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Biomedical subjects

L Chen

Publications and source records attributed to L Chen.

At least 631 records · Page 35Linked to original sources
PubMed1998

A 60 kd MDM2 isoform is produced by caspase cleavage in non-apoptotic tumor cells.

The MDM2 oncogene product is a regulator of the p53 tumor suppressor. MDM2 is cleaved by Caspase 3 (CPP32) during apoptosis after aspartic acid-361, generating a 60 kd fragment. Here we report that human tumor cell lines often express high levels of a 60 kd MDM2 isoform (p60) in the absence of apoptosis. We demonstrate that p60 is a product of caspase cleavage of full length MDM2 after residue 361. The protease that cleaves MDM2 in non-apoptotic cells appears to be distinct from the apoptosis-specific Caspase 3, since Caspase 3 substrate poly(ADP-ribose) polymerase (PARP) is not cleaved in cells producing p60. The p60 form of MDM2 is a significant fraction of the p53-bound MDM2 protein in certain tumor cells, suggesting that it functions in the regulation of p53. p60 is also detected in breast tumors overexpressing MDM2. These observations suggest that MDM2 is regulated by caspase processing in non-apoptotic cells, and may account for the MDM2 proteins of similar mobility seen in tumors and other cell lines.

Amino Acid Sequence↗
PubMed1998

Ex vivo fibroblast transduction in rabbits results in long-term (>600 days) factor IX expression in a small percentage of animals.

Delivery of human factor IX to the circulation was analyzed in rabbits by ex vivo fibroblast transduction followed by subcutaneous implantation. Kinetic studies of human factor IX in rabbits demonstrated a half-life of approximately 16 hr and a volume distribution of 22%, where intraperitoneal and subcutaneous bioavailability was three- to sevenfold lower than by intravenous administration. Ex vivo retroviral transduction of autologous fibroblasts was performed on 15 animals. After subcutaneous injection of fibroblast-collagen mixtures, the expression of human factor IX in rabbit plasma was followed by ELISA. Of 15 rabbits injected, expression of human factor IX was detected in 2 animals, and expression was long term (>600 days). One animal had stable levels of human factor IX, at 20 ng/ml, while the second animal had lower and gradually decreasing levels of human factor IX. There were no gross differences in pathology at the injection sites, when comparing animals with human factor IX in plasma and those without. Immunological studies demonstrated antibody formation in response to injection mixture components (including human factor IX), but again there was no correlation with immune response and long-term factor IX production in animals. Tissues at the implantation sites were positive for factor IX DNA by PCR analysis, regardless of whether there was detectable plasma factor IX or not. Small numbers of PCR-positive cells were detected in the internal organs of the long term-expressing rabbits while similar tissues were negative in nonexpressing animals.

Animals↗
PubMed1998

Characterization of diverse viral vector preparations, using a simple and rapid whole-virion dot-blot method.

A number of different viruses have been adapted as gene transfer vectors, including retroviruses, adenoviruses, adenoassociated viruses (AAVs), herpes simplex virus, SV40 viruses, and alphaviruses (both Semliki Forest and Sindbis viruses). One of the major rate-limiting and time-consuming steps in the characterization of these vectors is the process of determining the viral vector titers. In addition, there is no "universal" method that can be used to rapidly estimate the titer and the utility of viral vector preparations. We demonstrate here that supernatant from diverse classes of viral vectors, with either RNA or DNA genomes, can be rapidly evaluated by a simple virus dot-blot hybridization without prior extraction of nucleic acids. This system can provide a reliable screen for physical titer of viral vector supernatants in 1 day.

Adenoviridae↗
PubMed1998

The her-2/neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products.

Malignant transformation is associated with changes in the glycosylation of cell surface proteins. For example, the N-linked oligosaccharides containing the [GlcNAc beta(1,6)Man] branch are increased after transformation of many cell types by a number of tumor viruses and oncogenes which induce the expression of N-acetylglucosaminyl-transferase V (GlcNAc-T V), the enzyme that adds this branch. A large percentage of human breast carcinomas have increased N-linked beta(1,6) branches on glycoproteins, while up to 30% of breast carcinomas have amplified the oncogene her-2/neu (erb-B2). We tested the hypothesis that expression of her-2/neu stimulates GlcNAc-T V gene expression and increases the beta(1,6) branching of N-linked oligosaccharides. We found that neu-transformed NIH3T3 cells have a threefold increase in GlcNAc-T V enzyme activity and increased beta(1,6) branching on a specific set of glycoproteins. Promoter/reporter experiments showed that her-2/neu stimulates transcription from the human GlcNAc-T V promoter and that the her-2/neu response element was located about 400 bp 5' of the transcription initiation site and includes three Ets transcription factor binding sequences. Co-transfections with dominant-negative Raf and Ets expression plasmids demonstrated that the transcriptional activation of the GlcNAc-T V promoter by neu is mediated by the Ras-Raf-Ets signal transduction pathway.

3T3 Cells↗
PubMed1998

Intercalated cytosine motif and novel adenine clusters in the crystal structure of the Tetrahymena telomere.

The cytosine-rich strand of the Tetrahymena telomere consists of multiple repeats of sequence d(AACCCC). We have solved the crystal structure of the crystalline repeat sequence at 2.5 A resolution. The adenines form two different and previously unknown clusters (A clusters) in orthogonal directions with their counterparts from other strands, each containing a total of eight adenines. The clusters appear to be stable aggregates held together by base stacking and three different base-pairing modes. Two different types of cytosine tetraplexes are found in the crystal. Each four-stranded complex is composed of two intercalated parallel-stranded duplexes pointing in opposite directions, with hemiprotonated cytosine-cytosine (C.C+) base pairs. The outermost C.C+base pairs are from the 5'-end of each strand in one cytosine tetraplex and from the 3'-end of each strand in the other. The A clusters and the cytosine tetraplexes form two alternating stacking patterns, creating continuous base stacking in two perpendicular directions along the x - and z -axes. The adenine clusters could be organizational motifs for macromolecular RNA.

Adenine↗
PubMed1998

[Genetic polymorphisms of TH01 and VWA loci in Tibetans in China].

OBJECTIVE: To understand the genetic relationship between Han population and Tibetan population, the genetic polymorphisms of two STR loci, TH01 and VWA were studied in the Tibetans in China. METHODS: EDTA-blood specimens were collected from 89 healthy unrelated Tibetan individuals in Lasa. DNA was extracted using Chelex method. The DNA samples were amplified by PCR technique, and the PAGE horizontal electrophoresis were used to typing the PCR products. RESULTS: There were five alleles at TH01 locus in Tibetan population. The alleles frequencies were TH01*6:0.097, TH01*7:0.227, TH01*8:0.091, TH01*9:0.481 and TH01*9.3:0.104. A total of 12 genotypes were observed in 77 individuals for TH01. For VWA locus there were seven alleles and the allele frequencies were VWA *14:0.101, VWA*15:0.034, VWA*16:0.208, VWA*17:0.303, VWA*18:0.208, VWA*19:0.129 and VWA*20:0.017. A total of 20 genotypes for VWA were observed in 89 individuals. The results of test for Hardy-Weinberg equilibrium showed that the genotype distributions observed at both STR loci were correspondent with those expected(chi-square =7.421 df =8 P>0.05 for TH01; chi-square =19.61 df =14 P>0.05 for VWA). CONCLUSION: There is no significant difference between Han and Tibetan populations in allele frequencies at TH01 and VWA loci. The results of analysis using genetic distance and phylogenetic tree indicate that Tibetan population is identical with Han population at TH01 and VWA loci .

Asian People↗
PubMed1998

Cerebellar brain-derived neurotrophic factor-TrkB defect associated with impairment of eyeblink conditioning in Stargazer mutant mice.

In the spontaneous ataxic mutant mouse stargazer, there is a selective reduction of brain-derived neurotrophic factor (BDNF) mRNA expression in the cerebellum. BDNF protein levels in the cerebellum are reduced by 70%. Despite normal levels of full-length and truncated TrkB receptor, constitutive and neurotrophin-4/5-induced tyrosine phosphorylation was significantly reduced in several signal transduction molecules, including phospholipase-Cgamma1, erk1, and erk2. Morphological examination revealed an increased number of external granule cells at postnatal day 15 and the presence of abnormal neurons resembling immature granule cells in the adult. These abnormalities are associated with a severe impairment in the acquisition of classical eyeblink conditioning, indicating cerebellar malfunction. Our data suggest that normal BDNF expression and TrkB signal transduction in the cerebellum are necessary for learning and plasticity in this model.

Animals↗
PubMed1998

Antisense to MDR1 mRNA reduces P-glycoprotein expression, swelling-activated C1- current and volume regulation in bovine ciliary epithelial cells.

Native ciliary epithelial cells from the ciliary epithelium of the eye exhibit anti-P-glycoprotein (P-gp) immunofluorescence. We have used an antisense 'knock-down' approach to investigate the relationship between P-gp and the volume-activated chloride current (IC1,swell) and its role in volume regulation. An antisense oligonucleotide to the human multidrug resistance (MDR1) gene, taken up by the cells in a dose-dependent manner, reduced P-gp immunofluorescence, inhibited IC1,swell and significantly increased the latency of activation of IC1,swell. Increasing the hypotonic stress did not result in an increased activation of ICl,swell. MDR1 antisense 'knock-down' also reduced the ability of the cells to volume regulate following a hypotonic challenge. These cells are known to express at least two volume-activated chloride channels, and the data suggest that P-gp is involved in the activation pathway of a subset of channels that contribute to whole-cell IC1,swell and participate in volume regulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

The activity of topoisomerase I is modulated by large T antigen during unwinding of the SV40 origin.

When simian virus 40 (SV40) large T antigen binds to the virus origin of replication, it forms a double hexamer that functions as a helicase to unwind the DNA bidirectionally. We demonstrate in this report that T antigen can unwind and release an origin DNA single strand of less than full length in the presence of purified human topoisomerase I. The sites nicked by topoisomerase I in the strands released by T antigen during DNA unwinding were localized primarily to the "late" side of the origin, and the template for lagging strand synthesis was preferred significantly over the one for leading strand synthesis. Importantly, these sites were, for the most part, different from the sites nicked by topoisomerase I in the absence of T antigen. These data indicate that T antigen activates topoisomerase I nicking at discrete sites and releases these nicked strands during unwinding. We hypothesize that a single molecule of topoisomerase I can form a functional complex with a double hexamer of T antigen to simultaneously relax and unwind double-stranded origin-containing DNA.

Antigens, Viral, Tumor↗
PubMed1998

Thermodynamics of human DNA ligase I trimerization and association with DNA polymerase beta.

The interaction between human DNA polymerase beta (pol beta) and DNA ligase I, which appear to be responsible for the gap filling and nick ligation steps in short patch or simple base excision repair, has been examined by affinity chromatography and analytical ultracentrifugation. Domain mapping studies revealed that complex formation is mediated through the non-catalytic N-terminal domain of DNA ligase I and the N-terminal 8-kDa domain of pol beta that interacts with the DNA template and excises 5'-deoxyribose phosphate residue. Intact pol beta, a 39-kDa bi-domain enzyme, undergoes indefinite self-association, forming oligomers of many sizes. The binding sites for self-association reside within the C-terminal 31-kDa domain. DNA ligase I undergoes self-association to form a homotrimer. At temperatures over 18 degreesC, three pol beta monomers attached to the DNA ligase I trimer, forming a stable heterohexamer. In contrast, at lower temperatures (<18 degreesC), pol beta and DNA ligase I formed a stable 1:1 binary complex only. In agreement with the domain mapping studies, the 8-kDa domain of pol beta interacted with DNA ligase I, forming a stable 3:3 complex with DNA ligase I at all temperatures, whereas the 31-kDa domain of pol beta did not. Our results indicate that the association between pol beta and DNA ligase I involves both electrostatic binding and an entropy-driven process. Electrostatic binding dominates the interaction mediated by the 8-kDa domain of pol beta, whereas the entropy-driven aspect of interprotein binding appears to be contributed by the 31-kDa domain.

Chromatography, Affinity↗
PubMed1998

Cloning, characterization, and the complete 56.8-kilobase DNA sequence of the human NOTCH4 gene.

The first complete mammalian genomic sequence reported thus far in the Notch gene family, including a putative promoter region and 30 exons of the human NOTCH4 gene spanning 56.8 kb of DNA, were sequenced. The NOTCH4 locus contains a TATA-less promoter with two putative transcription initiation sites (Inr), three RBP-Jkappa sites, and two GATA recognition sites. Two cDNA isoforms, NOTCH4(L) and NOTCH4(S),were identified. Whereas the NOTCH4(S) isoform contains the entire coding sequence, the NOTCH4(L) isoform has two unspliced intronic sequences between exons 11 and 12 and exons 20 and 21 and a misspliced exon 6. Consistent with these results, two alternatively spliced isoforms of transcripts of approximately 9.3 and 6.7 kb were detected by Northern blot analysis. The predicted amino acid sequence of the NOTCH4 protein based on the NOTCH4(S) cDNA sequence contains 2003 amino acids and includes the predominant motifs of the Notch family: 29 epidermal growth factor (EGF)-like repeats, 3 Notch/lin-12 repeats, a transmembrane region, 6 cdc10/Ankyrin repeats, and a PEST domain.

Adult↗
PubMed1998

Electrophysiological study of chimeric sodium channels from heart and skeletal muscle.

The alpha-subunit cDNAs encoding voltage-sensitive sodium channels of human heart (hH1) and rat skeletal muscle (rSkM1) have been expressed in the tsA201 mammalian cell line, in which inactivation properties appear to be normal in contrast to Xenopus oocytes. A series of rSkM1/hH1 chimeric sodium channels has been evaluated to identify the domains of the alpha-subunits that are responsible for a set of electrophysiological differences between hH1 and rSkM1, namely, midpoints and slope factors of steady-state activation and inactivation, inactivation kinetics and recovery from inactivation kinetics and their voltage-dependence. The phenotype of chimeric channels in which each hH1 domain was successively introduced into a rSkM1 alpha-subunit framework confirmed the following conclusions. (i) The D4 and or/C-ter. are responsible for the slow inactivation of hH1 sodium channels. (ii) Concerning the other differences between rSkM1 and hH1: steady-state activation and inactivation, kinetics of recovery from inactivation, the phenotypes are determined probably by more than one domain of the alpha-subunit.

Animals↗
PubMed1998

Structure-function relationships and flexible tetramer assembly in pyruvate decarboxylase revealed by analysis of crystal structures.

The crystal structures of pyruvate decarboxylase from the yeast Saccharomyces uvarum and Saccharomyces cerevisiae have been determined at 2.4 and 2.3 A resolution, respectively. These structures provide details about the protein fold and domain assembly within subunits, about subunit assembly to form dimers and about dimer assembly to form tetramers. They also provide a clear picture of the active site centered on the thiamin diphosphate cofactor, and have allowed amino acids critical for catalysis and involved in stabilization of the unusual cofactor conformation to be identified. The structural information has enabled identification of the site of allosteric activation to be centered on Cys-221, and suggests that a six residue segment leading from the regulatory site to the catalytic site may be involved in transmission of a binding signal. The importance of several amino acids within this segment in the regulatory process, as well as some involved in stabilizing and activating the cofactor has been confirmed by analyzing the behavior of recombinant enzymes with single point mutations introduced at these sites. Additional structures have been determined for pyruvate decarboxylase in multiple crystal forms, some of which were obtained from crystals grown with known allosteric activators present in the media. Currently four distinct types of tetramers have been observed, with each showing a different mode of association of dimers to form the tetramers. In some of the cases involving the presence of allosteric activators drastic changes in the mode of dimer assembly to form tetramers is seen.

Allosteric Regulation↗
PubMed1998

Agrobacterium VirD2 protein interacts with plant host cyclophilins.

Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5' end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2-cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer.

Bacterial Proteins↗
PubMed1998

Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells.

Immunological unresponsiveness established by the elimination or anergy of self-reactive lymphocyte clones is of importance to immunization against tumor-associated antigens. In this study, we have investigated induction of immunity against the human MUC1 carcinoma-associated antigen in MUC1 transgenic mice unresponsive to MUC1 antigen. Immunization of adult MUC1 transgenic mice with irradiated MUC1-positive tumor cells was unsuccessful in reversing unresponsiveness to MUC1. By contrast, fusions of dendritic cells with MUC1-positive tumor cells induced cellular and humoral immunity against MUC1. Immunization with the dendritic cell fusions that express MUC1 resulted in the rejection of established metastases and no apparent autoimmunity against normal tissues. These findings demonstrate that unresponsiveness to the MUC1 tumor-associated antigen is reversible by immunization with heterokaryons of dendritic cells and MUC1-positive carcinoma cells.

Adult↗
PubMed1998

Low density lipoprotein receptor related protein gene exon 3 polymorphism association with Alzheimer's disease in Chinese.

Since apolipoprotein E4 (apoE4) is the major genetic risk for late onset Alzheimer's disease (AD), proteins that interact with apoE might be involved in AD pathogenesis. Low density lipoprotein receptor related protein (LRP) is an apoE receptor in the brain. In exon 3 of the LRP gene a polymorphism was found to be underrepresented in AD compared to normal Caucasian subjects (N). We examined this polymorphism in Chinese AD and N subjects. The polymorphism frequency in N was roughly half that reported for Caucasians. Compared to N, the frequency was significantly decreased in pathologically diagnosed, but not in clinically diagnosed AD patients. Thus, the role of the LRP exon 3 polymorphism in AD has now been demonstrated in two ethnic groups, suggesting the importance of LRP in AD pathogenesis.

Aged↗
PubMed1998

Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in balloon-injured carotid artery.

Endothelial cells in normal blood vessels might prevent the unscheduled proliferation of smooth muscle cells (SMCs) by the expression of cell migration and growth inhibitors. NO, a potent vasodilator, generated by endothelium-specific constitutive NO synthase (ecNOS) might be such an inhibitor. To test this hypothesis, we overexpressed human ecNOS in syngeneic rat arterial SMCs using retrovirus-mediated gene transfer. Compared with SMCs transduced with vector alone (LXSN SMCs), DNA synthesis and cell proliferation were inhibited in the ecNOS-expressing SMCs (LCNSN SMCs). Basal and stimulated (by the calcium ionophore A23187) secretion of NO and intracellular cGMP were increased in LCNSN SMCs. Nomega-Nitro-L-arginine (L-NA), an inhibitor of NO synthesis, enhanced the proliferation of LCNSN SMCs but had no effect on LXSN SMCs. LCNSN SMCs seeded onto the luminal surface of balloon-injured rat carotid arteries inhibited neointimal formation by 37% and induced marked dilatation (3-fold increase in vessel diameter) at 2 weeks compared with LXSN SMC-seeded arteries. Orally administered L-NA blocked these changes. Phosphorylation of vasodilator-stimulated phosphoprotein, which is regulated in part by NO, was elevated in LCNSN SMCs and in LCNSN SMC-seeded arteries. This study demonstrates that NO generation by ecNOS inhibits SMC proliferation in vitro and modulates vascular tone locally in vivo.

Angioplasty, Balloon↗