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G Chen

Publications and source records attributed to G Chen.

At least 379 records · Page 21Linked to original sources
PubMed1999

Phenotypic differentiation without permanent cell-cycle arrest by skeletal myocytes with deregulated E2F-1.

Skeletal muscle terminal differentiation includes expression of muscle cell-specific proteins and concomitant cell-cycle arrest. These two processes require functional retinoblastoma protein (RB). E2F-1 is an RB-associated transcriptional factor and an effector of RB in the regulation of G1 to S-phase transition. Here, we show that proper regulation of E2F-1 is crucial for differentiation-coupled cell-cycle arrest by skeletal myocytes. On induction to differentiate, C2 myoblasts constitutively expressing E2F-1 synthesized muscle cell-specific proteins, fused into myotubes, and upregulated the cdk inhibitor p21. However, unlike control cells, differentiated myocytes expressing exogenous E2F-1 incorporated bromodeoxyuridine into nuclei, indicating S-phase entry. This S-phase entry was accompanied by expression of cyclin A. Our results support the view that RB regulates cell-cycle arrest and muscle cell differentiation through separable mechanisms.

Carrier Proteins↗
PubMed1999

Enhancement of glutathione S-transferase placental-form positive liver cell foci development by microcystin-LR in aflatoxin B1-initiated rats.

The objective of this study was to elucidate whether microcystin-LR (MC-LR), a hepatotoxic blue-green algal toxin in drinking water, is carcinogenic or possesses the ability to modulate aflatoxin B1 (AFB1)-induced hepatocarcinogenicity. In a medium-term liver bioassay, male Fischer 344 rats were given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg) followed by an i.p. injection of MC-LR for 6 weeks after 2 weeks of DEN treatment. To study the synergism between AFB1 and MC-LR, DEN-treated rats were given an i.p. injection of AFB1 (0.5 mg/kg) dissolved in dimethyl sulfoxide (DMSO) followed by MC-LR at 2 weeks after the treatment. In a separate experiment, the rats were first given AFB1 (0.5 mg/kg) and 2 weeks later an i.p. injection of 1 or 10 microg/kg of MC-LR twice a week for 6 weeks. Most rats were subjected to a two-thirds partial hepatectomy (PH) at week 3 and were killed under anesthesia at week 8. Liver sections were analyzed for glutathione S-transferase placental form (GST-P) expression, and subjected to histopathological examination for phenotypic alteration of hepatocellular foci. In rats that did not receive DEN, MC-LR did not cause a significant increase in the numbers of GST-P-positive foci, whereas AFB1 induced a slight increase in GST-P-positive foci development. In rats given DEN, MC-LR enhanced the expression of GST-P-positive foci, as did AFB1 but no synergism was observed. Histopathological analysis revealed that the area of eosinophilic foci, a biomarker for preneoplastic liver lesion, markedly increased because of MC-LR. In rats given AFB1 as an initiator, treatment with MC-LR resulted in a synergistic increase in the development of GST-P-positive foci. These results suggest that the hepatocarcinogenicities of MC-LR and AFB1 can be predicted in experimental animals with a medium-term bioassay. Furthermore, tumor promoting activity of MC-LR was demonstrated in rats treated with AFB1.

Aflatoxin B1↗
PubMed1999

In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site.

For the first time, each specificity determining residue (SDR) in the binding site of an antibody has been replaced with every other possible single amino acid substitution, and the resulting mutants analyzed for binding affinity and specificity. The studies were conducted on a variant of the 26-10 antidigoxin single chain Fv (scFv) using in vitro scanning saturation mutagenesis, a new process that allows the high throughput production and characterization of antibody mutants [Burks,E.A., Chen,G., Georgiou,G. and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412-417]. Single amino acid mutants of 26-10 scFv were identified that modulated specificity in dramatic fashion. The overall plasticity of the antibody binding site with respect to amino acid replacement was also evaluated, revealing that 86% of all mutants retained measurable binding activity. Finally, by analyzing the physical properties of amino acid substitutions with respect to their effect on hapten binding, conclusions were drawn regarding the functional role played by the wild-type residue at each SDR position. The reported results highlight the value of in vitro scanning saturation mutagenesis for engineering antibody binding specificity, for evaluating the plasticity of proteins, and for comprehensive structure-function studies and analysis.

Amino Acids↗
PubMed1999

Large proportion of Epstein-Barr virus-associated small noncleaved cell lymphomas among children with non-Hodgkin's lymphoma at a single institution in Moscow, Russia.

To evaluate clinical and biologic features of children with non-Hodgkin's lymphoma (NHL) treated in Moscow, Russia, we studied 53 cases treated in the Republican Children's Hospital from 1990 to 1994. Histologic examination disclosed a predominance of the small noncleaved cell subtype (32 cases, 60%); a smaller percentage of the lymphoblastic and large-cell subtypes (14 cases, 26% and 7 cases, 13%, respectively) were identified. The frequencies of primary sites of involvement in descending order included 60% in abdomen, 19% in mediastinum, 15% in head/neck, and 4% in peripheral nodes. The majority of children presented with advanced stage disease (St. Jude stage III/IV/B-ALL, 92.5%). Of interest, 8 of 15 (53%) small noncleaved cell NHL cases examined contained Epstein-Barr virus (EBV). The high frequency of EBV association in this study suggests that this virus may play a more global role in NHL pathogenesis than previously recognized.

Adolescent↗
PubMed1999

Intelligent control of a planning system for astronaut training.

This work intends to design, analyze and solve, from the systems control perspective, a complex, dynamic, and multiconstrained planning system for generating training plans for crew members of the NASA-led International Space Station. Various intelligent planning systems have been developed within the framework of artificial intelligence. These planning systems generally lack a rigorous mathematical formalism to allow a reliable and flexible methodology for their design, modeling, and performance analysis in a dynamical, time-critical, and multiconstrained environment. Formulating the planning problem in the domain of discrete-event systems under a unified framework such that it can be modeled, designed, and analyzed as a control system will provide a self-contained theory for such planning systems. This will also provide a means to certify various planning systems for operations in the dynamical and complex environments in space. The work presented here completes the design, development, and analysis of an intricate, large-scale, and representative mathematical formulation for intelligent control of a real planning system for Space Station crew training. This planning system has been tested and used at NASA-Johnson Space Center.

Algorithms↗
PubMed1999

Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7-azido-4-methylcoumarin.

A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 +/- 0.05 mM). Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.

Amino Acid Sequence↗
PubMed1999

Signalling pathways in the brain: cellular transduction of mood stabilisation in the treatment of manic-depressive illness.

The long-term treatment of manic-depressive illness (MDI) likely involves the strategic regulation of signalling pathways and gene expression in critical neuronal circuits. Accumulated evidence has identified signalling pathways, in particular the family of protein kinase C (PKC) isozymes, as targets for the long-term action of lithium. Chronic lithium administration produces a reduction in the expression of PKC alpha and epsilon, as well as a major PKC substrate, MARCKS, which has been implicated in long-term neuroplastic events in the developing and adult brain. More recently, studies have demonstrated robust effects of lithium on another kinase system, GSK-3beta, and on neuroprotective/neurotrophic proteins in the brain. Given the key roles of these signalling cascades in the amplification and integration of signals in the central nervous system, these findings have clear implications not only for research into the neurobiology of MDI, but also for the future development of novel and innovative treatment strategies.

Adult↗
PubMed1999

Interaction of Leishmania gp63 with cellular receptors for fibronectin.

The most abundant protein on the surface of the promastigote form of the protozoan parasites Leishmania spp. is a 63-kDa molecule, designated gp63 or leishmanolysin. Because gp63 has been shown to possess fibronectin-like properties, we examined the interaction of gp63 with the cellular receptors for fibronectin. We measured the direct binding of Leishmania to human macrophages or to transfected mammalian cells expressing human fibronectin receptors. Leishmania expressing gp63 exhibited modest but reproducible adhesion to human macrophages and to transfected CHO cells expressing alpha4/beta1 fibronectin receptors. In both cases, this interaction depended on gp63 but occurred independently of the SRYD sequence of gp63, because parasites expressing gp63 with a mutated SRYD sequence bound to macrophages and alpha4/beta1 receptor-expressing cells as well as did wild-type parasites. The contribution of gp63 to parasite adhesion was more pronounced when the assays were performed in the presence of complement, suggesting that the receptors for complement and fibronectin may cooperate to mediate the efficient adhesion of parasites to macrophages. The interaction of gp63 with fibronectin receptors may also play an important role in parasite internalization by macrophages. Erythrocytes to which gp63 was cross-linked were efficiently phagocytized by macrophages, whereas control erythrocytes opsonized with complement alone bound to macrophages but remained peripherally attached to the outside of the cell. Similarly, parasites expressing wild-type gp63 were rapidly and efficiently phagocytized by resting macrophages, whereas parasites lacking gp63 were internalized more slowly. This rapid internalization of gp63-expressing parasites was dependent on the beta1 integrins, because pretreatment of macrophages with monoclonal antibodies to the beta1 integrins decreased the internalization of gp63-expressing parasites. These observations indicate that complement receptors are the primary mediators of parasite adhesion; however, maximal parasite adhesion and internalization may require the participation of the beta1 integrins, which recognize fibronectin-like molecules such as gp63 on the surface of the parasite.

Animals↗
PubMed1999

Nitric oxide is the predominant mediator of cerebellar hyperemia during somatosensory activation in rats.

Crus II is an area of the cerebellar cortex that receives trigeminal afferents from the perioral region. We investigated the mechanisms of functional hyperemia in cerebellum using activation of crus II by somatosensory stimuli as a model. In particular, we sought to determine whether stimulation of the perioral region increases cerebellar blood flow (BFcrb) in crus II and, if so, whether the response depends on activation of 2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-kainate receptors and nitric oxide (NO) production. Crus II was exposed in anesthetized rats, and the site was superfused with Ringer. Field potentials were recorded, and BFcrb was measured by laser-Doppler flowmetry. Crus II was activated by electrical stimulation of the perioral region (upper lip). Perioral stimulation evoked the characteristic field potentials in crus II and increased BFcrb (34 +/- 6%; 10 Hz-25 V; n = 6) without changing arterial pressure. The BFcrb increases were associated with a local increase in glucose utilization (74 +/- 8%; P < 0.05; n = 5) and were attenuated by the AMPA-kainate receptor antagonist 2, 3-dihydroxy-6-nitro-7-sulfamoylbenzo-[f]quinoxaline (-71 +/- 3%; 100 microM; P < 0.01; n = 5). The neuronal NO synthase inhibitor 7-nitroindazole (7-NI, 50 mg/kg; n = 5) virtually abolished the increases in BFcrb (-90 +/- 2%; P < 0.01) but did not affect the amplitude of the field potentials. In contrast, 7-NI attenuated the increase in neocortical cerebral blood flow produced by perioral stimulation by 52 +/- 6% (P < 0.05; n = 5). We conclude that crus II activation by somatosensory stimuli produces localized increases in local neural activity and BFcrb that are mediated by activation of glutamate receptors and NO. Unlike in neocortex, in cerebellum the vasodilation depends almost exclusively on NO. The findings underscore the unique role of NO in the mechanisms of synaptic function and blood flow regulation in cerebellum.

Animals↗
PubMed1999

Novel form of spreading acidification and depression in the cerebellar cortex demonstrated by neutral red optical imaging.

A novel form of spreading acidification and depression in the rat cerebellar cortex was imaged in vivo using the pH-sensitive dye, Neutral red. Surface stimulation evoked an initial beam of increased fluorescence (i.e., decreased pH) that spread rostrally and caudally across the folium and into neighboring folia. A transient but marked suppression in the excitability of the parallel fiber-Purkinje cell circuitry accompanied the spread. Characteristics differentiating this phenomenon from the spreading depression of Leao include: high speed of propagation on the surface (average of 450 microm/s), stable extracellular DC potential, no change in blood vessel diameter, and repeatability at short intervals. This propagating acidification constitutes a previously unknown class of neuronal processing in the cerebellar cortex.

Acids↗
PubMed1999

Mechanisms by which blood levels of interleukin-6 (IL-6) are elevated after intracerebroventricular injection of IL-1beta in the rat: neural versus humoral control.

Intracerebroventricular (icv) injection of interleukin-1beta (IL-1beta) in rats induces elevated IL-6 levels in peripheral blood, exceeding those induced by iv or ip injection. Two hypotheses postulated to explain this phenomenon were tested. Mediation by peripheral sympathetic activation was excluded by showing that agents that blocked preganglionic cholinergic synapses (chlorisondamine), beta-adrenergic receptors (propanalol, butoxamine), and alpha-adrenergic receptors (phentolamine) did not prevent the IL-6 response. That the peripheral response was due to passage of the injected IL-1beta into blood from the brain was supported by several observations. Immunoreactive IL-1beta appeared in peripheral blood by 10 min after icv injection and remained constant between 10-100 min after injection; values after icv injection were virtually identical to those after iv injection at 60 and 80 min. Radioiodine-labeled IL-1beta appeared in blood as early as 5 min, and by phamacokinetic analysis was found to be transferred from the brain at a rate greater than 2% of brain content per min(-1). IL-1beta infused iv in a pattern mimicking brain to blood transfer induced IL-6 levels that were more than double the values induced by a single bolus injection and were not significantly different from the values observed after icv injection. Sustained levels of IL-1beta in blood over time contribute to the high peripheral IL-6 response. This was shown by administering the same total dose iv as a single bolus of 100 ng or in two doses of 50 ng 1 h apart. Rats given a divided dose had 6-10 times higher blood IL-6 levels at 2 h than those given a single injection. The high levels of IL-6 in blood after icv injection of IL-1beta are best explained by the reservoir function of the brain IL-1beta pool and the self-priming effect of IL-1beta in peripheral tissues.

Adrenergic alpha-Antagonists↗
PubMed1999

Modulation of CNS signal transduction pathways and gene expression by mood-stabilizing agents: therapeutic implications.

In an attempt to find the key to reducing the excessive morbidity and mortality seen with mood disorders, our laboratory has been extensively investigating lithium's mechanisms of action in an integrated series of clinical and preclinical studies. We have found that the chronic administration of the 2 structurally highly dissimilar agents, lithium and valproate, brings about a strikingly similar reduction in protein kinase C (PKC) alpha and epsilon isozymes in rat frontal cortex and hippocampus. In view of PKC's critical role in regulating neuronal excitability and neurotransmitter release, we have postulated that PKC inhibition may have antimanic efficacy. In a small study, we have found that tamoxifen (which, in addition to its estrogen receptor blockade, is also a PKC inhibitor) has marked antimanic efficacy. These exciting preliminary results suggest that PKC inhibitors may represent a novel class of improved therapeutic agents for bipolar disorder, and this is under further investigation. The beneficial effects of mood stabilizers require a lag period for onset of action and are generally not immediately reversed upon drug discontinuation; such patterns of effects suggest alterations at the genomic level. We have therefore undertaken a series of studies to investigate the effects of these agents on the AP-1 family of transcription factors and have found that both drugs increase AP-1 DNA binding activity in areas of rodent brain ex vivo and in human neuronal cells in culture. Both treatments also increase the expression of a reporter gene driven by an AP-1-containing promoter, and mutations in the AP-1 sites of the reporter gene promoter markedly attenuate these effects. Both treatments also increase the expression of several endogenous proteins, whose genes are known to be regulated by AP-1. Although the precise mechanisms have not been fully elucidated, preliminary results suggest that these effects may be mediated, in part, by mitogen-activating protein kinases and glycogen synthase kinase 3beta. We have also utilized mRNA reverse transcription-polymerase chain reaction (RT-PCR) differential display to identify concordant changes in gene expression induced by the chronic administration of both lithium and valproate. We have identified concordant changes in a number of cDNA bands by both lithium and valproate. Cloning and characterizing of these genes is currently underway. The identification of the functions of these genes offers the potential not only for improved therapeutics for reducing the morbidity and mortality associated with mood disorders, but may also provide important clues about the underlying pathophysiology.

Bipolar Disorder↗
PubMed1999

[Polymorphisms of five short tandem repeat systems in Chinese Han population in Chengdu].

OBJECTIVE: To get preliminary genotype and allele frequency distributions of D6S366, D6S477, D12S375, D12S391 and PLA2A loci in Chinese Han population in Chengdu area and to validate more short Tandem repeat (STR) systems for forensic application. METHODS: The polymerase chain reaction (PCR) was used. Five STR systems (D6S366, D6S477, D12S375, D12S391 and PLA2A) were amplified on DNA samples, which were extracted from 108 unrelated individuals EDTA-blood by Phenol/Chloroform method. The PCR products were analyzed by PAG vertical electrophoresis. RESULTS: Seven alleles were found at D6S366 locus, nine alleles at D6S477 locus, five alleles at D12S375 locus, nine alleles at D12S391 locus and seven alleles at PLA2A locus. No deviations from Hardy-Weinberg equilibrium were observed. The heterozygosities observed were 0.69, 0.78, 0.81, 0.68 and 0.79 for D6S366, D6S477, D12S375, D12S391 and PLA2A respectively. The chances of exclusion were 0.3621, 0.6004, 0.4581, 0.7014 and 0.5589 and the discriminating powers were 0.79, 0.93, 0.86, 0.96 and 0.91. CONCLUSION: All of the five loci in this study were useful markers for individual identification and paternity test and for genetics purposes.

Alleles↗
PubMed1999

[The mutation of deletion for glutathione S-transferase M1 gene in the tissue of hepatocellular carcinoma].

OBJECTIVE: To Study whether the mutation of deletion for glutathione S-transferase M1 (GSTM1) gene occurred during the development of hepatocellular carcinoma (HCC). METHODS: The genotypes of GSTM1 of 46 pairs of HCC tissue and the noncancerous liver tissue were detected by polymerase chain reaction (PCR). RESULTS: The frequency of GSTM1 null genotype for HCC tissue was 78.26%, but 65.22% for the noncancerous liver tissue (P<0.05). The GSTM1 null genotypes of 6 HCC tissue were transformed from the non- null genotypes of the noncancerous liver tissue. According to Hardy-Weinberg law., the rate of deletion mutation for GSTM1 gene was inferred to be 38.89%. CONCLUSION: The results suggested that the mutation of deletion for GSTM1 gene had occurred during the development of HCC.

Carcinoma, Hepatocellular↗
PubMed1999

A high dose of ionizing radiation induces tissue-specific activation of nuclear factor-kappaB in vivo.

Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) is one of the important responses of cells to an external stress such as ionizing radiation. We studied radiation-induced NF-kappaB activation in vivo in male BALB/c mice. After the mice were exposed to 8.5 Gy total-body gamma irradiation, the spleen, mesenteric lymph nodes, thymus, liver, lung, colon, brain and bone marrow were harvested 1, 2.5, 5, 10 and 20 h postirradiation. NF-kappaB DNA-binding activity was analyzed in the nuclear protein extracts by a gel shift assay. When compared to the levels in untreated control mice, radiation induced activation of NF-kappaB in spleen, mesenteric lymph nodes and bone marrow but not in the other tissues examined. In contrast, an i.p. injection of a lethal dose (3 mg/kg) of lipopolysaccharide also increased activity of NF-kappaB in the liver and lung. The gel supershift assay with Nfkb1, Rela and/or Rel antibodies revealed that the specific molecular forms of NF-kappaB activated by radiation in the spleen were Nfkb1 homodimers and Nfkb1/Rela heterodimers. In mesenteric lymph nodes, the heterodimerized Rel/Rela NF-kappaB was also activated. In bone marrow, an NF-kappaB-like binding factor was induced that may be Nfkb1/Rela- and Rel/Rela-like heterodimers, but it exhibited a higher mobility than Nfkb1 homodimers. These results indicate that in vivo, ionizing radiation induces NF-kappaB activation that varies in both tissue distribution and moleoular composition.

Animals↗
PubMed1999

A transgenic animal model of osmotic cataract. Part 1: over-expression of bovine Na+/myo-inositol cotransporter in lens fibers.

PURPOSE: Intracellular osmotic stress is believed to be linked to the advancement of diabetic cataract. Although the accumulation of organic osmolytes (myo-inositol, sorbitol, taurine) is thought to protect the lens by maintaining osmotic homeostasis, the physiologic implication of osmotic imbalance (i.e., hyperosmotic stress caused by intracellular over-accumulation of organic osmolytes) on diabetic cataract formation is not clearly understood. Studies from this laboratory have identified several osmotic compensatory mechanisms thought to afford the lens epithelium, but not the lens fibers, protection from water stress during intervals of osmotic crisis. This model is founded on the supposition that the fibers of the lens are comparatively more susceptible to damage by osmotic insult than is the lens epithelium. To test this premise, several transgenic mouse lines were developed that over-express the bovine sodium/myo-inositol cotransporter (bSMIT) gene in lens fiber cells. METHODS: Of the several transgenic mouse lines generated, two, MLR14 and MLR21, were analyzed in detail. Transgenic mRNA expression was analyzed in adult and embryonic transgenic mice by a coupled reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization on embryonic tissue sections, respectively. Intralenticular myo-inositol content from individual mouse lenses was quantified by anion exchange chromatography and pulsed electrochemical detection. Ocular histology of embryonic day 15.5 (E15.5) embryos from both transgenic (TG) families was analyzed and compared to their respective nontransgenic (NTG) littermates. RESULTS: Both RT-PCR and in situ hybridization determined that transgene expression was higher in line MLR21 than in line MLR14. Consistent with this, intralenticular myo-inositol from MLR21 TG mice was markedly higher compared with NTG littermates or MLR14 TG mice. Histologic analysis of E15.5 MLR21 TG embryos disclosed a marked swelling in the differentiating fibers of the bow region and subcapsular fibers of the central zone, whereas the lens epithelium appeared morphologically normal. The lenticular changes, initiated early during lens development in TG MLR21 embryos, result in severe bilateral nuclear cataracts readily observable in neonates under normal rearing and dietary conditions. In contrast, TG MLR14 pups reared under standard conditions produced no lens opacity. CONCLUSIONS: Lens fiber swelling and related cataractous outgrowth positively correlated to the degree of lens bSMIT gene expression and intralenticular myo-inositol content. The affected (i.e., swollen) lens fibers appeared to be unable to cope with the water stress generated by the transgene-induced over-accumulation of myo-inositol and, as a result of this inability to osmoregulate, suffered osmotic damage due to water influx.

Animals↗
PubMed1999

[Gene transfer efficiency enhancement by inactivated Coxsakie virus B1 in primary skeletal muscle cells].

OBJECTIVE: To study the effect of inactivated Coxsackie virus B1(CVB1), which was considered to have high affinity to skeletal muscle cell, on gene transfer efficiency of reporter gene, pCDNA3-LacZ, in primary muscle cells. METHODS: Viruses were purified by polyethylene sedimentation followed by sucrose supercentrifugation. Virus, transferrin-polylysine(TfpL) and LacZ were designed as different concentrations and groups, then positive cells contained beta-galactosidase were calculated as gene transfer efficiency. RESULTS: Inactivated CVB1 made an enhancement of gene transfer efficiency from less than 1% in TfpL+LacZ group to 15-20 per cent in CVB1+TfpL+LacZ group. CONCLUSION: Like adenovirus, inactivated CVB1 can also increase gene transfer efficiency in muscle cells, which provides experimental basis for constructing new vector targeting to muscle cells.

Cells, Cultured↗