Biomedical subjects

F Chen

Publications and source records attributed to F Chen.

At least 361 records · Page 20Linked to original sources

PubMed1998

[Establishment of a method for detecting transforming growth factor beta 1 mRNA].

AIM: To establish a method to measure the TGF-beta 1 mRNA level for studying the mechanism of fibrogenesis caused by schistosomiasis japonica. METHODS: Reverse-transcription polymerase chain reaction and dot blot analysis were used. A plasmid of TGF-beta 1 was constructed for standardization, and beta-actin was used as control. Eight different concentrations of the plasmid and 11 double-tube of TGF-beta 1 mRNA in the peripheral blood mononuclear cell (PBMC) of different persons were measured. RESULTS: Quantitative results of 8 different dilutions of TGF-beta 1 plasmid had positive with the logarithm of the original concentration. The results of the 11 double-tube were 1.71 +/- 0.90 and 1.54 +/- 0.88. CONCLUSION: The duplicability and stability of the method showed it can be used to analyse the TGF-beta 1 mRNA level of the peripheral blood mononuclear cells.

Humans↗

PubMed1998

[The distribution of nitric oxide synthetase in Vcx and its relation with the expression of FOS induced by teeth movement in rats].

It was studied the central role of nitric oxide(NO) during experimental teeth movement and the relation between nitric oxide synthetase (NOS) positive neurons and FOS like immunoreactivity (FLN) with the NADPH-diaphorase histochemistry and immunocytochemical reaction method. Results indicated that NOS positive neurons and FLN showed typical distribution in Vcx and there was some overlap between them. It suggests that NO is involved in the central modulation of the stimulating message of teeth movement, and which further explains the central modulation mechanism of experimental teeth movement in rats.

Animals↗

PubMed1998

Butyrate attenuates BCLX(L) expression in human fibroblasts and acts in synergy with ionizing radiation to induce apoptosis.

Ionizing radiation is a poor inducer of apoptosis in many cell types. In this study we investigated what effect the differentiation agent butyrate had on the cellular levels of the apoptosis regulators BCL2, BCLX(L) and BAX and on radiation-induced apoptosis. It was found that butyrate significantly lowered the level of the apoptosis antagonist BCLX(L) in a time- and dose-dependent manner in diploid human fibroblasts. The reduction of the level of BCLX(L) protein by butyrate correlated with an increased induction of apoptosis in cells irradiated with ionizing radiation. Butyrate also acted in synergy with ultraviolet (UV) light and cisplatin to induce apoptosis in human fibroblasts. Radiosensitization was obtained when butyrate was added before and/or after irradiation, although the combination of treatment both before and after irradiation was the most effective. Our results suggest that butyrate acts in synergy with ionizing radiation, UV light and cisplatin to induce apoptosis, perhaps by lowering the level of the apoptosis antagonist BCLX(L) in cells.

Apoptosis↗

PubMed1998

Pertussis toxin modification of PC12 cells lowers cytoskeletal F-actin and enhances norepinephrine secretion: involvement of protein kinase C and protein phosphatases.

We have investigated the relationship between norepinephrine secretion and cytoskeletal F-actin in rat phaeochromocytoma PC12 cells. Stimulation of PC12 cells with extracellular ATP or high K+ caused both the release of norepinephrine and a decrease in F-actin. The stimulation of secretion and the decrease in F-actin were dependent on extracellular Ca2+. The addition of Ca2+ to digitonin-permeabilized PC12 cells also stimulated norepinephrine release and decreased F-actin. Modification of PC12 cells with pertussis toxin caused a 35% decrease in F-actin, and it enhanced ATP-stimulated and K+ stimulated norepinephrine secretion from intact cells and Ca(2+)-dependent norepinephrine secretion from permeabilized cells. After down regulation of protein kinase C, pertussis toxin still enhanced secretion, but it had no effect on F-actin indicating that the effect of pertussis toxin on F-actin was dependent on protein kinase C activity. The addition of okadaic acid, an inhibitor of serine/threonine protein phosphatases, to PC12 cells caused a decrease F-actin, but it had no effect on ATP-stimulated or K(+)-stimulated norepinephrine secretion. After down regulation of protein kinase C, much higher concentrations of okadaic acid were need to decrease F-actin. The similarity between the effects of pertussis toxin and low concentrations of okadaic acid suggest that the effect of pertussis toxin on cytoskeletal F-actin in PC12 cells may result from an inhibition of protein phosphatase 2A.

Actins↗

PubMed1997

[3H]zolpidem binding in alcohol-preferring and non-preferring rat brain.

The present study has employed in vitro autoradiography to study the distribution and density of [3H]zolpidem binding sites, which are regarded as an index of ethanol-sensitive gamma-aminobutyric acid (GABA)A receptors, in the brains of alcohol-preferring Fawn-Hooded (FH) rats compared to non-alcohol preferring Wistar-Kyoto (WKY) rats. Binding of [3H]zolpidem showed a similar distribution profile in both rat strains examined and included cerebellum, globus pallidus, nucleus of the solitary tract and a number of midbrain/hindbrain nuclei. Densitometric quantitation of binding revealed that FH rats possessed a significantly higher density of [3H]zolpidem binding compared to WKY rats in cortical regions, substantia nigra pars reticulata and the ventral pallidum. These data indicate that FH rats may have an increased number of ethanol-sensitive GABA(A) receptors in regions intimately involved in reward processes, and may partially explain the alcohol-seeking nature of the FH rat.

Animals↗

PubMed1997

Tanshinone production in Ti-transformed Salvia miltiorrhiza cell suspension cultures.

Transformed cell cultures of Salvia miltiorrhiza were established by infecting sterile plantlets with Agrobacterium tumefaciens strain C58. The transformed cells in suspension formed macroscopic clumps of cell aggregates up to 2-3 cm in size rather than homogeneous cell suspensions. These transformed cells grew well in hormone-free media. It was found that the B5 mediums supported the best growth while the 6,7-V medium promoted tanshinone production in the transformed cell suspension cultures. The effect of initial sucrose concentration on cell growth was also studied. The best growth was observed when cells were cultivated in the B5 medium containing 30 g l-1 sucrose. Although low levels of tanshinones were produced in fast growing cell cultures, there existed a rapid increase in tanshinone production when the cell aggregates were transferred to the fresh yeast-extract-containing medium. By this two-stage culture method, about 22 mg tanshinones were produced in 1 liter of medium. Green cell aggregates were formed when cells were cultured under illumination. Light was found to have an inhibitory effect on tanshinone biosynthesis. A sensitive high-performance liquid chromatographic method was developed for the measurement of tanshinones. Cryptotanshinone, tanshinone I and tanshinone IIA were identified from the transformed cultures.

Abietanes↗

PubMed1997

Biological characterization of Drosophila Rapgap1, a GTPase activating protein for Rap1.

The activity of Ras family proteins is modulated in vivo by the function of GTPase activating proteins, which increase their intrinsic rate of GTP hydrolysis. We have isolated cDNAs encoding a GAP for the Drosophila Rap1 GTPase. Drosophila Rapgap1 encodes an 850-amino acid protein with a central region that displays substantial sequence similarity to human RapGAP. This domain, when expressed in Escherichia coli, potently stimulates Rap1 GTPase activity in vitro. Unlike Rap1, which is ubiquitously expressed, Rapgap1 expression is highly restricted. Rapgap1 is expressed at high levels in the developing photoreceptor cells and in the optic lobe. Rapgap1 mRNA is also localized in the pole plasm in an oskar-dependent manner. Although mutations that completely abolish Rapgap1 function display no obvious phenotypic abnormalities, overexpression of Rapgap1 induces a rough eye phenotype that is exacerbated by reducing Rap1 gene dosage. Thus, Rapgap1 can function as a negative regulator of Rap1-mediated signaling in vivo.

Amino Acid Sequence↗

PubMed1997

Genetic interactions with Rap1 and Ras1 reveal a second function for the fat facets deubiquitinating enzyme in Drosophila eye development.

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential very early in eye development, prior to facet assembly, to limit the number of photoreceptor cells in each facet of the compound eye to eight. The Fat facets protein facilitates the production of a signal in cells outside the developing facets that inhibits neural development of particular facet precursor cells. Novel gain-of-function mutations in the Drosophila Rap1 and Ras1 genes are described herein that interact genetically with fat facets mutations. Analysis of these genetic interactions reveals that Fat facets has an additional function later in eye development involving Rap1 and Ras1 proteins. Moreover, the results suggest that undifferentiated cells outside the facet continue to influence facet assembly later in eye development.

Amino Acid Sequence↗

PubMed1997

Ku80 gene expression is Sp1-dependent and sensitive to CpG methylation within a novel cis element.

The Ku70/80 complex, known as Ku, constitutes the DNA end binding component of the DNA-dependent protein kinase (DNA-PK). We have characterized the promoter region of the mouse and human Ku80 genes to delineate transcriptional elements necessary for basal gene expression and proliferation-dependent regulation. Consensus Sp1 recognition elements were identified in both promoters, and were determined to be essential for basal expression. We further identified a near-perfect palindrome of 21 base pairs located immediately 5' to one Sp1 element. This sequence was present once within the mouse Ku80 promoter and seven times, in a head-to-tail tandem array, within the human Ku80 promoter. This sequence possessed homology with a methylation-sensitive promoter element, Enh2, present in the LTR of mouse intractisternal A-particles. Promoter deletion studies and expression analysis of in-vitro methylated reporter gene constructs provided strong evidence that, in vivo, this repeat sequence regulates Ku80 gene expression in cis, through a mechanism involving CpG methylation. Evidence is also presented, suggesting that Ku is directly involved in this regulatory process.

Animals↗

PubMed1997

Calpain contributes to silica-induced I kappa B-alpha degradation and nuclear factor-kappa B activation.

Both silica and lipopolysaccharide (LPS) induce a rapid degradation of I kappa B alpha, an intracellular inhibitor of the nuclear factor (NF)-kappa B transcription factor. In this report, we demonstrate that MG132, a relatively specific proteasome inhibitor, is capable of suppressing LPS-induced I kappa B alpha degradation and NF-kappa B activation in mouse macrophage line RAW 264.7 cells, but is unable to influence the same induction produced by silica. In contrast, the lysosome inhibitor chloroquine has little effect on I kappa B alpha degradation induced by either silica or LPS. In fact, chloroquine enhances the signal-induced nuclear expression of NF-kappa B p50/p65 heterodimer by inhibiting the resynthesis of I kappa B alpha. With the use of transient transfection of a plasmid that expresses calpastatin, a natural inhibitor for calpain, the silica-induced degradation of I kappa B alpha and NF-kappa B activation was attenuated. In contrast, no inhibition of LPS-induced I kappa B alpha degradation and NF-kappa B activation was observed by the overexpression of calpastatin. This suggests that calpain contributes to silica-induced I kappa B alpha degradation and NF-kappa B activation but not to LPS-induced I kappa B alpha degradation and NF-kappa B activation.

Animals↗

PubMed1997

Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance.

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob/ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db/db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.

Animals↗

PubMed1997

Botulinum neurotoxin B inhibits insulin-stimulated glucose uptake into 3T3-L1 adipocytes and cleaves cellubrevin unlike type A toxin which failed to proteolyze the SNAP-23 present.

Types A, B, and C1 botulinum neurotoxin (BoNT), a group of selective Zn2+-dependent endoproteases, have been instrumental in demonstrating that their respective substrates [synaptosomal-associated protein with Mr = 25 kDa (SNAP-25), synaptobrevin (Sbr), and syntaxin] are essential for regulated exocytosis from nerve terminals and neuroendocrine cells. The colocalization of Sbr, or its homologue cellubrevin (Cbr), in the majority of the glucose transporter-isotype 4 (GLUT4)-containing vesicles from adipocytes implicates their involvement in insulin-stimulated glucose uptake, which results in part from enhanced fusion of these vesicles with the plasmalemma. In this study, exposure of cultured 3T3-L1 adipocytes to BoNT/B in a low-ionic strength medium was found to block insulin-evoked glucose uptake by up to 64%. BoNT/B was shown by immunoblotting to cause extensive proteolysis of Cbr and Sbr resulting in a significant blockade of the insulin-stimulated translocation of GLUT4 to the plasmalemma. This establishes that these two toxin substrates contribute to the insulin-regulated fusion of GLUT4-containing vesicles with the plasmalemma, at least in this differentiated 3T3-L1 clone. Although SNAP-25 was not detectable in the differentiated adipocytes, its functional homologue SNAP-23 is abundant and largely confined to the plasmalemma. SNAP-23 proved to be resistant to cleavage by BoNT/A. Consistent with these results, type A did not block insulin-induced glucose uptake, precluding a demonstration of its likely importance in this process.

3T3 Cells↗

PubMed1997

Epstein-Barr virus (EBV) gene expression in lymphoid B cells during acute infectious mononucleosis (IM) and clonality of the directly growing cell lines.

We examined the patterns of viral gene expression in acute infectious mononucleosis (IM) patients and the clonality of the directly growing EBV-carrying cell lines. Both low- and high-density EBV-carrying B cells obtained from the patients' tonsils expressed EBNA1, EBNA2 and LMP1. Like LCLs and immunoblastic B-cell lymphomas, the in vivo EBV-carrying low-density cells used only the latency III program for viral gene expression. The in vivo EBV-carrying high-density B cells used both the latency I program, as indicated by the QUK-, and the latency III program, as indicated by the YUK-EBNA1. This suggests that the lymphoid tissues contained not only proliferating immunoblasts but also cells programmed for latent viral persistence in vivo. EBV-carrying cells that grew directly into permanent cell lines in the presence of virus-neutralizing antibody and a late viral inhibitor were polyclonal, as indicated by JH rearrangement. Two of the high-density-derived lines had identical JH and TR patterns, indicating a common parental origin. Our investigation indicates that EBV-carrying cells divide and survive in a fully competent immune system during the outbreak of acute IM.

Antigens, Viral↗

PubMed1997

Phosducin and betagamma-transducin interaction I: effects of post-translational modifications.

The interaction between phosducin and betagamma-transducin plays regulatory roles in light adaptation of photoreceptors. Both phosducin and betagamma-transducin undergo post-translational modifications, with phosducin modified by phosphorylation and the gamma subunit of betagamma-transducin by farnesylation and carboxylmethylation. In this study we exploited the electrophoretic mobilities of these native proteins to develop a micro binding assay and examined the effects of post-translational modifications on binding affinities. It was found that decarboxylmethylation of gamma-transducin increased the mobility of betagamma-transducin during native gel electrophoresis, but decreased the apparent affinity for phosducin by about 2-fold. Phosphorylation of phosducin by protein kinase A increased the mobility but decreased the apparent affinity for betagamma-transducin by at least 3-fold.

Animals↗

PubMed1997

Characterization of two DNA double-stranded break repair-deficient cell lines that express inactive DNA-dependent protein kinase catalytic subunits.

The DNA-dependent protein kinase (DNA-PK) is a trimeric enzyme consisting of a 460-kDa catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex called Ku, which is comprised of 70 (Ku70) and 86 (Ku80) kDa subunits. Mutations that affect the expression of the catalytic or Ku80 subunits of DNA-PK disrupt both V(D)J recombination and DNA double-stranded break repair pathways. In this report, we show that two previously uncharacterized rodent cell lines that are defective in DNA double-stranded break repair express catalytically inactive DNA-PK. The DNA-PKcs from the DNA double-stranded break repair mutant cell lines IRS-20 and SX-9 assembles on double-stranded DNA but fails to function as a protein kinase. In addition to the kinase defect, the abundance of the DNA-PKcs from both of these cell lines is reduced relative to wild-type controls. These results suggest that the DNA-PKcs gene from each of these cell lines contains mutations that inactivate the enzymatic activity and the expression or stability of the gene product. These data further strengthen the hypothesis that DNA-PK-mediated protein phosphorylation is a necessary component of the DNA double-stranded break repair pathway.

Animals↗

PubMed1997

One-electron reduction of chromium(VI) by alpha-lipoic acid and related hydroxyl radical generation, dG hydroxylation and nuclear transcription factor-kappaB activation.

Reaction of chromium(VI) with alpha-lipoic acid (reduced form, also called 1,2-dithiolane-3-pentanoic acid) generated Cr(V) and hydroxyl radical (*OH) as measured by electron spin resonance and ESR spin trapping. 5,5-Dimethyl-1-pyrroline was used as a spin trapping agent. Catalase inhibited the *OH generation and enhanced the Cr(V) formation. Superoxide dismutase had an opposite effect. H2O2 enhanced the *OH generation and decreased the Cr(V) formation in a dose-dependent manner. Metal chelators, EDTA, diethylenetriaminepentaacetic acid, deferoxamine, and 1, 10-phenanthroline inhibited *OH radical generation in the order of EDTA > 1,10-phenanthroline > DTPA > deferoxamine. Oxygen consumption measurements indicated that molecular oxygen was used to generate *OH radical in the mixture of Cr(VI) and alpha-lipoic acid. H2O2 and superoxide radical (O2-) were involved as reactive intermediates. The *OH radical was generated via Cr(V)-mediated Fenton-like reaction (Cr(V) + H2O2 --> Cr(VI) + OH- + *OH). HPLC measurements show that the *OH radical generated by this reaction is capable of generating 8-hydroxyl-2'-deoxyguanosine from 2-deoxyguanosine. Incubation of Cr(VI) with cultured Jurkat cells resulted in an activation of DNA binding activity of the nuclear factor (NF)-kappaB. Addition of alpha-lipoic acid enhanced the NF-kappaB activation, while the *OH radical scavenger, sodium formate, inhibited it, showing that alpha-lipoic acid enhanced Cr(VI)-induced NF-kappaB activation via free radical reactions. The results indicate that while alpha-lipoic acid is considered to be an antioxidant, it may be a cellular one-electron Cr(VI) reductant and could be involved in the mechanism of Cr(VI)-induced carcinogenesis.

Antioxidants↗

PubMed1997

Analysis of IgM structures involved in J chain incorporation.

J chain is associated with pentameric IgM and polymeric IgA. In IgM, J chain is disulfide bonded to the C575 residue of the mu-chain, located in the mu tail piece (mu tp). Previous studies indicated that mu tp is not sufficient to mediate J chain incorporation into polymeric Ig. In this study, we analyzed which other C mu domains are involved in J chain incorporation. Three altered forms of mouse IgM were analyzed: IgM lacking the C mu 1 domain, IgM in which the C mu 2 and C mu 3 domains were replaced by the hinge region and the C gamma 2 domain of IgG2b, and IgM, in which the C mu 4 domain was replaced by C gamma 3. We found that neither C mu 1, C mu 2, nor C mu 3 was absolutely required for J chain incorporation. The importance of C mu 4 could not be fully analyzed because the C gamma 3 replacement mutant failed to form polymers. Also, we found that the glycosylation site at asparagine 563 of mu tp was important for J chain incorporation. Disruption of this site by replacement of either asparagine 563 by tyrosine or serine 565 by phenylalanine resulted in diminished J chain incorporation and increased production of hexameric IgM. These results demonstrate the importance of structural elements located close to mu tp in the incorporation of J chain into IgM.

Amino Acid Sequence↗

PubMed1997

Tetrandrine inhibits signal-induced NF-kappa B activation in rat alveolar macrophages.

Tetrandrine is a bisbenzylisoquinoline alkaloid isolated from a natural Chinese herbal medicine. While this alkaloid has been shown to exhibit antifibrotic and anti-inflammatory activities, its mechanism of action is unknown. The present study was designed to investigate the inhibitory effect of tetrandrine on NF-kappa B activation in the alveolar macrophage. Three different provocative stimuli were used to activate NF-kappa B in these cells. The results indicate that tetrandrine can inhibit the activation of NF-kappa B and NF-kappa B-dependent reporter gene expression by LPS, PMA, and silica in a dose-dependent manner. In contrast, at the doses used, tetrandrine did not interfere with Sp-1 DNA binding activity or Sp-1-dependent reporter gene expression in these cells. Western blot analysis suggests that the inhibitory effect of tetrandrine on NF-kappa B activation can be attributed to its ability to suppress signal-induced degradation of I kappa B alpha, a cytoplasmic inhibitor of the NF-kappa B transcription factor.

Alkaloids↗