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Z Luo

Publications and source records attributed to Z Luo.

At least 19 recordsLinked to original sources
PubMed2001

Enhancement of Fas ligand-induced inhibition of neointimal formation in rabbit femoral and iliac arteries by coexpression of p35.

Adenovirus-mediated gene transfer of Fas ligand (FasL) inhibits neointimal formation in balloon-injured rat carotid arteries. Vascular smooth muscle (VSM) cells coexpressing murine FasL and p35, a baculovirus gene that inhibits caspase activity, are not susceptible to FasL-mediated apoptosis in vitro but are capable of inducing apoptosis of VSM cells that do not express p35. We reasoned that coexpression of p35 in FasL-transduced VSM cells in vivo would promote their survival, enhance FasL-induced apoptosis of adjacent VSM cells, and thereby facilitate a greater inhibition of neointimal formation. In balloon-injured rabbit femoral arteries, either Ad2/FasL/p35 or Ad2/FasL was infused into the injured site and withdrawn 20 min later. Both vectors induced a dose-dependent reduction (p < 0.05) of the neointima-to-media ratio when assessed 14 days later. However, Ad2/FasL/p35 exhibited a significantly greater inhibition of neointimal formation than Ad2/FasL. In a more clinically relevant model of restenosis, rabbit iliac arteries were injured with an angioplasty catheter under fluoroscopic guidance. Adenoviral vectors were delivered locally to the injured site over a period of 2 min, using a porous infusion balloon catheter. Twenty-eight days after gene transfer angiographic and histologic assessments indicated a significant (p < 0.05) inhibition of iliac artery lumen stenosis and neointimal formation by Ad2/FasL/p35 (5 x 10(11) particles per artery). The extent of inhibition was comparable to that achieved with Ad2/TK, an adenoviral vector encoding thymidine kinase (5 x 10(11) particles per artery) and coadministration of ganciclovir for 7 days. These data suggest that coexpression of p35 in FasL-transduced VSM cells is more potent at inhibiting neointimal formation and as such represents an improved gene therapy approach for restenosis.

Adenoviruses, Human↗
PubMed2001

Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies.

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.

Aspartic Acid↗
PubMed2001

Ultraviolet radiation alters the phosphorylation of RNA polymerase II large subunit and accelerates its proteasome-dependent degradation.

It has been shown that ultraviolet (UV) radiation induces the ubiquitination of the large subunit of RNA polymerase II (RNAP II-LS) as well as its proteasomal degradation. Studies in mammalian cells have indicated that highly phosphorylated forms of RNAP II-LS are preferentially ubiquitinated, but studies in Saccharomyces cerevisiae have provided evidence that unphosphorylated RNAP II-LS is an equally suitable substrate. In the present study, an antibody (ARNA-3) that recognizes all forms of RNAP II-LS, regardless of the phosphorylation status of its C-terminal domain (CTD), was utilized to evaluate the degradation of total cellular RNAP II-LS in human fibroblasts under basal conditions or after UV-C (10J/m(2)) irradiation. It was found that UV radiation rapidly shifted the phosphorylation profile of RNAP II-LS from a mixture of dephosphorylated and phosphorylated forms to entirely more phosphorylated forms. This shift in phosphorylation status was not blocked by pharmacologic inhibition of either the ERK or p38 pathways, both of which have been implicated in the cellular UV response. In addition to shifting the phosphorylation profile, UV radiation led to net degradation of total RNAP II-LS. UV-induced degradation of RNAP II-LS was also greatly reduced in the presence of the transcriptional and CTD kinase inhibitor DRB. Using a panel of protease inhibitors, it was shown that the bulk of UV-induced degradation is proteasome-dependent. However, the UV-induced loss of hypophosphorylated RNAP II-LS was proteasome-independent. Lastly, UV radiation induced a similar shift to all hyperphosphorylated RNAP II-LS in Cockayne syndrome (CS) cells of complementation groups A or B (CSA or CSB) when compared to appropriate controls. The UV-induced degradation rates of RNAP II-LS were not significantly altered when comparing CSA or CSB to repair competent control cells. The implications for the cellular UV response are discussed.

Cell Line↗
PubMed2001

Fluorous Boc ((F)Boc) carbamates: new amine protecting groups for use in fluorous synthesis.

The first fluorous variants of the Boc (tert-butyloxycarbonyl) group have been prepared and tested for their suitability as nitrogen protecting groups. A group with two fluorous chains and an ethylene spacer, (RfCH2CH2)2(CH3)COC(O)-, was readily attached to a representative amine but was difficult to cleave. In contrast, groups with two fluorous chains and a propylene spacer, (RfCH2CH2CH2)2(CH3)COC(O)-, or one fluorous chain and an ethylene spacer, (RfCH2CH2)(CH3)2COC(O)-, were readily formed and cleaved. The fluorous alcohol component of the (F)Boc group can be removed by evaporation and can be recovered and reused. The utility of the new (F)Boc group (C8F17CH2CH2)(CH3)2COC(O)- was demonstrated in 16 and 96 compound library synthesis exercises. Separations can be achieved either by manual, parallel fluorous solid-phase extraction, or automated, serial fluorous chromatography. The results provide additional confirmation of the value of "light" fluorous synthesis techniques, and the new fluorous Boc groups expand the applicability of fluorous synthesis techniques to many classes of nitrogen-containing organic compounds.

Amines↗
PubMed2001

Fas ligand overexpression on allograft endothelium inhibits inflammatory cell infiltration and transplant-associated intimal hyperplasia.

Despite recent advances in immunosuppressive therapy, accelerated coronary atherosclerosis remains a major problem in the long-term survival of transplant recipients. Chronic graft vasculopathy is believed to result from recipient inflammatory responses, and it is characterized by early mononuclear cell infiltration of the transplanted vessel. Here we show that endothelial cells can be genetically modified to overexpress functional, cell-surface Fas ligand (FasL) by adenovirus-mediated gene transfer without undergoing self-destruction. In a rodent model of transplant graft vasculopathy, endothelial overexpression of FasL attenuated T cell and macrophage infiltration at 1 wk posttransplantation. These vessels also displayed reduced neointima formation at one and 2 mo posttransplantation. These results indicate that inhibition of the early inflammatory response to allografted vessels by endothelial cell-specific overexpression of FasL may have utility in the treatment of transplant arteriosclerosis.

Animals↗
PubMed2001

Microtubule integrity regulates Pak leading to Ras-independent activation of Raf-1. insights into mechanisms of Raf-1 activation.

Growth factors activate Raf-1 by engaging a complex program, which requires Ras binding, membrane recruitment, and phosphorylation of Raf-1. The present study employs the microtubule-depolymerizing drug nocodazole as an alternative approach to explore the mechanisms of Raf activation. Incubation of cells with nocodazole leads to activation of Pak1/2, kinases downstream of small GTPases Rac/Cdc42, which have been previously indicated to phosphorylate Raf-1 Ser(338). Nocodazole-induced stimulation of Raf-1 is augmented by co-expression of small GTPases Rac/Cdc42 and Pak1/2. Dominant negative mutants of these proteins block activation of Raf-1 by nocodazole, but not by epidermal growth factor (EGF). Thus, our studies define Rac/Cdc42/Pak as a module upstream of Raf-1 during its activation by microtubule disruption. Although it is Ras-independent, nocodazole-induced activation of Raf-1 appears to involve the amino-terminal regulatory region in which the integrity of the Ras binding domain is required. Surprisingly, the Raf zinc finger mutation (C165S/C168S) causes a robust activation of Raf-1 by nocodazole, whereas it diminishes Ras-dependent activation of Raf-1. We also show that mutation of residues Ser(338) to Ala or Tyr(340)-Tyr(341) to Phe-Phe immediately amino-terminal to the catalytic domain abrogates activation of both the wild type and zinc finger mutant Raf by both EGF/4beta-12-O-tetradecanoylphorbol-13-acetate and nocodazole. Finally, an in vitro kinase assay demonstrates that the zinc finger mutant serves as a better substrate of Pak1 than the wild type Raf-1. Collectively, our results indicate that 1) the zinc finger exerts an inhibitory effect on Raf-1 activation, probably by preventing phosphorylation of (338)SSYY(341); 2) such inhibition is first overcome by an unknown factor binding in place of Ras-GTP to the amino-terminal regulatory region in response to nocodazole; and 3) EGF and nocodazole utilize different kinases to phosphorylate Ser(338), an event crucial for Raf activation.

Amino Acid Substitution↗
PubMed2001

Fluorous mixture synthesis: a fluorous-tagging strategy for the synthesis and separation of mixtures of organic compounds.

The solution-phase synthesis of organic compounds as mixtures rather than in individual pure form offers efficiency advantages that are negated by the difficulty in separating and identifying the components of the final mixture. Here, a strategy for mixture synthesis that addresses these separation and identification problems is presented. A series of organic substrates was tagged with a series of fluorous tags of increasing fluorine content. The compounds were then mixed, and multistep reactions were conducted to make enantiomers or analogs of the natural product mappicine. The resulting tagged products were then demixed by fluorous chromatography (eluting in order of increasing fluorine content) to provide the individual pure components of the mixture, which were detagged to release the final products.

Alkaloids↗
PubMed2001

[Protective effect of c-fos antisense oligonucleotides on brain damage induced by glutamate].

OBJECTIVE: To investigate the relation between glutamate neurotoxicity and c-fos gene expression. METHODS: c-fos antisense oligonucleotides (AS ODN) was injected into the right lateral ventricles of 9 SD rats to block the c-fos gene expression in brain tissue. c-fos sense oligonucleotides (S ODN) was used a control. The numbers and morphology of neurons in both cerebral cortex and hippocampal CA1 were detected by MIAS-300 image analysing instrument. c-fos gene expression in brain was observed by immunohistochemical method. The content of water and electrolytes in the brain tissue and Ca(2+) in the synapse were measured. RESULTS: The c-fos AS ODN blocked the c-fos gene expression and reduced the content of both water and sodium in brain tissue and Ca(2+) in symptosome, thus alleviating the morphological damage in neuron. S ODN did not have such effect. CONCLUSION: c-fos gene expression plays an important role in mediating the effect of glutamate neurotoxicity. Blocking the c-fos gene expression could antagonize glutamate neurotoxicity.

Animals↗
PubMed2001

Microarray applications in neuroscience.

Advances in all facets of technology from molecular biology to imaging and computational biology offer unprecedented opportunities for improving our understanding of the brain in health and disease. Oligonucleotide and cDNA microarray analysis, using a variety of "DNA chips," is a recently developed high-throughput technique that allows for tour-de-force analysis of gene expression. We review this powerful technique, developed in genetics laboratories, with reference to applications in neurologic diseases in humans and the use of animal models. The typical microarray experiment is multistaged and includes preparation or purchase of arrays, preparation of target DNA and probe, target DNA hybridization, microarray scanning, and image analysis. The power and pitfalls of this technology are discussed in the context of neuroscience paradigms. Since unprecedented amounts of data are produced from microarray experiments, bioinformatics and modeling expertise are increasingly becoming critical components of this approach.

Animals↗
PubMed2001

Effect of multiple mutations in the core promoter and pre-core/core region of hepatitis B virus genome on the response to interferon in e antigen-positive chronic hepatitis B.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) genomic mutations may be one of the factors that influence the efficacy of interferon (IFN) therapy. The aim of this study was to investigate the effects of mutations in different parts of the HBV genome on IFN therapy. METHODS: We studied the baseline clinical, biochemical, serologic and virologic parameters in 17 patients with e antigen-positive chronic hepatitis B. The DNA sequence of the X gene and pre-core/core gene in serum samples of these patients was analyzed before the initiation of IFN therapy. RESULTS: All five patients with the T1762-A1764 mutation were IFN responsive, while among the 12 remaining patients, only two responded to therapy. Among five patients with both a pre-core A1896 mutation and a mutation in the epitope aa 107-118 of the core region, four were non-responders whereas the fifth responded to therapy. In three other patients with A1896 mutations, one with simultaneous mutations in five lymphocytic epitopes did not respond to therapy; the two remaining patients with concomitant mutations in one or two epitopes were responsive. Serum HBV-DNA levels were lower and titers of antibody to hepatitis B virus core antigen-immunoglobin M (anti-HBc-IgM) were higher in the responders than in the non-responders. Hepatitis B virus genotypes B and C were found to be in all these Chinese patients. CONCLUSIONS: These results suggest that HBV genomic mutations, serum viral loads and titers of anti-HBc-IgM might be predictive of the efficacy of IFN therapy. These clinical findings should be further investigated by in vivo and in vitro experiments.

Adult↗
PubMed2001

Spatio-temporal EEG power spectral patterns during a short daytime nap.

This is an approach to investigate topographic changes in electroencephalographic (EEG) spectral power during pre- and post-nap wakefulness as well as stages 1 (S1) and 2 (S2) NREM sleep in 12 subjects. Delta- and theta-band power significantly increased in the frontal and central regions during S1 and S2 with an increase in inter- and intra-hemispheric correlations. Beta-band power significantly increased in the frontal, central and parietal regions during S2 with an increase in interhemispheric correlation. In contrast, alpha-band power significantly decreased in the parietal-occipital regions during S1 and S2 with a decrease in interhemispheric correlation. Thus, daytime nap modulated spatio-temporal patterns of EEG power spectral patterns in wide scalp regions.

Adult↗
PubMed2001

Extracellular ATP stimulates an inhibitory pathway towards growth factor-induced cRaf-1 and MEKK activation in astrocyte cultures.

ATP, acting via P2Y, G protein-coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances fibroblast growth factor-2 (FGF-2)-induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF-2 cotreatment on the main components of the extracellular-signal regulated protein kinase (ERK) cascade, cRaf-1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf-1 by FGF-2 in primary cultures of rat cortical astrocytes. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf-1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf-1 by other growth factors, epidermal growth factor and platelet-derived growth factor, as well as other MEK1 activators stimulated by FGF-2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF-2-induced cRaf-1 activation, thereby indicating specificity in the ATP-induced inhibitory cross-talk. These findings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor-induced activation of cRaf-1 and MEKKS: Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y-GPCRS:

Adenosine Triphosphate↗
PubMed2001

Regulation of flavin-containing monooxygenase 1 expression by ying yang 1 and hepatic nuclear factors 1 and 4.

The flavin-containing monooxygenases (FMOs) are important for the oxidation of a variety of environmental toxicants, natural products, and therapeutics. Consisting of six family members (FMO1-5), these enzymes exhibit distinct but broad and overlapping substrate specificity and are expressed in a highly tissue- and species-selective manner. Corresponding to previously identified regulatory domains, a YY1 binding site was identified at the major rabbit FMO1 promoter, position -8 to -2, two overlapping HNF1alpha sites, position -132 to -105, and two HNF4alpha sites, position -467 to -454 and -195 to -182. Cotransfection studies with HNF1alpha and HNF4alpha expression vectors demonstrated a major role for each of these factors in enhancing FMO1 promoter activity. In contrast, YY1 was shown by site-directed mutagenesis to be dispensable for basal promoter activity but suppressed the ability of the upstream domains to enhance transcription. Finally, comparisons between rabbit and human FMO1 demonstrated conservation of each of these regulatory elements. With the exception of the most distal HNF4alpha site, each of the orthologous human sequences also was able to compete with rabbit FMO1 cis-elements for specific protein binding. These data are consistent with these same elements being important for regulating human FMO1 developmental- and tissue-specific expression.

Animals↗
PubMed2001

Inhibition of insulin signaling and glycogen synthesis by phorbol dibutyrate in rat skeletal muscle.

Numerous studies have shown a correlation between changes in protein kinase C (PKC) distribution and/or activity and insulin resistance in skeletal muscle. To investigate which PKC isoforms might be involved and how they affect insulin action and signaling, studies were carried out in rat soleus muscle incubated with phorbol esters. Muscles preincubated for 1 h with 1 microM phorbol 12,13-dibutyrate (PDBu) showed an impaired ability of insulin to stimulate glucose incorporation into glycogen and a translocation of PKC-alpha, -betaI, -theta, and -epsilon, and probably -betaII, from the cytosol to membranes. Preincubation with 1 microM PDBu decreased activation of the insulin receptor tyrosine kinase by insulin and to an even greater extent the phosphorylation of Akt/protein kinase B and glycogen synthase kinase-3. However, it failed to diminish the activation of phosphatidylinositol 3'-kinase by insulin. Despite these changes in signaling, the stimulation by insulin of glucose transport (2-deoxyglucose uptake) and glucose incorporation into lipid and oxidation to CO2 was unaffected. The results indicate that preincubation of skeletal muscle with phorbol ester leads to a translocation of multiple conventional and novel PKC isoforms and to an impairment of several, but not all, events in the insulin-signaling cascade. They also demonstrate that these changes are associated with an inhibition of insulin-stimulated glycogen synthesis but that, at the concentration of PDBu used here, glucose transport, its incorporation into lipid, and its oxidation to CO2 are unaffected.

Animals↗
PubMed2001

Ras activation of the Raf kinase: tyrosine kinase recruitment of the MAP kinase cascade.

A continuing focus of our work has been an effort to understand the signal transduction pathways through which insulin achieves its cellular actions. In the mid-1970s, we and others observed that insulin promoted an increase in Ser/Thr phosphorylation of a subset of cellular proteins. This finding was unanticipated, inasmuch as nearly all of the actions of insulin then known appeared to result from protein dephosphorylation. In fact, nearly 15 years elapsed before any physiologic response to insulin attributable to stimulated (Ser/Thr) phosphorylation was established. Nevertheless, based on the hypothesis that insulin-stimulated Ser/Thr phosphorylation reflected the activation of protein (Ser/Thr) kinases downstream of the insulin receptor, we sought to detect and purify these putative, insulin-responsive protein (Ser/Thr) kinases. Our effort was based on the presumption that an understanding of the mechanism for their activation would provide an entry into the biochemical reactions through which the insulin receptor activated its downstream effectors. To a degree that, in retrospect, is surprising, this goal was accomplished, much in the way originally envisioned. It is now well known that receptor tyrosine kinases (RTKs) recruit a large network of protein (Ser/Thr) kinases to execute their cellular programs. The first of these insulin-activated protein kinase networks to be fully elucidated was the Ras-Raf-mitogen-activated protein kinase (MAPK) cascade. This pathway is a central effector of cellular differentiation in development; moreover, its inappropriate and continuous activation provides a potent promitogenic force and is a very common occurrence in human cancers. Conversely, this pathway contributes minimally, if at all, to insulin's program of metabolic regulation. Nevertheless, the importance of the Ras-MAPK pathway in metazoan biology and human malignancies has impelled us to an ongoing analysis of the functions and regulation of Ras and Raf. This chapter will summarize briefly the way in which work from this and other laboratories on insulin signaling led to the discovery of the mammalian MAP kinase cascade and, in turn, to the identification of unique role of the Raf kinases in RTK activation of this protein (Ser/Thr) kinase cascade. We will then review in more detail current understanding of the biochemical mechanism through which the Ras proto-oncogene, in collaboration with the 14-3-3 protein and other protein kinases, initiates activation of the Raf kinase.

14-3-3 Proteins↗
PubMed2001

Molecular regulation of genes encoding xenobiotic-metabolizing enzymes: mechanisms involving endogenous factors.

It is widely recognized that xenobiotic-metabolizing enzymes play a fundamental role in the basic processes of carcinogenesis and toxicity on one hand, and chemoprevention and drug efficacy on the other. Realization that different factors can profoundly affect the expression of these enzymes at the genome level has resulted in an enhanced appreciation of the importance these genes play in our modern industrialized age. There continues to be rapid proliferation of studies addressing the molecular regulation of these genes. The discovery of common signal transduction pathways and transcription factors that dictate tissue and developmental-specific expression, as well as variation in expression within a given tissue, suggest that there may be significant interaction among these various regulatory systems. This report is a summary of a symposium that was part of the Structure, Function and Regulation of Cytochromes P450 and Xenobiotic Metabolizing Enzymes satellite meeting of the 2000 joint meeting of the American Society for Biochemistry and Molecular Biology, the American Society for Pharmacology and Experimental Therapeutics, the French Pharmacological Society, and the Pharmacological Society of Canada held in Boston, Massachusetts. This symposium brought together several speakers who addressed specific receptor-mediated signal transduction pathways involved in the regulation of xenobiotic-metabolizing enzymes, as well as other molecular mechanisms whereby endogenous factors are involved in controlling tissue- and developmental-specific expression.

Enzymes↗
PubMed2001

[Significance of detection of transfusion transmitted virus in extrahepatic tissues].

OBJECTIVE: To investigate the location and distribution of transfusion transmitted virus (TTV) in the liver and extrahepatic tissues. METHODS: TTV DNA was detected in paraffin-embedded autopsy liver and extrahepatic tissues, including liver, pancreas, kidney, spleen, testicle, and heart from 13 patients with non A-G hepatitis by nested-PCR and in situ hybridization (ISH) techniques. RESULTS: Positive hybridization signals of TTV nucleus acid were observed in 5 liver, 3 kidney, 2 pancreas and spleen tissues, respectively. TTV DNA was detected in one from each two samples of testicle and heart. Positive signal was mainly located in nucleoli of the liver and extrahepatic parenchymal cells. There was not obvious pathological damage in those extrahepatic tissues. The positive rate of PCR approximately corresponded to ISH. CONCLUSIONS: TTV can infect hepatic and extrahepatic tissues and its infection in extrahepatic tissues might be responsible for a state of reinfection and higher prevalence in different population.

Adult↗