Biomedical subjects

Y Shi

Publications and source records attributed to Y Shi.

At least 271 records · Page 15Linked to original sources

PubMed1999

Function for p300 and not CBP in the apoptotic response to DNA damage.

The cellular response to ionizing radiation (IR) includes the induction of apoptosis. The p300/CBP proteins possess histone acetyltransferase activity and function as transcriptional coactivators of p53. We have prepared cells deficient in p300 or CBP to define the roles of these proteins in the cellular response to DNA damage. The present results demonstrate that p300, but not CBP, contributes to IR sensitivity of cells. The results also demonstrate that IR-induced apoptosis is impaired in the p300-, but not CBP-, deficient cells. These findings indicate that p300 functions in the apoptotic response to DNA damage.

Adenocarcinoma↗

PubMed1999

[Influence of IL-4 on the expression of TNF-alpha and IL-1 alpha mRNA by PBMCs from patients with subfulminant viral hepatitis before and after treatment].

OBJECTIVE: The aim of this study was to compare the influence of IL-4 on the expression of TNF-alpha and IL-1 alpha mRNA by PBMCs from patients with subfulminant viral hepatitis(SFH) before and after IL-4 treatment. METHODS: The expression levels of TNF-alpha and IL-1 alpha mRNA by PBMCs were assessed by semiquantitative RT-PCR. RESULTS: IL-4 suppressed the expression of both TNF-alpha and IL-1 alpha mRNA by PBMCs from SFH patients before and after treatment dose-dependently. However, their dose-response curves showed significant differences. The inhibitory effect could be observed at a concentration of 100 U/ml and was near to maximum at 1,000 U/ml in acute phase, whereas the same suppressive action of IL-4 was reached at 100 U/ml in recovery phase. When PBMCs were cultured in the presence of IL-4 at a concentration of 100 U/ml, IL-4 down-regulated the production of both TNF-alpha and IL-1 alpha mRNA by acute phase PBMCs only to 18.18-21.98% of control cells(in absence of IL-4), while the inhibitory rates were all near to 50% in recovery phases. Moreover, it was also found that the suppressive effect of IL-4 on the expression of PBMCs TNF-alpha and IL-1 alpha mRNA in the patients with endotoxinemia and HBeAg was significantly decreased in acute phase. CONCLUSIONS: The inhibitory effect of IL-4 on the expression of PBMCs TNF-alpha and IL-1 alpha mRNA is markedly decreased in acute phase as compared with that in recovery phase and this declined response may be related to endotoxinemia and viremia.

Adult↗

PubMed1999

Structure-based ligand design by dynamically assembling molecular building blocks at binding site.

A structure-based ligand design method is proposed and tested. The method is based on stochastic dynamics simulation of multiple copies of molecular building blocks in the presence of a receptor molecule. The molecular building blocks are assembled into candidate compounds "on the fly" at given intervals during the simulation. In the algorithm, a special effort is made to explore different possible combinations of building blocks and to select an optimum combination. By repeating the cycle of deconstruction and reconstruction in a single simulation, a set of candidate compounds that can be built from the building blocks evolves and is dynamically optimized. The method was tested by breaking two known flexible human immunodeficiency virus type 1 protease inhibitors into building blocks and reassembling them in the active site of the enzyme. For the inhibitor L700417, a set of conformations was generated by the calculation. Among these, the original compound was recovered with the lowest energy at the experimentally observed binding site and in the correct conformation. For pepstatin, the experimentally observed binding mode of the backbone of the inhibitor was reproduced by a calculation in which the building blocks corresponding to the side-chain groups were omitted. Proteins 1999;36:462-470.

Algorithms↗

PubMed1999

Computational EST database analysis identifies a novel member of the neuropoietic cytokine family.

A novel member of the neuropoietic cytokine family has been cloned and the protein expressed and characterized. In an effort to identify novel secreted proteins, an algorithm incorporating neural network algorithms was applied to a large EST database. A full-length clone was identified that is 1710 bp in length and has a single open reading frame of 225 amino acids. This new cytokine is most homologous to cardiotrophin-1, having a similarity and an identity of 46 and 29%, respectively, and therefore we have named it cardiotrophin-like cytokine (CLC). Northern hybridization analysis identified a 1.4-kb messenger RNA that is highly expressed in spleen and peripheral leukocytes. Purified recombinant CLC induced the activation of NFkappaB and SRE reporter constructs in the TF-1, U937, and M1 cell lines. Furthermore, the signal transduction pathway for CLC was characterized in the neuroblastoma cell line SK-N-MC and found to involve tyrosine phosphorylation of gp130 and STAT-1.

Amino Acid Sequence↗

PubMed1999

Different anesthetic sensitivities of skeletal and cardiac isoforms of the Ca-ATPase.

We have previously shown that low levels of the volatile anesthetic halothane activate the Ca-ATPase in skeletal sarcoplasmic reticulum (SR), but inhibit the Ca-ATPase in cardiac SR. In this study, we ask whether the differential inhibition is due to (a) the presence of the regulatory protein phospholamban in cardiac SR, (b) different lipid environments in skeletal and cardiac SR, or (c) the different Ca-ATPase isoforms present in the two tissues. By expressing skeletal (SERCA 1) and cardiac (SERCA 2a) isoforms of the Ca-ATPase in Sf21 insect cell organelles, we found that differential anesthetic effects in skeletal and cardiac SR are due to differential sensitivities of the SERCA 1 and SERCA 2a isoforms to anesthetics. Low levels of halothane inhibit the SERCA 2a isoform of the Ca-ATPase, and have little effect on the SERCA 1 isoform. The biochemical mechanism of halothane inhibition involves stabilization of E2 conformations of the Ca-ATPase, suggesting direct anesthetic interaction with the ATPase. This study establishes a biochemical model for the mechanism of action of an anesthetic on a membrane protein, and should lead to the identification of anesthetic binding sites on the SERCA 1 and SERCA 2a isoforms of the Ca-ATPase.

Anesthetics, Inhalation↗

PubMed1999

[Observation on effect of chongcao shenkang capsules in preventing and treating acute renal failure in cases of hemorrhagic fever with renal syndrome].

OBJECTIVE: To observe the effect of Chongcao shenkang capsules in preventing and treating acute renal failure in cases of hemorrhagic fever with renal syndrome (HFRS). METHODS: 150 cases of HFRS were randomly divided into two groups: the treatment group and the control group. The former contained 76 cases and the latter the 74 cases. All cases in both groups were given a combined therapy mainly composed of balanced salt solution and ribavirin, cases in treatment group additionally accepted Chongcao shenkang capsules 2.7 grams tid, till the end of polyuric phase. RESULTS: The results showed that in the treatment group the occurrence rate of cases with oliguric phase was 13.0% (4/46) and the oliguric and polyuric periods were 2.7 +/- 1.6 days and 6.8 +/- 2.9 days respectively while those in the control group were 31.1% (4/45), 4.3 +/- 2.1 dnys and 8.2 +/- 3.4 days respectively (P < 0.01 or P < 0.05). The urinary protein of the treatment group disappeared within 5.1 +/- 2.3 days and their serum creatinine restored within 10.9 +/- 6.2 days, but the urinary protein disappearing and the serum creatinine restoring periods of the control group were 6.8 +/- 2.7 days and 13.7 +/- 7.4 days respectively (P < 0.01 or P < 0.05). The serum and urine beta 2-microglobulin levels of the treatment group were remarkably lower than those of the control group on the fifth and tenth days after treatment (P < 0.01 or P < 0.05). Compared with those of the control group of 33.8% (25/74) and 87.8% (65/74), the occurrence rate of severe complications and curative rate of the treatment group were 18.4% (14/76) and 94.7% (72/76) respectively (the former P < 0.05, but the latter P > 0.05). CONCLUSION: It was concluded that Chongcao shenkang capsules is capable of reducing the occurrence rate of acute renal failure in HFRS patients, shortening the duration of acute renal failure, decreasing their needs for dialysis, lessening their complications and increasing their curative rate. Its administration is safe and convenient and therefore it is worthy to be popularized.

Acute Kidney Injury↗

PubMed1999

Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis. Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA.

Porphyromonas gingivalis produces arginine-specific cysteine proteinase (Arg-gingipain, RGP) and lysine-specific cysteine proteinase (Lys-gingipain, KGP) in the extracellular and cell-associated forms. Two separate genes (rgpA and rgpB) and a single gene (kgp) have been found to encode RGP and KGP, respectively. We constructed rgpA rgpB kgp triple mutants by homologous recombination with cloned rgp and kgp DNA interrupted by drug resistance gene markers. The triple mutants showed no RGP or KGP activity in either cell extracts or culture supernatants. The culture supernatants of the triple mutants grown in a rich medium had no proteolytic activity toward bovine serum albumin or gelatin derived from human type I collagen. Moreover, the mutants did not grow in a defined medium containing bovine serum albumin as the sole carbon/energy source. These results indicate that the proteolytic activity of P. gingivalis toward bovine serum albumin and gelatin derived from human type I collagen appears to be attributable to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for hemagglutination and hemoglobin binding that are also located in the C-terminal regions of rgpA and kgp. A rgpA kgp hagA triple mutant constructed in this study exhibited no hemagglutination using sheep erythrocytes or hemoglobin binding activity, as determined by a solid-phase binding assay with horseradish peroxidase-conjugated human hemoglobin, indicating that the adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin, leading to heme acquisition.

Adhesins, Bacterial↗

PubMed1999

p16/pRb pathway alterations are required for bypassing senescence in human prostate epithelial cells.

The cell cycle regulatory genes p16/CDKN2 and RB are frequently deleted in prostate cancers. In this study, we examined the role of alterations in p16 and pRb during growth, senescence, and immortalization in vitro of human prostate epithelial cells (HPECs). HPECs are established from normal prostate tissues and cultured on collagen-coated dishes. Our results show that p16 is reproducibly elevated at senescence in HPECs. HPECs are immortalized using human papilloma virus 16 E6 and/or E7 as molecular tools to inactivate p53 and/or pRb, respectively. Immortalization occurs infrequently in this system and only after a latent period during which additional genetic/epigenetic changes are thought to occur. Notably, all of the E6-immortalized HPEC lines but none of the E7 lines show inactivation of p16/CDKN2 (by deletion, methylation, or mutation) in association with immortalization. In contrast, E7 lines, in which pRb function is abrogated by E7 binding, retain the high levels of p16 observed at senescence. Thus, all lines show either a p16 or pRb inactivation. Analysis of six independent lines from metastatic prostate cancers reveals a similar loss of either p16 or pRb. Comparative genomic hybridization of HPECs shows that gains of chromosomes 5q, 8q, and 20 are nonrandomly associated with bypassing senescence (probability = 0.95). These results suggest that high levels of the cyclin-dependent kinase inhibitor p16 mediate senescence G1 arrest in HPECs and that bypassing this block by a p16/pRb pathway alteration is required for immortalization in vitro and possibly tumorigenesis in vivo. Our results further indicate that inactivation of the p16/pRb pathway alone is not sufficient to immortalize HPECs and that additional genetic alterations are required for this process.

Aged↗

PubMed1999

Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1.

Caspase-9-mediated apoptosis (programmed cell death) plays a central role in the development and homeostasis of all multicellular organisms. Mature caspase-9 is derived from its procaspase precursor as a result of recruitment by the activating factor Apaf-1. The crystal structures of the caspase-recruitment domain of Apaf-1 by itself and in complex with the prodomain of procaspase-9 have been determined at 1.6 and 2.5 A resolution, respectively. These structures and other evidence reveal that each molecule of Apaf-1 interacts with a molecule of procaspase-9 through two highly charged and complementary surfaces formed by non-conserved residues; these surfaces determine recognition specificity through networks of intermolecular hydrogen bonds and van der Waals interactions. Mutation of the important interface residues in procaspase-9 or Apaf-1 prevents or reduces activation of procaspase-9 in a cell-free system. Wild-type, but not mutant, prodomains of caspase-9 completely inhibit catalytic processing of procaspase-9. Furthermore, analysis of homologues from Caenorhabditis elegans indicates that recruitment of CED-3 by CED-4 is probably mediated by the same set of conserved structural motifs, with a corresponding change in the specificity-determining residues.

Amino Acid Sequence↗

PubMed1999

Metallopeptide approach to the design of biologically active ligands: design of specific human neutrophil elastase inhibitors.

A series of compounds was designed to inhibit human neutrophil elastase utilizing a modified metallopeptide scheme developed in our laboratory termed metal-ion induced distinctive array of structures (MIDAS). These metallopeptides, synthesized by solution and solid-phase methods, exhibited excellent structural diversity and specificity in inhibiting human neutrophil elastase.

Binding Sites↗

PubMed1999

Cloning of an L-3-hydroxyacyl-CoA dehydrogenase that interacts with the GLUT4 C-terminus.

Evidence indicates that the carboxy-terminal cytoplasmic domain of glucose transporter 4 (GLUT4) is important for the regulation of GLUT4 in muscle and adipocytes. We cloned from a human skeletal muscle cDNA library a 34-kDa protein which interacts with GLUT4 C-terminal cytoplasmic domain in a two-hybrid system and also with GLUT4 C-terminus synthetic peptide in an in vitro binding assay. This protein, called YP10, showed a high degree (>90%) of sequence homology with l-3-hydroxyacyl-CoA dehydrogenase (HAD) and had a dehydrogenase activity similar to pig heart HAD, which was inhibited by GLUT4 C-terminus synthetic peptide. An antiserum raised against pig heart HAD also reacted with YP10. Western blot analysis using this antiserum revealed abundant immunoreactivity only in the mitochondria- and plasma membrane-enriched fractions of rat adipocytes. Northern blots revealed that YP10 mRNA is most abundant in skeletal and heart muscle. These findings suggest that YP10, a HAD isoform, interacts with GLUT4 at the plasma membrane and may play a role in cross-talk between glucose transport and fatty acid metabolism.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1999

Effect of mutations at the monomer-monomer interface of cAMP receptor protein on specific DNA binding.

To determine the thermodynamic role of binding of an operon to cAMP receptor protein (CRP) in the activation of transcription, isothermal titration calorimetry measurements were performed on the binding of three 40-base pair DNA sequences to the cyclic nucleoside complexes of CRP and its mutants at 296 K. The three 40-base pair sequences consisted of a consensus DNA (conDNA) duplex derived from the CRP-binding site sequences of the operons activated by CRP and two DNA sequences based on the CRP-binding site sequences of the lac operon (lacDNA) and of the gal operon (galDNA). The mutants of CRP consisted of a T127L mutant, a S128A mutant, and a mutant containing both mutations (CRP*) which not only alter the transcriptional activity of the CRP complexes but also are involved in the monomer-monomer interfacial interactions of the CRP dimer. The binding reactions of the DNA duplexes to the fully cNMP-ligated CRP-mutant complexes were endothermic with binding constants as high as 6.6 +/- 1.1 x 10(6) M-1 (conDNA.CRP(cAMP)2). ConDNA binding to the unligated T127L and CRP* mutants was observed as well as conDNA and lacDNA binding to CRP with cAMP bound to only one monomer. The reduction of the binding constants with increase in KCl concentration indicated the formation of two ion pairs for the cAMP-ligated CRP and S128A complexes and four ion pairs for the cAMP-ligated T127L and CRP* complexes. Reduction of the DNA binding constants upon substitution of D2O for H2O in the buffer, the large heat capacity changes, and the enthalpy-entropy compensation exhibited by the binding reactions indicate the importance of dehydration in the binding reaction. Small angle neutron scattering measurements on the lacDNA.CRP(cAMP)2 complex in D2O/H2O mixtures show that the DNA is bent around the cAMP-ligated protein in solution.

Base Sequence↗

PubMed1999

Molecular mimicry between a viral peptide and a myelin oligodendrocyte glycoprotein peptide induces autoimmune demyelinating disease in mice.

Semliki Forest Virus (SFV) induces an encephalomyelitis followed by demyelination in the brains of C57Bl6/J (B6) mice. To investigate the role of molecular mimicry in the pathogenesis of postviral demyelination, alignment algorithms were used and amino acid homologies between immunogenic epitopes of SFV and myelin autoantigens, myelin basic protein (MBP), myelin proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) were identified. Immunization of B6 mice with SFV proteins induced significant lymphocyte proliferation to SFV E2 peptides and to MOG peptide, 18-32 (which had molecular mimicry with E2 115-129), but not to MBP or PLP peptides. Both MOG 18-32 and E2 115-129, induced a later-onset chronic EAE-like disease that correlated with the presence of multifocal vacuolation in the CNS white matter. This histopathology was reminiscent of the secondary demyelination seen following SFV infection. Serum antibody responses to the peptides appeared late after immunizations and some samples cross-reacted with other myelin peptides, as well as with the mimicked MOG peptides. These findings suggest that following a CNS viral infection, antibody response to an epitope of virus that exhibits molecular mimicry with a peptide of MOG may contribute to autoimmune mediated injury to CNS myelin.

Acute Disease↗

PubMed1999

Characterization of a mutant pancreatic eIF-2alpha kinase, PEK, and co-localization with somatostatin in islet delta cells.

Phosphorylation of eukaryotic translation initiation factor-2alpha (eIF-2alpha) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2alpha kinases including mammalian double-stranded RNA-dependent eIF-2alpha kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2alpha kinases including phosphorylation of eIF-2alpha in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2alpha kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.

Amino Acid Sequence↗

PubMed1999

[Pathogenic gene linkage analysis and hemopoietic characteristics in a kindred with sideroblastic anemia].

OBJECTIVE: Analysis of pathogenic gene linkage and hemopoietic characteristics in a kindred with sideroblastic anemia. METHODS: PCR amplification of the microsatellites DXS991,DXS1199 in chromosome Xp11.22 linked gene ALAS2 and of the microsatellite DXS1226 in Xp22. 13 linked another irrelevant gene and analysis of gene linkage in a kindred with 2 patients and 7 normal persons. The bone marrow hemopoietic cells from 2 patients were cultured in condition culture matrix with various cytokines added in and the CFU-E, CFU-GM and CFU-Meg formations were observed at different times. RESULTS: The kindred study revealed that pathogenic gene linked with DXS991 and DXS1199 but did not link with DXS1226.Hemopoietic cell culture showed that erythroid colonies of the two patients grew more vigorously than controls and they could grew in the absence of Epo except in common condition matrix. The erythroid colonies withered after a week and were smaller than the controls after 13 days. CONCLUSION: The kindred is subject to an X-linked sideroblastic anemia(XLSA) with the pathogenic gene ALAS2 involved. In XLSA,the function of stem cells is primarily normal before erythropoiesis, then the erythroid progenitors become dysplasia.

Adult↗

PubMed1999

Decreased expression of the pro-apoptotic protein Par-4 in renal cell carcinoma.

Par-4 is a widely expressed leucine zipper protein that confers sensitization to apoptosis induced by exogenous insults. Because the expression of genes that promote apoptosis may be down-regulated during tumorigenesis, we sought to examine the expression of Par-4 in human tumors. We present here evidence that Par-4 protein levels were severely decreased in human renal cell carcinoma specimens relative to normal tubular cells. Replenishment of Par-4 protein levels in renal cell carcinoma cell lines conferred sensitivity to apoptosis. Because apoptosis may serve as a defense mechanism against malignant transformation or progression, decreased expression of Par-4 may contribute to the pathophysiology of renal cell carcinoma.

Apoptosis↗

PubMed1999

Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels.

DNA sequencing separations have been performed in microfabricated electrophoresis channels with the goal of determining whether high-quality sequencing is feasible with these microdevices. The separation matrix, separation temperature, channel length and depth, injector size, and injection parameters were optimized. DNA fragment sizing separations demonstrated that 50-micron-deep channels provide the best sensitivity for our detection configuration. One-color sequencing separations of single-stranded M13mp18 DNA on 3% linear polyacrylamide (LPA) were used to optimize the twin-T injector size, injection conditions, and temperature. The best one-color separations were observed with a 250-micron twin-T injector, an injection time of 60 s, and a temperature of 35 degrees C. The first 500 bases appeared in 9.2 min with a resolution of > 0.5, and the separation extended to 700 bases. The best four-color sequencing separations were performed using 4% LPA, a temperature of 40 degrees C, and a 100-micron twin-T injector. These four-color runs were complete in only 20 min, could be automatically base-called using BaseFinder to over 600 bp after the primer, and were 99.4% accurate to 500 bp. These results significantly advance the quality of microchip-based electrophoretic sequencing and indicate the feasibility of performing high-speed genomic sequencing with microfabricated electrophoretic devices.

Base Sequence↗

PubMed1999

Role for p300 in stabilization of p53 in the response to DNA damage.

The nuclear p300/CBP proteins function as coactivators of gene transcription. Here, using cells deficient in p300 or CBP, we show that p300, and not CBP, is essential for ionizing radiation-induced accumulation of the p53 tumor suppressor and thereby p53-mediated growth arrest. The results demonstrate that deficiency of p300 results in increased degradation of p53. Our findings suggest that p300 contributes to the stabilization and transactivation function of p53 in the cellular response to DNA damage.

CREB-Binding Protein↗