Biomedical subjects

W Chen

Publications and source records attributed to W Chen.

At least 397 records · Page 22Linked to original sources

PubMed1999

Expression of the human cholesterol 7alpha-hydroxylase gene in transgenic mice.

We have generated transgenic mice expressing human CYP7A1 transgenes. Only 1.5 kilobases (kb) of 5' upstream sequence and 6.5 kb of 3' sequence were sufficient for hepatic transcription of the transgenes. However, the 5' end segment alone was not sufficient to direct liver expression, suggesting that additional hepatic regulatory elements reside in the 3' extension or within introns. The level of expression of these transgenes was low in comparison to the levels of the endogenous mouse CYP7A1 mRNA. To generate mice expressing higher levels of CYP7A1 mRNA, we injected a large human genomic PAC clone, extending up to -105 kb 5' of the structural gene and about 50 kb 3' of the gene. These transgenic mice expressed CYP7A1 mRNA at higher levels, suggesting that additional hepatic regulatory elements are found either 5' of -1520 or beyond 6.5 kb 3' of the gene.

Animals↗

PubMed1999

Activation of p38 MAP kinase and ERK are required for ultraviolet-B induced c-fos gene expression in human keratinocytes.

The effects of p38 MAP kinase and ERK on UVB induced c-fos gene expression were studied in a human keratinocyte cell line, FL30. UVB significantly increased c-fos gene expression at both the transcriptional and protein levels. p38 and ERK were also significantly activated after UVB irradiation. Treating the cells with p38 inhibitor SB202190 inhibited p38 activation, but not ERK; treating the cells with MEK-1 inhibitor PD98059 inhibited ERK activation without suppressing p38 activation. The kinase activation was determined by Western blots using phospho-p38 or ERK antibodies, or an in vivo p38 activity assay. Further studies demonstrated that blocking p38 almost completely abrogated UVB induced c-fos gene transcription and c-Fos protein synthesis. Inhibiting ERK partially abrogated UVB induced c-fos transcriptional and protein levels. Suppression of both p38 and ERK not only completely blocked UVB induced c-fos expression, but also decreased c-fos gene basal expression. Our data indicated that p38 may play a more important role than ERK in UVB induced c-fos expression in human keratinocytes. Since c-fos expression may play an important role in UVB induced AP-1 activation, and AP-1 activation is known to play a role in tumor promotion, both p38 and ERK could be potential targets for chemoprevention of skin cancer.

Cell Line↗

PubMed1999

Metal ligation by Walker homology B aspartate betaD262 at site 3 of the latent but not activated form of the chloroplast F(1)-ATPase from Chlamydomonas reinhardtii.

Site-directed mutations D262C, D262H, D262N, and D262T were made to the beta subunit Walker Homology B aspartate of chloroplast F(1)-ATPase in Chlamydomonas. Photoautotrophic growth and photophosphorylation rates were 3-14% of wild type as were ATPase activities of purified chloroplast F(1) indicating that betaD262 is an essential residue for catalysis. The EPR spectrum of vanadyl bound to Site 3 of chloroplast F(1) as VO(2+)-ATP gave rise to two EPR species designated B and C in wild type and mutants. (51)V-hyperfine parameters of species C, present exclusively in the activated enzyme state, did not change significantly by the mutations examined indicating that it is not an equatorial ligand to VO(2+), nor is it hydrogen-bonded to a coordinated water at an equatorial position. Every mutation changed the ratio of EPR species C/B and/or the (51)V-hyperfine parameters of species B, the predominant conformation of VO(2+)-nucleotide bound to Site 3 in the latent (down-regulated) state. The results indicate that the Walker Homology B aspartate coordinates the metal of the predominant metal-nucleotide conformation at Site 3 in the latent state but not in the conformation present exclusively upon activation and elucidates one of the specific changes in metal ligation involved with activation.

Animals↗

PubMed1999

A large-scale insertional mutagenesis screen in zebrafish.

It is estimated that approximately 2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared approximately 36, 000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed approximately 80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F(5). We screened an estimated 760 insertions among F(3) progeny from 92 F(2) families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.

Alleles↗

PubMed1999

Transformation of polychlorinated biphenyls by a novel BphA variant through the meta-cleavage pathway.

The transformation of 20 polychlorinated biphenyls (PCBs) through the meta-cleavage pathway by recombinant Escherichia coli cells expressing the bphEFGBC locus from Burkholderia cepacia LB400 and the bphA genes from different sources was compared. The analysis of PCB congeners for which hydroxylation was observed but no formation of the corresponding yellow meta-cleavage product demonstrated that only lightly chlorinated congeners including one tetrachlorobiphenyl (2,2',4,4'-CB) were transformed into their corresponding yellow meta-cleavage products. Although many other tetrachlorobiphenyls (2, 2',5,5'-CB, 2,2',3,5'-CB, 2,4,4',5-CB, 2,3',4',5-CB, 2,3',4,4'-CB) and one pentachlorobiphenyl (2,2',4,5,5'-CB) tested were depleted from resting cell suspensions, no yellow meta-cleavage products were observed. For most of these congeners, dihydrodiol compounds accumulated as the endproducts, indicating that the bphB-encoded biphenyl-2,3-dihydrodiol-2,3-dehydrogenase is a key limiting step for further degradation of highly chlorinated congeners. These results suggest that engineering the biphenyl dioxygenase alone is insufficient for an improved removal of PCB. Rather, improved degradation of PCBs is more likely to be achieved with recombinant strains containing metabolic pathways not only specifically engineered for expanding the initial dioxygenation but also for the mineralization of PCBs.

Biotransformation↗

PubMed1999

Decreased selective uptake of high density lipoprotein cholesteryl esters in apolipoprotein E knock-out mice.

Scavenger receptor BI (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CE) by cells, i.e., the uptake of CE without degradation of HDL protein. Mice with attenuated expression of SR-BI, because of targeted gene mutation (SR-BIatt mice), have increased plasma HDL levels as a result of decreased selective uptake in the liver. To further evaluate the role of SR-BI in lipoprotein metabolism, compound apolipoprotein E knock-out (apoE0)/SR-BIatt mice were bred. Hepatic SR-BI protein was increased (2.3-fold) in apoE0 mice compared with wild type (wt) and was reduced significantly in apoE0/SR-BIatt mice. However, the plasma lipoprotein profile of apoE0 and apoE0/SR-BIatt mice was identical. This was explained by HDL turnover studies that revealed that the selective clearance of HDL CE by the liver and adrenal was already profoundly impaired in apoE0 mice compared with wt (28% of wt in liver). A similar decrease in selective uptake was seen when apoE0 HDL was incubated with isolated apoE0 hepatocytes. The results suggest that apoE plays a major role in the selective clearance of HDL CE by the liver and adrenal gland, possibly by facilitating the presentation of HDL to SR-BI at the cell surface.

Animals↗

PubMed1999

Cardiovascular risk factors clustering features of insulin resistance syndrome (Syndrome X) in a biracial (Black-White) population of children, adolescents, and young adults: the Bogalusa Heart Study.

Recently, independent factors representing different features of insulin resistance syndrome (Syndrome X) have been identified by factor analysis in middle-aged and elderly adult populations. In this study, factor analysis was applied to the clustering characteristics of Syndrome X in a biracial (Black-White) community-based population of 4,522 children (ages 5-11 years), adolescents (ages 12-17 years), and young adults (ages 18-38 years) from the Bogalusa Heart Study who were screened during 1988-1996. Ponderal index (weight (kg)/height (m)3), levels of insulin, glucose, triglycerides, and high density lipoprotein cholesterol, and systolic and diastolic blood pressure were used as measures of components of Syndrome X. No evidence was found to support a one-factor hypothesis for this syndrome, but factor analysis yielded two uncorrelated factors (factor 1: insulin/lipids/glucose/ponderal index; factor 2: insulin/blood pressure). These two factors explained 54.6% of the total variance in the entire sample. The factor loading patterns were very similar in all race and age groups, based on high values of coefficients of congruence (0.89-1.0). These results suggest that Syndrome X is characterized by the linking of a metabolic entity (hyperinsulinemia/insulin resistance, dyslipidemia, and obesity) to a hemodynamic factor (hypertension) through shared correlation with hyperinsulinemia/insulin resistance, and that the clustering features are independent of sex and age in both Black and White populations.

Adolescent↗

PubMed1999

Perfusion of kidneys with unformulated "naked" intercellular adhesion molecule-1 antisense oligodeoxynucleotides prevents ischemic/reperfusion injury.

BACKGROUND: We have previously shown that phosphorothioate intercellular adhesion molecule (ICAM)-1 antisense oligodeoxynucleotide (oligo) IP-9125 blocks the expression of rat ICAM-1 mRNA in rat L2 cells. A single ex situ perfusion of grafts with unformulated IP-9125, suspended in Euro-Collins solution, prolonged the survival of kidney allografts in rats. The present experiments examined whether perfusion of kidneys with unformulated IP-9125 prevents ischemic/reperfusion injury. METHODS: Kidneys were perfused ex situ with 2 ml of Euro-Collins solution without or with IP-9125 and exposed to 30-min cold (4 degrees C storage time) and 30-min warm (anastomosis time) ischemia. Kidneys were then transplanted to syngeneic nephrectomized recipients. RESULTS: Within 24 hr after transplantation, the glomerular filtration rate values were reduced by almost 60% to 0.49+/-0.14 ml/min from 1.20+/-0.27 ml/min in normal kidneys (P<0.001). Kidney perfusion with 10 mg of either IP-12140 (0.41+/-0.07 ml/min) or IP-13944 (0.47+/-0.07 ml/min) control oligo was ineffective. In contrast, perfusion with 10 mg of IP-9125 significantly improved kidney function (0.8+/-0.18 ml/min; P<0.005), whereas the lower doses of 2 mg (0.47+/-0.13 ml/min; NS) or 4 mg (0.54+/-0.04 ml/min; NS) had no significant effect. The glomerular filtration rate results were confirmed by measurements of blood creatinine (CR) levels at 24 hr after grafting: untreated recipients had a twofold higher CR value (0.70+/-0.14 mg/dl) compared with normal controls (0.65+/-0.07 mg/dl; P<0.001). Although perfusion with 10 mg of control IP-12140 (0.80+/-0.14 mg/dl) or IP-13944 (0.65+/-0.07 mg/dl) did not affect CR levels, perfusion with 10 mg of IP-9125 (0.45+/-0.07 mg/dl) lowered CR levels. The Western blots or reverse transcription-polymerase chain reaction experiments performed in kidney transplants within 24 hr after grafting showed that 10 mg of IP-9125 (but not control IP-12140) reduced the expression of ICAM-1 protein and ICAM-1 mRNA, respectively. CONCLUSIONS: Perfusion of grafts with unformulated ICAM-1 antisense oligo specifically reduces intragraft ICAM-1 protein expression and prevents ischemic/reperfusion injury.

Animals↗

PubMed1999

Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death.

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein synthesized and secreted by retinal pigment epithelial (RPE) cells in early embryogenesis and has been shown to be present in the extracellular matrix between the RPE cells and the neural retina. It induces neuronal differentiation and promotes survival of neurons of the central nervous system from degeneration caused by serum withdrawal or glutamate cytotoxicity. Because the role of PEDF in the retina is still unknown, we examined its ability to protect cultured retinal neurons against hydrogen peroxide (H(2)O(2))-induced cell death. Retinas of 0-2-day-old Sprague-Dawley rats were isolated and dissociated, and the neurons were maintained for 2 weeks in a synthetic serum-free medium. Immunocytochemical labeling showed that 50-60% of the cultured cells were rod photoreceptors. Treatment with H(2)O(2) induced significant death of retinal neurons in a dose- and time-dependent manner. Pretreatment with PEDF prior to insult greatly attenuated H(2)O(2)-induced cytotoxicity, and its effect was shown to be dose dependent. Cytotoxicity was determined by 3,(4,5-dimethylthiazol-2-yl)2, 5-diphenyl-tetrazolium bromide and lactate dehydrogenase assays, and apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay. The present study also showed that H(2)O(2)-induced retinal neuron death was by apoptosis that could be inhibited by PEDF. Combination of PEDF with basic fibroblast growth factor, brain-derived neurotrophic factor, or ciliary neurotrophic factor improves the protection. These data strongly suggest that PEDF is a potential neuroprotective agent in the treatment of retinal degeneration.

Animals↗

PubMed1999

B cell apoptosis triggered by antigen receptor ligation proceeds via a novel caspase-dependent pathway.

In contrast to positive signaling leading to proliferation, the mechanisms involved in negative signaling culminating in apoptosis after B cell Ag receptor (BCR) ligation have received little study. We find that apoptosis induced by BCR cross-linking on EBV-negative mature and immature human B cell lines involves the following sequential, required events: a cyclosporin A-inhibitable, likely calcineurin-mediated step; and activation of caspase-2, -3, and -9. Caspase-2 is activated early and plays a major role in the apoptotic pathway, while caspase-9 is activated later in the apoptotic pathway and most likely functions to amplify the apoptotic signal. Caspase-8 and -1, which are activated by ligation of the CD95 and TNF-R1 death receptors, are not involved. Apoptosis induced by BCR ligation thus proceeds via a previously unreported intracellular signaling pathway.

Animals↗

PubMed1999

Anti-recoverin antibodies cause the apoptotic death of mammalian photoreceptor cells in vitro.

We developed an in vitro model to study the effect of anti-recoverin antibodies on retinal cells and the mechanism(s) by which they kill photoreceptors in cancer-associated retinopathy (CAR). Rat retinal cells were grown in a defined medium, and cell types were identified by using antibodies against rhodopsin, recoverin, syntaxin, and thy-1. Purified immunoglobulin (IgG) against recoverin was added to the cultures at different concentrations for 24, 48, or 72 hr, and the survival of the cells was determined by fluorescence microscopy. Preimmune IgG and normal medium were used as controls. The cell death detection enzyme-linked immunosorbent assay and the terminal deoxyuridine triphosphate nick-end labeling assay were used to demonstrate cells undergoing apoptosis. Double labeling was used to visualize cell types and apoptotic death. Rods, amacrine cells, and ganglion cells were identified in the cultures. Rod cells, but not ganglion cells and amacrine cells, markedly decreased in the presence of 200 microg/ml of anti-recoverin IgG for 24, 48, and 72 hr. Anti-recoverin antibodies caused apoptosis in rod cells but not in amacrine cells. Almost all cells were shown to take up IgG from the medium. In conclusion, our retinal cell cultures provide a system for investigating antibody-mediated photoreceptor cell death and demonstrate that anti-recoverin antibodies cause the apoptotic death of rod cells, with no effect on amacrine cells. The results suggest that anti-recoverin antibodies play a key role in the apoptotic death of photoreceptors in CAR.

Animals↗

PubMed1999

Role of synaptic vesicle proton gradient and protein phosphorylation on ATP-mediated activation of membrane-associated brain glutamate decarboxylase.

Previously, we have shown that the soluble form of brain glutamic acid decarboxylase (GAD) is inhibited by ATP through protein phosphorylation and is activated by calcineurin-mediated protein dephosphorylation (Bao, J., Cheung, W. Y., and Wu, J. Y. (1995) J. Biol. Chem. 270, 6464-6467). Here we report that the membrane-associated form of GAD (MGAD) is greatly activated by ATP, whereas adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a non-hydrolyzable ATP analog, has no effect on MGAD activity. ATP activation of MGAD is abolished by conditions that disrupt the proton gradient of synaptic vesicles, e.g. the presence of vesicular proton pump inhibitor, bafilomycin A1, the protonophore carbonyl cyanide m-chorophenylhydrazone or the ionophore gramicidin, indicating that the synaptic vesicle proton gradient is essential in ATP activation of MGAD. Furthermore, direct incorporation of (32)P from [gamma-(32)P]ATP into MGAD has been demonstrated. In addition, MGAD (presumably GAD65, since it is recognized by specific monoclonal antibody, GAD6, as well as specific anti-GAD65) has been reported to be associated with synaptic vesicles. Based on these results, a model linking gamma-aminobutyric acid (GABA) synthesis by MGAD to GABA packaging into synaptic vesicles by proton gradient-mediated GABA transport is presented. Activation of MGAD by phosphorylation appears to be mediated by a vesicular protein kinase that is controlled by the vesicular proton gradient.

Adenosine Triphosphate↗

PubMed1999

Selective A1-adenosine receptor antagonists identified using yeast Saccharomyces cerevisiae functional assays.

Evaluation of a biased "library" of pyrrolo[2,3-d]pyrimidines using yeast-based functional assays expressing human A1- and A2a-adenosine receptors, led to the A1 selective antagonist 4b. A direct correlation between yeast functional activity and binding data was established. Practical compounds with polar residues at C-4 of the pyrrolopyrimidine system required H-bond donor functionality for high potency.

Binding, Competitive↗

PubMed1999

Isolation and characterisation of the cDNA encoding a glycosylated accessory protein of pea chloroplast DNA polymerase.

The cDNA encoding p43, a DNA binding protein from pea chloroplasts (ct) that binds to cognate DNA polymerase and stimulates the polymerase activity, has been cloned and characterised. The characteristic sequence motifs of hydroxyproline-rich glyco-proteins (HRGP) are present in the cDNA corres-ponding to the N-terminal domain of the mature p43. The protein was found to be highly O-arabinosylated. Chemically deglycosylated p43 (i.e. p29) retains its binding to both DNA and pea ct-DNA polymerase but fails to stimulate the DNA polymerase activity. The mature p43 is synthesised as a pre-p43 protein containing a 59 amino acid long transit peptide which undergoes stromal cleavage as evidenced from the post-translational in vitro import of the precursor protein into the isolated intact pea chloroplasts. Surprisingly, p43 is found only in pea chloroplasts. The unique features present in the cloned cDNA indicate that p43 is a novel member of the HRGP family of proteins. Besides p43, no other DNA-polymerase accessory protein with O-glycosylation has been reported yet.

Amino Acid Sequence↗

PubMed1999

Functional mapping in the human brain using high magnetic fields.

An avidly pursued new dimension in magnetic resonance imaging (MRI) research is the acquisition of physiological and biochemical information non-invasively using the nuclear spins of the water molecules in the human body. In this trial, a recent and unique accomplishment was the introduction of the ability to map human brain function non-invasively. Today, functional images with subcentimetre resolution of the entire human brain can be generated in single subjects and in data acquisition times of several minutes using 1.5 tesla (T) MRI scanners that are often used in hospitals for clinical purposes. However, there have been accomplishments beyond this type of imaging using significantly higher magnetic fields such as 4 T. Efforts for developing high magnetic field human brain imaging and functional mapping using MRI (fMRI) were undertaken at about the same time. It has been demonstrated that high magnetic fields result in improved contrast and, more importantly, in elevated sensitivity to capillary level changes coupled to neuronal activity in the blood oxygenation level dependent (BOLD) contrast mechanism used in fMRI. These advantages have been used to generate, for example, high resolution functional maps of ocular dominance columns, retinotopy within the small lateral geniculate nucleus, true single-trial fMRI and early negative signal changes in the temporal evolution of the BOLD signal. So far these have not been duplicated or have been observed as significantly weaker effects at much lower field strengths. Some of these high-field advantages and accomplishments are reviewed in this paper.

Brain↗

PubMed1999

Hydroperoxide dependence and cooperative cyclooxygenase kinetics in prostaglandin H synthase-1 and -2.

Prostaglandin H synthase isoform-1 (PGHS-1) cyclooxygenase activity has a cooperative response to arachidonate concentration, whereas the second isoform, PGHS-2, exhibits saturable kinetics. The basis for the cooperative PGHS-1 behavior and for the difference in cooperativity between the isoforms was unclear. The two cyclooxygenase activities have different efficiencies of feedback activation by hydroperoxide. To determine whether the cooperative kinetics were governed by the feedback activation characteristics, we examined the cyclooxygenase activities under conditions where feedback activation was either assisted (by exogenous peroxide) or impaired (by replacement of heme with mangano protoporphyrin IX to form MnPGHS-1 and -2). Heme replacement increased PGHS-1 cyclooxygenase cooperativity and changed PGHS-2 cyclooxygenase kinetics from saturable to cooperative. Peroxide addition decreased or abolished cyclooxygenase cooperativity in PGHS-1, MnPGHS-1, and MnPGHS-2. Kinetic simulations predicted that cyclooxygenase cooperativity depends on the hydroperoxide activator requirement and initial peroxide concentration, consistent with observed behavior. The results indicate that PGHS-1 cyclooxygenase cooperativity originates in the feedback activation kinetics and that the cooperativity difference between the isoforms can be explained by the difference in feedback activation loop efficiency. This linkage between activation efficiency and cyclooxygenase cooperativity indicates an interdependence between fatty acid and hydroperoxide levels in controlling the synthesis of potent prostanoid mediators.

Arachidonic Acid↗

PubMed1999

Genetic studies on a family with acute myelogenous leukemia.

Seven cases of myelogenous leukemia--two acute erythroleukemia (AEL), four acute myelogenous leukemia (AML), and one acute myelomonocytic leukemia (AMMoL)--were found in 22 members of three consecutive generations of a family in the past 16 years (1973-1989). By using cytogenetic, hematologic, and biochemical analyses of those surviving in this family, we also found four members who might develop leukemia in the future. Southern blot analysis of one of the four members and her father (an acute leukemia patient) with a v-ERBB probe showed that the gene abnormalities consisted of a c-ERBB rearrangement (hereditary) and a rearrangement/amplification of the same gene.

Blotting, Southern↗

PubMed1999

[Analysis of TTV infection in patients of hemodialysis and bone marrow transplantation].

OBJECTIVE: To make clear the infection of TT virus(TTV) in patients of hemodialysis and bone marrow-transplantation and to evaluate the virulence. METHODS: The TTV infection were investigated in 37 cases of hemodialysis and 90 cases of bone marrow-transplantation by means of review method. RESULTS: The positive rate of TTV was 16.22% (6/37) in 37 cases of hemodialysis and 3.33% (3/90) in 90 cases of bone marrow-transplantation. The difference between them was significant(P < 0.01). Of the six TTV-positive patients, 5 had blood transfusion and 4 of them had bone marrow-transplantation. Infection of TTV had nothing to do with the infection of HBV and HCV. There were no significant differences in the results of liver function tests between TTV positive and TTV negative patients. CONCLUSION: These results suggest that blood transfusion might be one of the critical routes to transmit TTV. The virulence of TTV might be mild. Whether TTV is a hepatitis viral remains further study.

Adult↗