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Biomedical subjects

L Wang

Publications and source records attributed to L Wang.

At least 685 records · Page 38Linked to original sources
PubMed2000

The MITE family heartbreaker (Hbr): molecular markers in maize.

Transposable elements are ubiquitous in plant genomes, where they frequently comprise the majority of genomic DNA. The maize genome, which is believed to be structurally representative of large plant genomes, contains single genes or small gene islands interspersed with much longer blocks of retrotransposons. Given this organization, it would be desirable to identify molecular markers preferentially located in genic regions. In this report, the features of a newly described family of miniature inverted repeat transposable elements (MITEs) (called Heartbreaker), including high copy number and polymorphism, stability, and preference for genic regions, have been exploited in the development of a class of molecular markers for maize. To this end, a modification of the AFLP procedure called transposon display was used to generate and display hundreds of genomic fragments anchored in Hbr elements. An average of 52 markers were amplified for each primer combination tested. In all, 213 polymorphic fragments were reliably scored and mapped in 100 recombinant inbred lines derived from a cross between the maize inbreds B73 x Mo17. In this mapping population, Hbr markers are distributed evenly across the 10 maize chromosomes. This procedure should be of general use in the development of markers for other MITE families in maize and in other plant and animal species where MITEs have been identified.

Base Sequence↗
PubMed2000

Insulin-like growth factor I retards apoptotic signaling induced by ethanol in cardiomyocytes.

Apoptosis of cardiac muscle cells may contribute to the development of cardiomyopathy and heart failure. Alcohol (ethanol) abuse is a major cause of cardiomyopathy, but its underlying mechanism remains unknown. To determine whether ethanol causes apoptosis in cardiac muscle and whether insulin-like growth factor I (IGF 1) improves cardiac muscle survival upon ethanol exposure, we have defined the effects of ethanol and IGF I in primary cardiomyocytes. Ethanol decreased cell viability in dose-response manner from 0.2% to 1%. In contrast, ethanol (0.2-1%) did not alter viability of cardiac fibroblasts. To assess the occurrence of apoptosis, DNA fragmentation was determined with quantitation of nucleosomes. Nucleosomes were increased in ethanol-treated cells, thus confirming the apoptotic effects of ethanol. The pro-apoptotic Bax protein and Caspase 3 are important proteins of apoptotic signaling. The content of Bax and the activities of Caspase 3 were increased upon ethanol exposure. IGF I partially suppressed Bax induction, Caspase 3 activation, DNA fragmentation, and increased cardiomyocyte survival. The effects of IGF I on ethanol-induced apoptosis can be inhibited with a chemical inhibitor of PI 3 Kinase (LY-294002), suggesting that anti-apoptotic actions of IGF I involves PI 3 Kinase. These results may have important implications on further understanding the pathogenesis of alcoholic heart disease and the development of new strategies to treat alcoholic cardiomyopathy.

Animals↗
PubMed2000

Synergistic interaction between CCK and leptin to regulate food intake.

Leptin administered (either intracerebroventricularly, icv, or intraperitoneally, ip) acts in synergy with CCK to suppress food intake and body weight in lean mice or rats. The potentiating effect induced by the co-injection of ip CCK and leptin to inhibit food consumption in mice is mediated by the CCK-A receptor and capsaicin sensitive afferents. In vitro, studies in rats showed that a subset of gastric vagal afferent fibers responded to leptin injected directly into the gastric artery only after a prior intra-arterial CCK injection. Moreover, the tonic activity of gastric-related neurons in the nucleus tractus solitarius (NTS) increased when leptin was delivered into the gastric chamber of an in vitro stomach-brainstem preparation. CCK co-injected with leptin potentiated Fos expression selectively in the area postrema, NTS and paraventricular nucleus of the hypothalamus (PVN), which points to the PVN as part of the afferent and efferent limbs of the circuitry involved in the synergistic interaction between leptin and CCK. The dampening of CCK or leptin inhibitory action on ingestive behavior when either factor is not present or their receptors are non functional supports the notion that such leptin-CCK interaction may have a physiological relevance. These observations provide a mean through which leptin and CCK integrate short- and mid-term meal-related input signals into long-term control of energy balance.

Animals↗
PubMed2000

Membrane binding of peptides containing both basic and aromatic residues. Experimental studies with peptides corresponding to the scaffolding region of caveolin and the effector region of MARCKS.

We have studied the binding of peptides containing both basic and aromatic residues to phospholipid vesicles. The peptides caveolin(92-101) and MARCKS(151-175) both contain five aromatic residues, but have 3 and 13 positive charges, respectively. Our results show the aromatic residues insert into the bilayer and anchor the peptides weakly to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC). Incorporation of a monovalent acidic lipid (e.g., phosphatidylserine, PS) into the vesicles enhances the binding of both peptides via nonspecific electrostatic interactions. As predicted from application of the Poisson-Boltzmann equation to atomic models of the peptide and membranes, the enhancement is larger (e.g., 10(4)- vs 10-fold for 17% PS) for the more basic MARCKS(151-175). Replacing the five Phe with five Ala residues in MARCKS(151-175) decreases the binding to 10:1 PC/PS vesicles only slightly (6-fold). This result is also consistent with the predictions of our theoretical model: the loss of the attractive hydrophobic energy is partially compensated by a decrease in the repulsive Born/desolvation energy as the peptide moves away from the membrane surface. Incorporating multivalent phosphatidylinositol 4, 5-bisphosphate (PIP(2)) into PC vesicles produces dramatically different effects on the membrane binding of the two peptides: 1% PIP(2) enhances caveolin(92-101) binding only 3-fold, but increases MARCKS(151-175) binding 10(4)-fold. The strong interaction between the effector region of MARCKS and PIP(2) has interesting implications for the cellular function of MARCKS.

Amino Acids, Cyclic↗
PubMed2000

A HER2/NEU-derived peptide, a K(d)-restricted murine tumor rejection antigen, induces HER2-specific HLA-A2402-restricted CD8(+) cytotoxic T lymphocytes.

We have identified an H-2K(d)-binding peptide, HER2p780 (PYVSRLLGI), derived from murine HER2/neu (HER2), that can induce HER2-specific murine cytotoxic T lymphocytes (CTL). Weekly vaccination of BALB/c mice by syngeneic dendritic cells pulsed with HER2p780 peptide, entirely common to murine and human HER2, suppressed growth of pretransplanted HER2-expressing syngeneic tumors. A HER2-expressing human cancer cell line SKOV3 transfected with murine H-2K(d) cDNA could also be lysed by HER2p780-specific murine CTLs, indicating that human HER2-expressing cancer cells can process and present the cognate peptide in the context of H-2K(d). Since H-2K(d) and HLA-A2402 molecules have similar anchor motifs, the possibility of inducing HER2-specific CTL activity with HER2p780 in HLA-A2402 individuals was examined. CD8(+) CTL clones specific for HER2-expressing cancer cell lines were established from peripheral blood lymphocytes with HLA-A2402 by repeatedly sensitizing with peptide-pulsed autologous dendritic cells as well as peripheral blood mononuclear cells. Detailed analysis of their specificity revealed that the cytotoxicity of CTL clones is specific for the cognate peptide with HLA-A2402 restriction. The results suggest that HER2p780 is a unique peptide that may function as a tumor rejection antigen peptide in HLA-A2402 individuals, as it was directly proven here to function in a murine tumor system.

Animals↗
PubMed2000

The docking of primed vacuoles can be reversibly arrested by excess Sec17p (alpha-SNAP).

Homotypic vacuole fusion occurs in ordered stages of priming, docking, and fusion. Priming, which prepares vacuoles for productive association, requires Sec17p (the yeast homolog of alpha-SNAP), Sec18p (the yeast NSF, an ATP-driven chaperone), and ATP. Sec17p is initially an integral part of the cis-SNARE complex together with vacuolar SNARE proteins and Sec18p (NSF). Previous studies have shown that Sec17p is rapidly released from the vacuole membrane during priming as the cis-SNARE complex is disassembled, but the order and causal relationship of these subreactions has not been known. We now report that the addition of excess recombinant his(6)-Sec17p to primed vacuoles can block subsequent docking. This inhibition is reversible by Sec18p, but the reaction cannot proceed to the tethering and trans-SNARE pairing steps of docking while the Sec17p block is in place. Once docking has occurred, excess Sec17p does not inhibit membrane fusion per se. Incubation of cells with thermosensitive Sec17-1p at nonpermissive temperature causes SNARE complex disassembly. These data suggest that Sec17p can stabilize vacuolar cis-SNARE complexes and that the release of Sec17p by Sec18p and ATP allows disassembly of this complex and activates its components for docking.

Carrier Proteins↗
PubMed2000

BRCA1 is associated with a human SWI/SNF-related complex: linking chromatin remodeling to breast cancer.

Germline mutations in the tumor suppressor gene, BRCA1, predispose individuals to breast and ovarian cancers. Using a combination of affinity- and conventional chromatographic techniques, we have isolated a predominant form of a multiprotein BRCA1-containing complex from human cells displaying chromatin-remodeling activity. Mass spectrometric sequencing of components of this complex indicated that BRCA1 is associated with a SWI/SNF-related complex. We show that BRCA1 can directly interact with the BRG1 subunit of the SWI/SNF complex. Moreover, p53-mediated stimulation of transcription by BRCA1 was completely abrogated by either a dominant-negative mutant of BRG1 or the cancer-causing deletion in exon 11 of BRCA1. These findings reveal a direct function for BRCA1 in transcriptional control through modulation of chromatin structure.

BRCA1 Protein↗
PubMed2000

Mice deficient in the polysialyltransferase ST8SiaIV/PST-1 allow discrimination of the roles of neural cell adhesion molecule protein and polysialic acid in neural development and synaptic plasticity.

Functional properties of the neural cell adhesion molecule (NCAM) are strongly influenced by polysialylation. We used gene-targeting to generate mice lacking ST8SiaIV/PST-1, one of the polysialyltransferases responsible for addition of polysialic acid (PSA) to NCAM. Mice homozygous for the null mutation reveal normal development of gross anatomical features. In contrast to NCAM-deficient mice, olfactory precursor cells in the rostral migratory stream express PSA and follow their normal pathway. Furthermore, delamination of mossy fibers in the hippocampal CA3 region, as found in NCAM-deficient mice, does not occur in ST8SiaIV mutants. However, during postnatal development these animals show a decrease of PSA in most brain regions compared to wild-type animals. Loss of PSA in the presence of NCAM protein but in the absence of obvious histological changes allowed us to directly address the role of PSA in synaptic plasticity. Schaffer collateral-CA1 synapses, which express PSA in wild types, showed impaired long-term potentiation (LTP) and long-term depression (LTD) in adult mutants. This impairment was age-dependent, following the time course of developmental disappearance of PSA. Contrary to NCAM mutant mice, LTP in ST8SiaIV mutants was undisturbed at mossy fiber-CA3 synapses, which do not express PSA in wild-type mice. The results demonstrate an essential role for ST8SiaIV in synaptic plasticity in hippocampal CA1 synapses, whereas PSA produced by different polysialyltransferase or polysialyltransferases at early stages of differentiation regulates migration of neural precursor cells and correct lamination of mossy fibers. We suggest that NCAM but not PSA is likely to be important for LTP in the hippocampal CA3 region.

Aging↗
PubMed2000

Epiregulin is Up-regulated in pancreatic cancer and stimulates pancreatic cancer cell growth.

Epiregulin belongs to the epidermal growth factor (EGF) family of polypeptides. Previous studies have underscored the important role of the EGF family of ligands and receptors in the pathology of pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). It is not known, however, whether epiregulin may also have a role in these diseases. Therefore, in the present study we investigated the expression and function of epiregulin in five pancreatic cancer cell lines and in PDAC and CP tissue samples. Epiregulin mRNA was present at high (MIA-PaCa-2 cells) or moderate levels (ASPC-1, CAPAN-1, and T3M4) in most cells, but was below detection levels in PANC-1 cells. All the cell lines exhibited a dose-dependent increase in growth in response to recombinant human epiregulin. Epiregulin mRNA levels were increased 2.1-fold in PDAC samples (P < 0.01) and 1.7-fold in CP samples (P < 0.01), when compared with the normal controls. There was no correlation between epiregulin mRNA levels and tumor stage or grade. By in situ hybridization, a moderate to intense epiregulin mRNA signal was present in most pancreatic cancer cells in PDAC. In contrast, only a weak (normal pancreas) to moderate (CP) signals were present in the ductal and acinar cells in CP. These findings suggest that epiregulin may contribute to the pathobiology of PDAC, and may also have a role in CP.

Adenocarcinoma↗
PubMed2000

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

Animals↗
PubMed2000

Expression of GMP-140 (P-selectin) correlates with graft viability in cold-preserved rat livers.

BACKGROUND: Ischemia/reperfusion injury is an inflammatory process involving cytokine release, Kupffer cell activation, and sinusoidal endothelial cell activation. GMP-140 is synthesized by endothelial cells. METHODS: We analyzed by Western blotting the expression of GMP-140 in a syngeneic rat liver transplantation model using grafts preserved for different periods of time. RESULTS: Compared with prereperfusion samples, expression did not change significantly in freshly harvested and 4-hr preserved livers. In grafts preserved for 24 hr (100% survival), GMP-140 levels increased dramatically at 1 hr, then returned to baseline at 24 hr after transplantation. Forty-eight hour preserved grafts (0% survival) showed a decreasing expression. To identify possible mediators, the effects of tumor necrosis factor-alpha and interleukin-1beta on GMP-140 expression in primary sinusoidal endothelial cells were analyzed. These cytokines increased both the percentage of stained cells as well as their mean staining fluorescence. CONCLUSIONS: The absence of increase in 48-hr grafts suggests that GMP-140 may distinguish viable from nonviable livers.

Animals↗
PubMed2000

Functional analysis of the OCA-B promoter.

OCA-B was identified as a B cell-specific coactivator that functions with either Oct-1 or Oct-2 to mediate efficient cell type-specific transcription via the octamer site (ATGCAAAT) both in vivo and in vitro. Mice lacking OCA-B exhibit normal Ag-independent B cell maturation. In contrast, Ag-dependent functions, including production of secondary Ig isotypes and germinal center formation, are greatly affected. To better understand OCA-B expression and, ultimately, the defects observed in the OCA-B knockout mice, we have cloned the OCA-B promoter and examined its function in both transformed and primary B cells. We show here that the OCA-B promoter is developmentally regulated, with activity increasing throughout B cell differentiation. Through physical and functional assays, we have found an activating transcription factor/cAMP response element binding protein binding site (or cAMP response element) that is crucial for OCA-B promoter activity. Furthermore, we demonstrate that IL-4 and anti-CD40 induce both the OCA-B promoter and octamer-dependent promoters, thus implicating OCA-B in B cell signaling events in the nucleus.

Activating Transcription Factor 4↗
PubMed2000

CCAAT/enhancer binding protein beta mediates the activation of the murine alpha1(I) collagen promoter by acetaldehyde.

Acetaldehyde was previously shown to activate the alpha1(I) and alpha2(I) collagen promoters and to increase collagen production in activated stellate cells. Also, CCAAT/enhancer binding protein beta (C/EBPbeta) binds and activates the mouse alpha1(I) collagen promoter. This study investigates the role of C/EBPbeta in mediating the activation of the alpha1(I) collagen promoter by acetaldehyde. Nuclear extracts isolated from cultured activated rat hepatic stellate cells formed four protein-DNA complexes on electrophoretic mobility shift assay with an oligonucleotide including the C/EBP binding site between -365 and -335 in the alpha1(I) collagen promoter. The four complexes were identified to represent C/EBPbeta binding to the oligonucleotide by supershift with C/EBPbeta antibody. The principal C/EBP isoform found in the nuclear extracts from stellate cells was C/EBPbeta, with very low amounts of C/EBPalpha detected. Acetaldehyde (200 microM) increased C/EBPbeta protein in stellate nuclear extracts, increased its binding to the promoter, and activated the alpha1(I) collagen promoter in transfected stellate cells. Mutation of the C/EBPbeta binding site markedly decreased nuclear protein binding. A transfected promoter, mutated at the C/EBP binding site, had decreased basal activity, was not activated by acetaldehyde, and was not activated when cotransfected with a C/EBPbeta expression vector. This study shows that C/EBPbeta is the predominant C/EBP isoform found in activated stellate cells and that increased C/EBPbeta protein and C/EBPbeta binding to a proximal C/EBP binding site in the promoter mediates the activating effect of acetaldehyde.

Acetaldehyde↗
PubMed2000

Modulation of HIV-1 replication by a novel RhoA effector activity.

The RhoA GTPase is involved in regulating actin cytoskeletal organization, gene expression, cell proliferation, and survival. We report here that p115-RhoGEF, a specific guanine nucleotide exchange factor (GEF) and activator of RhoA, modulates HIV-1 replication. Ectopic expression of p115-RhoGEF or Galpha13, which activates p115-RhoGEF activity, leads to inhibition of HIV-1 replication. RhoA activation is required and the inhibition affects HIV-1 gene expression. The RhoA effector activity in inhibiting HIV-1 replication is genetically separable from its activities in transformation of NIH3T3 cells, activation of serum response factor, and actin stress fiber formation. These findings reveal that the RhoA signal transduction pathway regulates HIV-1 replication and suggest that RhoA inhibits HIV-1 replication via a novel effector activity.

Amino Acid Sequence↗
PubMed2000

Increased insulin receptor substrate-1 and enhanced skeletal muscle insulin sensitivity in mice lacking CCAAT/enhancer-binding protein beta.

CCAAT/enhancer-binding protein beta (C/EBPbeta) controls gene transcription and metabolic processes in a variety of insulin-sensitive tissues; however, its role in regulating insulin responsiveness in vivo has not been investigated. We performed hyperinsulinemic-euglycemic clamps in awake, non-stressed, chronically catheterized adult mice homozygous for a deletion in the gene for C/EBPbeta (C/EBPbeta(-/-)). Fasting plasma insulin, glucose, and free fatty acid (FFA) levels were significantly lower in C/EBPbeta(-/-) mice compared with wild-type (WT) controls. Acute hyperinsulinemia (4 h) suppressed hepatic glucose production, phosphoenolpyruvate carboxykinase mRNA, and plasma FFA to a similar extent in WT and C/EBPbeta(-/-) mice, suggesting that C/EBPbeta deletion does not alter the metabolic and gene regulatory response to insulin in liver and adipose tissue. In contrast, using submaximal (5 milliunits/kg/min) and maximal (20 milliunits/kg/min) insulin infusions, whole-body glucose disposal was 77% (p < 0.01) and 33% (p < 0.05) higher in C/EBPbeta(-/-) mice, respectively, compared with WT mice. Maximal insulin-stimulated 3-O-methylglucose uptake in isolated soleus muscle was 54% greater in C/EBPbeta(-/-) mice (p < 0.05). Furthermore, insulin-stimulated insulin receptor and Akt Ser(473) phosphorylation and phosphatidylinositol 3-kinase activity were 1.6-2.5-fold greater in skeletal muscle from C/EBPbeta(-/-) mice compared with WT mice. The level of insulin receptor substrate-1 protein was increased 2-fold in skeletal muscle from C/EBPbeta(-/-) mice. These results demonstrate that C/EBPbeta deletion decreases plasma FFA levels and increases insulin signal transduction specifically in skeletal muscle, and both contribute to increased whole-body insulin sensitivity.

Adipose Tissue↗
PubMed2000

Ornithine decarboxylase activity and its gene expression are increased in benign hyperplastic prostate.

BACKGROUND: Ornithine decarboxylase (ODC) is the first key enzyme in the polyamine biosynthesis pathway. Polyamine is believed to participate in cellular proliferation and differentiation. To study the relationship between ODC and the pathogenesis of benign prostatic hyperplasia (BPH), the polyamine levels, ODC activities, and expression of ODC mRNA in benign hyperplastic and normal human prostates were assayed. METHODS: Polyamine contents and ODC activities in tissue extracts were determined by reverse-phase high-performance liquid chromatography and spectrophotometric procedures, respectively. The ODC mRNA levels were assayed by Northern blot analysis. RESULTS: The contents of putrescine, spermidine, and spermine in BPH tissues were 2.2, 3.4, and 6.0 times higher than those in normal tissues, respectively; the ODC activity of BPH tissue was about 3.2 times higher than in normal tissue; the expression level of ODC mRNA in the BPH tissues was greater than that of normal tissues. CONCLUSIONS: The findings imply that 1) the increased ODC activity and polyamine content in prostatic tissue may correlate with the pathogenesis of BPH, and 2) the high level of ODC activity is induced by the overexpression of ODC mRNA.

Adult↗
PubMed2000

Rotavirus VP7 epitope mapping using fragments of VP7 displayed on phages.

cDNA copies of the complete porcine rotavirus CRW-8 VP7 gene were randomly digested to fragments of about 30-60 or 30-500 base pairs by DNase1 in the presence of Mn(2+). The fragments were cloned and expressed in a filamentous phage fd-tet-derived vector to create specific-gene-related peptide libraries. Polyclonal antibodies were then used to pan the SGRP libraries for antibody-binding phages. Analysis of the phage isolates revealed that the majority (86%) of them only had a single insert. However, phages displaying composite inserts containing the VP7 antigenic regions A, B, and C, originally defined by neutralising monoclonal antibody escape mutants, were also isolated. Inserts containing A or C region peptide were found to contain extra sequences from the C region, while the B region epitope was linear and had additional sequence from either upstream or downstream. In addition a dominant and possibly non-neutralising VP7 epitope was identified around amino acids 263-270. One of the recreated antigenic epitopes has also been fused to the outer membrane protein A (OmpA) of Escherichia coli and shown to maintain its antigenicity. The results in this study may have significant implication for recreation of conformational epitopes and vaccine development.

Amino Acid Sequence↗