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Biomedical subjects

H Chen

Publications and source records attributed to H Chen.

At least 577 records · Page 32Linked to original sources
PubMed1999

Development of New Chiral P,N Ligands and Their Application in the Cu-Catalyzed Enantioselective Conjugate Addition of Diethylzinc to Enones.

High enantioselectivity (up to 98 % ee) has been achieved for the conjugate addition of diethylzinc to acyclic enones utilizing Cu(I) complexes of the novel chiral P,N ligands 1. The high enantioselectivities are best achieved using [Cu(OTf)](2) small middle dotC(6)H(6) as the copper catalyst precursor in nonpolar solvents such as toluene or Cl(CH(2))(2)Cl. R=H, CH(3); Tf=F(3)CSO(2).

Journal Article↗
PubMed1999

Brain insulin receptors and spatial memory. Correlated changes in gene expression, tyrosine phosphorylation, and signaling molecules in the hippocampus of water maze trained rats.

Evidence accumulated from clinical and basic research has indirectly implicated the insulin receptor (IR) in brain cognitive functions, including learning and memory (Wickelgren, I. (1998) Science 280, 517-519). The present study investigates correlative changes in IR expression, phosphorylation, and associated signaling molecules in the rat hippocampus following water maze training. Although the distribution of IR protein matched that of IR mRNA in most forebrain regions, a dissociation of the IR mRNA and protein expression patterns was found in the cerebellar cortex. After training, IR mRNA in the CA1 and dentate gyrus of the hippocampus was up-regulated, and there was increased accumulation of IR protein in the hippocampal crude synaptic membrane fraction. In the CA1 pyramidal neurons, changes in the distribution pattern of IR in particular cellular compartments, such as the nucleus and dendritic regions, was observed only in trained animals. Although IR showed a low level of in vivo tyrosine phosphorylation, an insulin-stimulated increase of in vitro Tyr phosphorylation of IR was detected in trained animals, suggesting that learning may induce IR functional changes, such as enhanced receptor sensitivity. Furthermore, a training-induced co-immunoprecipitation of IR with Shc-66 was detected, along with changes in in vivo Tyr phosphorylation of Shc and mitogen-activated protein kinase, as well as accumulation of Shc-66, Shc-52, and Grb-2 in hippocampal synaptic membrane fractions following training. These findings suggest that IR may participate in memory processing through activation of its receptor Tyr kinase activity, and they suggest possible engagement of Shc/Grb-2/Ras/mitogen-activated protein kinase cascades.

3T3 Cells↗
PubMed1999

The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module.

Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. Epsin 1 and 2 are most similar in their NH(2)-terminal region, which represents a module (epsin NH(2) terminal homology domain, ENTH domain) found in a variety of other proteins of the data base. The multiple DPW motifs, typical of the central region of epsin 1, are only partially conserved in epsin 2. Both proteins, however, interact through this central region with the clathrin adaptor AP-2. In addition, we show here that both epsin 1 and 2 interact with clathrin. The three NPF motifs of the COOH-terminal region of epsin 1 are conserved in the corresponding region of epsin 2, consistent with the binding of both proteins to Eps15. Epsin 2, like epsin 1, is enriched in brain, is present in a brain-derived clathrin-coated vesicle fraction, is concentrated in the peri-Golgi region and at the cell periphery of transfected cells, and partially colocalizes with clathrin. High overexpression of green fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.

Adaptor Protein Complex alpha Subunits↗
PubMed1999

Effect of low frequency ultrasound on the in vitro percutaneous absorption of clobetasol 17-propionate.

The effect of low frequency ultrasound (20 kHz) on the permeation of clobetasol 17-propionate (CP) through skin (sonophoresis) was studied. The ultrasound was applied at either continuous or discontinuous modes and at different intensities. The results showed that low frequency ultrasound significantly enhanced the permeability of CP across hairless mouse skin in vitro. Delivering the same amount of ultrasonic energy in different modes of application markedly influenced the flux and skin residual of CP. The on/off discontinuous ultrasound had greater enhancement on CP permeation than the continuous ultrasound. The results of skin histopathology and permeation experiment using various membranes demonstrate that both disordering of stratum corneum and convective flow resulted from the cavitation effect were responsible for sonophoretic enhancement of CP. The permeation of CP through hair follicles and sweat ducts was susceptible to the application of ultrasound.

Administration, Topical↗
PubMed1999

Molecular cloning and functional characterization of the promoter region of the human uncoupling protein-2 gene.

As a member of the uncoupling protein family, UCP2 is ubiquitously expressed in rodents and humans, implicating a major role in thermogenesis. To analyze promoter function and regulatory motifs involved in the transcriptional regulation of UCP2 gene expression, 3.3 kb of 5'-flanking region of the human UCP2 (hUCP2) gene have been cloned. Sequence analysis showed that the promoter region of hUCP2 lacks a classical TATA or CAAT box, however, appeared GC-rich resulting in the presence of several Sp-1 motifs and Ap-1/-2 binding sites near the transcription initiation site. Functional characterization of human UCP2 promoter-CAT fusion constructs in transient expression assays showed that minimal promoter activity was observed within 65 bp upstream of the transcriptional start site (+1). 75 bp further upstream (from nt -141 to -66) a strong cis-acting regulatory element (or enhancer) was identified, which significantly enhanced basal promoter activity. The regulation of human UCP2 gene expression involves complex interactions among positive and negative regulatory elements distributed over a minimum of 3.3 kb of the promoter region.

Animals↗
PubMed1999

Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates.

In Drosophila, plexin A is a functional receptor for semaphorin-1a. Here we show that the human plexin gene family comprises at least nine members in four subfamilies. Plexin-B1 is a receptor for the transmembrane semaphorin Sema4D (CD100), and plexin-C1 is a receptor for the GPI-anchored semaphorin Sema7A (Sema-K1). Secreted (class 3) semaphorins do not bind directly to plexins, but rather plexins associate with neuropilins, coreceptors for these semaphorins. Plexins are widely expressed: in neurons, the expression of a truncated plexin-A1 protein blocks axon repulsion by Sema3A. The cytoplasmic domain of plexins associates with a tyrosine kinase activity. Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. We conclude that plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and trigger a novel signal transduction pathway controlling cell repulsion.

3T3 Cells↗
PubMed1999

[Variation of primary structure in nonstructural region 5 b of hepatitis C virus].

OBJECTIVE: To analyze the sequence variation in the nonstructural region 5 b(NS5 b) of hepatitis C virus (HCV) of China. METHODS: Complementary DNA fragments from sera of 49 Chinese patients were amplified by reverse transcription nested polymerase chain reaction (RT-nested-PCR) and the products were cloned and sequenced. RESULTS: 33 clones belonged to genotype 1 b and 16 clones were genotype 2 a. Several conserved nucleotide sites were found to be different from those of Japanese isolates though they belonged to an identical genotype. Comparison of average homology among the 33 clones of genotype 1 b and the 16 clones of genotype 2 a indicated that the average homology among genotype 2 a was lower than that among genotype 1 b. A deletion of three nucleotides and a frame-shift, resulting from the introduction of an in-frame stop codon, were firstly observed in the NS5 b region. CONCLUSION: The nucleotide sequences of different HCV isolates of a same subtype may possibly possess the characteristics for different geographical distribution and so a definite epidemic HCV strain may emerge from a definite area.

Base Sequence↗
PubMed1999

Lps(d)/Ran of endotoxin-resistant C3H/HeJ mice is defective in mediating lipopolysaccharide endotoxin responses.

C3H/HeJ inbred mice are defective in that they are highly resistant to endotoxic shock as compared with normal responder mice. Their B cells and macrophages do not respond significantly when exposed to lipopolysaccharide (LPS), whereas cells from the responder mice do. Using a functional assay, we previously isolated a cDNA, which encodes for Ran/TC4 GTPase. We now show that this gene is mutated in C3H/HeJ mice, which accounts for their resistance to endotoxin stimulation. Sequence analysis of independent mutant Lps(d)/Ran cDNAs isolated from splenic B cells of C3H/HeJ mice reveals a consistent single base substitution at position 870, where a thymidine is replaced with a cytidine. In situ hybridization maps the Lps(d)/Ran cDNA to mouse chromosome 4. By retroviral gene transfer, the wild-type Lps(n)/Ran cDNA but not the mutant Lps(d)/Ran cDNA can restore LPS responsiveness of C3H/HeJ cells. Adenoviral gene transfer in vivo with the mutant Lps(d)/Ran cDNA but not the wild-type Lps(n)/Ran cDNA rescues endotoxin-sensitive mice from septic shock. Thus Lps/Ran is an important target for LPS-mediated signal transduction, and the Lps(d)/Ran gene may be useful as a therapeutic sequence in gene therapy for endotoxemia and septic shock.

3' Untranslated Regions↗
PubMed1999

Regulation of hormone-induced histone hyperacetylation and gene activation via acetylation of an acetylase.

Nuclear receptors have been postulated to regulate gene expression via their association with histone acetylase (HAT) or deacetylase complexes. We report that hormone induces dramatic hyperacetylation at endogenous target genes through the HAT activity of p300/CBP. Unexpectedly, this hyperacetylation is transient and coincides with attenuation of hormone-induced gene activation. In exploring the underlying mechanism, we found that the acetylase ACTR can be acetylated by p300/CBP. The acetylation neutralizes the positive charges of two lysine residues adjacent to the core LXXLL motif and disrupts the association of HAT coactivator complexes with promoter-bound estrogen receptors. These results provide strong in vivo evidence that histone acetylation plays a key role in hormone-induced gene activation and define cofactor acetylation as a novel regulatory mechanism in hormonal signaling.

Acetylation↗
PubMed1999

The Epstein-Barr virus latency BamHI-Q promoter is positively regulated by STATs and Zta interference with JAK/STAT activation leads to loss of BamHI-Q promoter activity.

In Epstein-Barr virus (EBV)-associated tumors in nonimmunocompromised patients, EBV gene expression is highly restricted. EBV-encoded nuclear antigen (EBNA)-1 is expressed, whereas the immunogenic and proliferative EBNAs are not. This pattern of EBNA expression is generated by usage of the BamHI-Q promoter (Qp). We have determined that the JAK/STAT pathway positively regulates Qp activity. In transient-transfection assays, a Qp-CAT reporter was activated by cotransfected JAK-1 and by treatment of cells with the cytokine IL-6. The ability of Qp to bind signal transducer and activator of transcription (STAT) proteins was directly demonstrated by electrophoretic mobility-shift assay, and mutation of potential STAT-binding sites reduced Qp responsiveness to Janus kinase (JAK)-1. Consistent with a role for STATs in Qp function, Qp using Burkitt's lymphoma Rael cells and cultured nasopharyngeal carcinoma (NPC) cells contained nuclear STAT protein. We investigated whether the inability to maintain EBV-positive NPC cell lines in culture was related to Qp activity. Passaging of the NPC cell line HK666 led to activation of expression of BZLF1, which encodes Zta and loss of Qp function. Transient expression assays linked Zta expression to the down-regulation of Qp. Cotransfection of Zta reduced Qp activity in reporter assays. This negative regulation required Zta DNA-binding activity. We provide evidence that Zta up-regulation of p53 leads to p53-mediated interference with JAK/STAT activation of Qp. The data imply that JAK/STAT signaling has a role in EBV-associated malignancies.

B-Lymphocytes↗
PubMed1999

The cartilage-specific (V+C)- fibronectin isoform exists primarily in homodimeric and monomeric configurations.

Fibronectin is an extracellular-matrix glycoprotein encoded by a single gene, but with significant protein heterogeneity introduced through alternative RNA splicing and post-translational modifications. The (V+C)(-) splice variant, in which nucleotides encoding protein segments III-15 and I-10 are deleted along with the entire variable region, is unique in that expression is restricted to cartilaginous tissues. All known fibronectin splice variants retain the two C-terminal cysteine residues essential for dimerization, but cellular and/or structural constraints appear to influence homo- and heterodimerization patterns. Dimerization patterns of the (V+C)(-) isoform were studied under native conditions within canine articular cartilage and experimentally in COS-7, NIH-3T3 and CHO-K1 cell cultures. In all systems, (V+C)(-) fibronectin secretion was predominantly in a homodimeric configuration. Lower levels of (V+C)(-) monomers were also present. Heterodimers of (V+C)(-) with V(+),C(+) (V120) isoforms were not detected. Heterodimers of (V+C)(-) with V(-),C(+) (V0) subunits were detected only at low levels. Functional properties may differ significantly among monomers, homodimers and heterodimers. The unique dimerization pattern of (V+C)(-) fibronectin is consistent with this isoform having specialized functional properties in situ that are important for either the structural organization and biomechanical properties of cartilage matrix or regulation of a chondrocytic phenotype.

Animals↗
PubMed1999

Association of the NAD(P)H:quinone oxidoreductase 609C-->T polymorphism with a decreased lung cancer risk.

The NAD(P)H:quinone oxidoreductase gene, NQO1, often carries a C-->T transition at bp 609, which has been associated with a reduced enzymatic activity and which may result in altered metabolic activation of tobacco smoke procarcinogens. We tested the association of this polymorphism with lung cancer risk in a population-based case-control study of 327 cases and 440 controls of Caucasian, Japanese, or Native Hawaiian ancestry in Hawaii. We found a notable difference in the frequency of the variant allele among Japanese (38%), Caucasians (20%), and Hawaiians (22%). Overall, the variant allele was less frequent in cases than in controls (P = 0.03). A significant inverse association was found in Japanese, with adjusted odds ratios of 0.8 (95% confidence interval, 0.4-1.5) and 0.3 (0.1-0.7) for the heterozygous and homozygous variant genotypes, respectively, compared with the homozygous wild-type genotype (P for genetic trend, 0.02). The association did not reach statistical significance in Caucasians and Hawaiians but was in the same direction.

Alleles↗
PubMed1999

Constitutive cell surface association between CD4 and CCR5.

HIV-1 entry into cells involves formation of a complex between gp120 of the viral envelope glycoprotein (Env), a receptor (CD4), and a coreceptor. For most strains of HIV, this coreceptor is CCR5. Here, we provide evidence that CD4 is specifically associated with CCR5 in the absence of gp120 or any other receptor-specific ligand. The amount of CD4 coimmunoprecipitated with CCR5 was significantly higher than that with the other major HIV coreceptor, CXCR4, and in contrast to CXCR4 the CD4-CCR5 coimmunoprecipitation was not significantly increased by gp120. The CD4-CCR5 interaction probably takes place via the second extracellular loop of CCR5 and the first two domains of CD4. It can be inhibited by CCR5- and CD4-specific antibodies that interfere with HIV-1 infection, indicating a possible role in virus entry. These findings suggest a possible pathway of HIV-1 evolution and development of immunopathogenicity, a potential new target for antiretroviral drugs and a tool for development of vaccines based on Env-CD4-CCR5 complexes. The constitutive association of a seven-transmembrane-domain G protein-coupled receptor with another receptor also indicates new possibilities for cross-talk between cell surface receptors.

3T3 Cells↗
PubMed1999

Multiple integrase functions are required to form the native structure of the human immunodeficiency virus type I intasome.

Mu-mediated polymerase chain reaction footprinting was used to investigate the protein-DNA structure of human immunodeficiency virus type I (HIV-I) preintegration complexes. Preintegration complexes were partially purified from cells after using an established coculture infection technique as well as a novel technique using cell-free supernatant from transfected cells as the source of virus. Footprinting revealed that bound proteins protected the terminal 200-250 base pairs of each viral end from nuclease attack. Bound proteins also caused strong transpositional enhancements near each end of HIV-I. In contrast, regions of viral DNA internal to the ends did not show evidence of strong protein binding. The end regions of preintegrative HIV-I apparently form a unique nucleoprotein structure, which we term the intasome to distinguish it from the greater preintegration complex. Our novel system also allowed us to analyze the structure and function of preintegration complexes isolated from cells infected with integrase mutant viruses. Complexes were derived from viruses defective for either integrase catalysis, integrase binding to the viral DNA substrate, or an unknown function in the carboxyl-terminal domain of the integrase protein. None of these mutant complexes supported detectable integration activity. Despite the presence of the mutant integrase proteins in purified samples, none of these nucleoprotein complexes displayed the native intasome structure detected in wild-type preintegration complexes. We conclude that multiple integrase functions are required to form the native structure of the HIV-I intasome in infected cells.

Cloning, Molecular↗
PubMed1999

Gas chromatographic-mass spectrometric analysis of perillyl alcohol and metabolites in plasma.

Perillyl alcohol (POH), a metabolite of d-limonene and a component of the lavender oil, is currently in Phase I clinical trials both as a chemopreventative and chemotherapeutic agent. In vivo, POH is metabolized to less active perillic acid (PA) and cis- and trans-dihydroperillic acids [DHPA, 4-(1'-methylethenyl)-cyclohexane-1-carboxylic acid]. Previous pharmacokinetic studies using a GC-MS method detected POH metabolites but not POH itself; thus these studies lacked information on the parent drug. The present report describes a sensitive GC-MS method for the quantitation of POH and metabolites using stable-isotopically labeled internal standards. The residue obtained from CH2Cl2 extraction of a plasma sample was silylated. The products were separated on a capillary column and analyzed by an ion-trap GC-MS using NH3 chemical ionization. POH-d3 was used as the internal standard for POH while 13C-PA-d2 was used as the internal standards for the metabolites. The quantitation limits for POH, PA, cis- and trans-DPA were <10 ng/ml using 1-2 ml plasma. The assay was validated in rat and human plasma. The assay was linear from 2 to 2000 ng/ml for POH, 10 to 1000 ng/ml for PA and trans-DHPA, and 20 to 1000 ng/ml for cis-DHPA monitored. The within-run and between-run coefficients of variation were all <8%. Preliminary pharmacokinetic data from a rat following i.v. administration of POH at 23 mg/kg and from a patient receiving POH at 500 mg/m2 p.o. was also provided. Intact POH, PA, cis- and trans-DHPA were all detected in plasma in both cases. Two new major metabolites were found in human and one in the rat plasma.

Animals↗
PubMed1999

A quantitative approach to signal transduction.

The high affinity receptor for IgE (FcepsilonRI), is one of a family of immunoreceptors whose antigen-induced clustering leads to a variety of cellular responses. The signaling pathways are enormously complex but by focusing on only the most initial steps, it is now possible to sketch plausible molecular models that relate the interaction of multivalent antigens with the receptor-bound IgE to the earliest cellular events. In this paper, we describe how we have combined quantitative experimentation and mathematical modeling to probe this system further. We also discuss some of the formidable challenges that remain before we can claim reasonably complete understanding of even these early events.

Animals↗