (PsmR) antibody on the membrane currents of motor nerve ending was investigated in mouse intercostal nerve triangularis sterni preparations by perineurial recording. After inhibition of both the fast K+ current and Ca(2+)-dependent K+ current by 30 mM Tetraethyl-ammonium (TEA) unmasked the voltage dependent fast Ca2+ current and the "Ca plateau", which was contributed by the voltage-dependent slow Ca2+ current and slow K+ current. Application of the MG plasma caused further prolongation and increase of the Ca plateau, due to blockage of the slow K+ current. This effect was observed immediately after the application and could be partially reversed by washing, whereas no change was found by addition of the plasma from healthy persons. When K+ current was completely blocked by 30 mM TEA and 300 microM 3,4-diaminopyridine (3,4-DAP), the fast Ca2+ current and the slow Ca2+ current were revealed. Neither the fast nor the slow Ca2+ current could be affected by the MG plasma; It was also shown that the MG plasma was devoid of noticeable effect on the voltage dependent Na+ current, fast K+ current as well as the Ca(2+)-dependent K+ current. So the effect of the MG plasma with antibody to PsmR was concluded to inhibit the slow K+ current selectively. As we knew, the beta-bungarotoxin binding protein was a kind of K+ channel, these results further confirmed that the beta-bungarotoxin binding protein should be the target of the antibody to PsmR found in the plasma of some patients suffering from MG.

Adult↗
PubMed1995

Vaccination of sheep against Schistosoma japonicum with either glutathione S-transferase, keyhole limpet haemocyanin or the freeze/thaw schistosomula/BCG vaccine.

The protective potential of glutathione S-transferase (GST), keyhole limpet haemocyanin (KLH) and the freeze/thaw (F/T) schistosomula/BCG vaccine was evaluated against Schistosoma japonicum in the natural sheep host. Groups of ten sheep each were vaccinated as follows: Group I: 2 x F/T 30,000 schistosomula+BCG 3 x 10(8) organisms, with a 2 week interval between vaccinations (F/T 'Low'). Group II: 3 x F/T 20,000 schistosomula+BCG 3 x 10(8), with 4 week interval (F/T 'High'). Group III: 2 x GST 0.24 mg+FCA (Freund's complete adjuvant) with 2 week interval (GST 'Low'). Group IV: 3 x GST 0.24 mg+FCA, with 4 week interval (GST 'High'). Group V: 2 x KLH 1.0 mg in phosphate-buffered saline (PBS), with 2 week interval (KLH 'Low'). Group VI: 3 x KLH 1.0 mg in PBS, with 4 week interval (KLH 'High'). Group VII: control (not vaccinated). Specific antibody, detected by GST-enzyme-linked immunosorbent assay (ELISA) and KLH-ELISA on the day after the last vaccination and 1, 2 and 3 weeks post-challenge, was found in all GST- or KLH-vaccinated groups. The same was found in F/T schistosomula-vaccinated groups against crude adult worm antigen (AWA). In Western blotting all GST-vaccinated sera recognized 26 kDa and 28 kDa bands on the challenge day and at 3 and 11 weeks post-challenge. Mean faecal egg counts between Weeks 6 and 10 post-challenge were reduced in a statistically significant way at five time points in the four groups, i.e. 83.38% (P < 0.005) in Group II, 49.29% (P < 0.025) in Group III, 47.9% (P < 0.05) and 71.15% (P < 0.01) in Group IV, 52.0% (P < 0.025) and 66.38% (P < 0.025) in Group VI. On autopsy and perfusion 1 week after the last faecal count, adult worm reductions were obtained of 40.36% (P < 0.05) in Group I, 37.26% (P < 0.025) in Group II, 24.73% (not significant) in Group III, 35.93% (P < 0.025) in Group IV, 27.46% (P < 0.05) in Group V and 33.81% (P < 0.01) in Group VI. Mean tissue egg densities were also reduced significantly in Groups III, IV and VI, especially in Group IV vaccinated animals. Mean liver egg granuloma diameters of the vaccinated groups were found to be less than those of the controls but there was no statistical significance.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

A direct comparison of the properties of natural and designed zinc-finger proteins.

BACKGROUND: Zinc-finger proteins of the Cys2His2 type constitute an important family of DNA-binding proteins. Each zinc-finger domain has three residues that are thought to be important in determining DNA binding site specificity. Proteins have been designed previously by combining zinc-finger domains with a fixed sequence framework with different DNA-contacting residues. RESULTS: We compared the DNA-binding properties of the DNA-binding domain from the human transcription factor Sp1, which contains three zinc fingers, with designed proteins in which the sequences of the structural framework were greatly modified but the presumed DNA-contacting residues were retained. Frameworks based on a zinc-finger consensus sequence and on a minimalist sequence consisting largely of alanine residues were studied. The preference for binding to the target sequence, 5'-(G,T)GG G(C,A)G GG(G,T)-3', was retained in all cases tested. The consensus framework-based protein was found to be superior to the natural one in terms of overall DNA-binding affinity, the degree of sequence discrimination, and the resistance to inactivation by chelating agents. CONCLUSIONS: Our observations provide direct evidence that the residues previously observed to interact with the DNA bases are indeed the most important residues for determining DNA-binding specificity. We have also shown that these domains can tolerate considerable sequence variation while retaining function as well as three-dimensional structure. Finally, they show that framework modification can be used to generate proteins that have normal or enhanced DNA-binding activity but have different metal-binding properties.

Amino Acid Sequence↗
PubMed1995

Effect of different volatile anaesthetics on suxamethonium-induced jaw muscle contracture in rats.

Previous work has demonstrated that the interaction of hyperthermia and halothane may greatly increase the jaw muscle contracture produced by suxamethonium. We have compared the interaction of temperature and suxamethonium in the presence of halothane with the suxamethonium/temperature interaction of two other volatile anaesthetics, isoflurane and desflurane. Rats were anaesthetized with 1.35 MAC of halothane, isoflurane or desflurane. The jaw area was heated to 36-41 degrees C by a heating lamp while rectal temperature was maintained at 37 degrees C. Isometric tension was recorded from the jaw muscles. Suxamethonium 750 micrograms kg-1 i.v. induced a transient jaw muscle contracture (JMC) during halothane, isoflurane and desflurane anaesthesia. JMC exhibited significant dependence on jaw muscle temperature with all three volatile anaesthetics. Increasing the temperature of the jaw area from 37 degrees C to 41 degrees C increased JMC 8.7-fold with halothane, 8.8-fold with isoflurane and 3.1-fold with desflurane. The difference between halothane and desflurane was significant. While suxamethonium-induced JMC was dependent on temperature for all three volatile anaesthetic, the temperature dependence appeared to be less with desflurane.

Anesthetics↗