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Biomedical subjects

F Chen

Publications and source records attributed to F Chen.

At least 469 records · Page 26Linked to original sources
PubMed1995

Cooperative binding of transforming growth factor (TGF)-beta 2 to the types I and II TGF-beta receptors.

TGF-beta 1 binds with high affinity (KD = 25-50 pM) directly to the TGF-beta type II receptor serine-threonine kinase (T beta-RII) in the absence of expression of the TGF-beta type I or III receptors (T beta-RI and T beta-RIII). The serine-threonine kinase T beta-RI is essential for TGF-beta 1 signaling but not for binding to T beta-RII. TGF-beta 2, in contrast, does not bind directly to T beta-RII, although coexpression of T beta-RIII does allow binding and cross-linking of TGF-beta 2 to T beta-RII. Here we show that in transfected COS cells binding and cross-linking of 125I-TGF-beta 2 to T beta-RI or T beta-RII requires expression of both receptors. In cells transfected with the c-myc-tagged human T beta-RII cDNA, only low amounts of 125I-TGF-beta 2 cross-linked to T beta-RI and T beta-RII were detected even with high concentrations (700 pM) of ligand. Cotransfection of the influenza-hemagglutinin-tagged human T beta-RI cDNA dramatically increased the binding of TGF-beta 2 to T beta-RII; the concentration of 125I-TGF-beta 2 required for half-maximal binding and cross-linking to T beta-RI and T beta-RII was approximately 40 pM. Coimmunoprecipitation studies showed that the high affinity receptor for TGF-beta 2 is composed of a hetero-oligomer of T beta-RI and T beta-RII. Thus TGF-beta 1 and -beta 2 bind to TGF-beta receptors in different ways; TGF-beta 1 binds directly to T beta-RII, while binding of TGF-beta 2 to T beta-RII requires coexpression of T beta-RI or T beta-RIII.

Activin Receptors, Type I↗
PubMed1995

The G-rich auxiliary downstream element has distinct sequence and position requirements and mediates efficient 3' end pre-mRNA processing through a trans-acting factor.

A downstream G-rich sequence (GRS), GGGGGAGGUGUGGG, has been previously shown to influence the efficiency of 3' end processing of the SV40 late polyadenylation signal. We have now defined several important parameters for GRS-mediated polyadenylation. The ability of the GRS to influence 3' end processing efficiency was sensitive to individual and multiple point mutations within the element, as well as the position of the element in the downstream region. Competition analysis indicated that the GRS functioned through a titratable trans-acting factor. The GRS-specific DSEF-1 protein was found to be bound to the same population of RNAs as the 64 kDa protein of the general polyadenylation factor CstF, indicating that DSEF-1 is associated with RNA substrates undergoing 3' end processing. Furthermore, an association was obtained between the relative strength of DSEF-1 protein binding to GRS variants and the relative ability of the GRS variants to mediate efficient cleavage in vitro. Finally, mutations in the GRS affected the efficiency of cross-linking of the 64 kDa protein of CstF. These data define a novel class of auxiliary downstream element and suggest an important role for DSEF-1 in 3' end processing.

Base Sequence↗
PubMed1995

Cytotoxicity, metabolism, and mechanisms of action of 2',2'-difluorodeoxyguanosine in Chinese hamster ovary cells.

The emerging clinical success of gemcitabine (2',2'-difluorodeoxycytidine) stimulated interest in the synthesis and evaluation of purine congeners. The cytotoxicity, metabolism, and mechanisms of action of the lead candidate, 2',2'-difluorodeoxyguanosine (dFdGuo), were studied in Chinese hamster ovary cells. Unlike the natural nucleoside deoxyguanosine (dGuo), dFdGuo was not a substrate for purine nucleoside phosphorylase. Wild-type Chinese hamster ovary cells and a mutant line deficient in deoxycytidine (dCyd) kinase were similarly affected by dFdGuo (50% inhibitory concentration, 7.5 and 6.5 microM, respectively), suggesting that unlike gemcitabine, dCyd kinase was not responsible for activation of dFdGuo. This was further confirmed by separation of nucleoside kinases (adenosine kinase, dGuo kinase, and dCyd kinase) of Chinese hamster ovary cells on DEAE-cellulose column chromatography. The kinase activity that phosphorylated dGuo also converted dFdGuo to its monophosphate, suggesting that dGuo kinase activated dFdGuo. Consistent with this result, coincubation with dGuo spared the dFdGuo-mediated toxicity; however, addition of up to 10 mM dCyd did not reverse the toxicity of dFdGuo. Intracellularly, dFdGuo was phosphorylated to its mono-, di-, and triphosphates; dFdGuo triphosphate (dFdGTP) was the major metabolite and accumulated to 45 microM after a 6-h incubation with 30 microM dFdGuo. The elimination of dFdGTP was monophasic with a t1/2 of about 6 h. Deoxynucleotides were decreased in cells incubated with dFdGuo, suggesting that ribonucleotide reductase was inhibited. dATP, which decreased 78% after a 4-h incubation with 30 microM dFdGuo, was most affected. dFdGuo was a potent inhibitor of DNA synthesis. Extension of a DNA primer over a defined template in the presence of dFdGTP revealed that dFdGTP was a good substrate for incorporation opposite C sites of the template by DNA polymerase alpha. dFdGTP incorporation caused DNA polymerase alpha to pause after the polymerization of one additional deoxynucleotide. This pattern of inhibition, which is shared by gemcitabine, distinguishes 2',2'-difluoronucleosides from arabinosylnucleosides which halt primer extension at the incorporation site. dGTP competed effectively with dFdGTP for incorporation by DNA polymerase alpha. The unique activation requirements and patterns of inhibition of DNA synthesis distinguish this promising new antimetabolite from other nucleoside analogues.

Adenosine Triphosphate↗
PubMed1995

Biochemical evidence for the autophosphorylation and transphosphorylation of transforming growth factor beta receptor kinases.

Transforming growth factor beta (TGF-beta) signals through a receptor complex containing the type I (TGF-beta RI) and type II (TGF-beta RII) receptors. We describe here biochemical studies on early events in the TGF-beta signaling pathways. TGF-beta RII is highly phosphorylated when expressed alone in COS-1 cells; its autophosphorylation occurs via an intramolecular (cis) mechanism that is independent of ligand binding. TGF-beta RI is also highly phosphorylated when expressed alone in COS-1 cells. Both wild-type TGF-beta RI and a kinase-deficient mutant thereof are transphosphorylated by the coexpressed TGF-beta RII kinase in a ligand-independent fashion in these cells. We propose that the association of TGF-beta RI and TGF-beta RII, induced by ligand binding or over-expression, leads to transphosphorylation of the TGF-beta RI by the TGF-beta RII kinase. This represents a mechanism of activation of receptors distinct from that of tyrosine kinase receptors and may apply to other serine/threonine kinase receptors.

Cells, Cultured↗
PubMed1995

Inhibition of cellular and SV40 DNA replication by the adeno-associated virus Rep proteins.

In order to define the mechanism used by the adeno-associated virus replication (rep) gene to mediate inhibition of cell proliferation, we have studied its effects on SV40 and cellular DNA replication. SV40 DNA replication was inhibited by the presence of the rep gene in human 293 cells, and the inhibition was not linked to suppression of SV40 early gene expression. Using double-immunofluorescence assays that measured both rep gene expression and bromodeoxyuridine incorporation, we found that the presence of the Rep78 and Rep68 proteins correlated with inhibition of cellular DNA synthesis in NIH3T3 cells. This links the rep gene's anti-proliferative effects to either: (i) a direct inhibition of DNA synthesis or (ii) a possible cell cycle block.

3T3 Cells↗
PubMed1995

Germline mutations in the von Hippel-Lindau disease tumor suppressor gene: correlations with phenotype.

von Hippel-Lindau disease (VHL) is an inherited neoplastic disease characterized by a predisposition to develop retinal angiomas, central nervous system hemangioblastomas, renal cell carcinomas, pancreatic cysts, and pheochromocytomas. The VHL gene was recently isolated by positional cloning. The cDNA encodes 852 nucleotides in 3 exons. The VHL gene is unrelated to any known gene families. We identified germline mutations in 85/114 (75%) of VHL families. Clinical heterogeneity is a well-known feature of VHL. VHL families were classified into 2 types based on the presence or absence of pheochromocytoma. The types of mutations responsible for VHL without pheochromocytoma (VHL type 1) differed from those responsible for VHL with pheochromocytoma (VHL type 2). Fifty-six % of the mutations responsible for VHL type 1 were microdeletions/insertions, nonsense mutations, or deletions; 96% of the mutations responsible for VHL type 2 were missense mutations. Specific mutations in codon 238 accounted for 43% of the mutations responsible for VHL type 2. The mutations identified in these families will be useful in presymptomatic diagnosis. The identification of mutations associated with phenotypes contributes to the understanding of fundamental genetic mechanisms of VHL disease.

Adrenal Gland Neoplasms↗
PubMed1995

Steady-state mRNA levels of G protein subunits in developing rabbit myocardium.

Cardiac responsiveness to beta-adrenergic stimulation changes with age. Developmental changes in expression of guanine nucleotide-binding coupling protein (G protein) subunits may account for these physiologic changes. We measured steady-state levels of mRNA encoding the alpha-subunit of the specific G protein that stimulates adenylyl cyclase (Gs alpha) and three isoforms of beta-subunit of G proteins (G beta) in developing myocardium. Total RNA prepared from the right and left ventricles of fetal, neonatal, juvenile, and adult rabbits was size-fractionated, blotted, and probed with 32P-labeled cDNAs encoding rat Gs alpha, bovine G beta-1, human G beta-2, and human G beta-3. For standardization, these blots were subsequently hybridized with a 32P-labeled cDNA encoding glyceraldehyde 3-phosphate dehydrogenase (GAPD). Two-dimensional densitometric analysis of autoradiographs was used to quantify relative hybridization intensities. An age-dependent decrease in mRNAs encoding Gs alpha, G beta-1, and G beta-2 relative to mRNA encoding GAPD was observed in both ventricles, while G beta-3 mRNA was not detected. At all ages studied, levels of Gs alpha and G beta-1 mRNA were similar in the two ventricles. However, G beta-2 mRNA declined more in the left ventricle than in the right ventricle during maturation. Our results demonstrate developmental control in heart for mRNAs encoding several G protein subunits. In addition, differential declines in G beta-1 and G beta-2 mRNA in the right ventricle suggest that these G beta isoforms are regulated uniquely and may reflect functional roles for these G beta isoforms in different signaling cascades.

Adenylyl Cyclases↗
PubMed1995

Ionic and haemodynamic changes influence the release of the excitatory amino acid glutamate in the posterior hypothalamus.

The push-pull technique was used to investigate the release of the excitatory amino acid glutamate in the posterior hypothalamic area of the conscious rat. The hypothalamus was superfused through the push-pull cannula with artificial cerebrospinal fluid (CSF), and the superfusate was collected in time periods of 10 min when ionic conditions in the CSF were changed, or in short periods of 3 min when blood pressure changes were evoked. The mean glutamate release rate was 2.8 +/- 0.7 pmol/min. Depolarization by hypothalamic superfusion with CSF containing 50 mM K+ enhanced the release of glutamate in the presence of Ca2+. The K(+)-induced release was attenuated by 40% when the hypothalamus was superfused with Ca(2+)-free CSF. Replacement of Ca2+ by Mg2+ abolished the K(+)-induced release of glutamate. Hypovolaemia elicited by haemorrhage enhanced the release rate of glutamate. Similarly, a hypotension elicited by i.v. injection of chlorisondamine (3 mg/kg) led to a pronounced and permanent enhancement in glutamate release. The effects of hypovolaemia and chlorisondamine on glutamate release were abolished in aortic denervated rats, indicating that this response is due to a decrease of impulse generation in baroreceptors. A hypervolaemia elicited by blood infusion did not affect the release of glutamate. Similarly, a pronounced pressor response to phenylephrine (15 micrograms/kg per minute) infused intravenously for 9 min was ineffective. The results show that the K(+)-induced release of glutamate in the hypothalamus is dependent on the presence of Ca2+. The increase in glutamate release rate by hypovolaemia or chlorisondamine suggests that the glutamatergic neurons in the posterior hypothalamic area respond to unloading of aortic baroreceptors and possess a counteracting, hypertensive function.

Animals↗
PubMed1995

Von Hippel-Lindau (VHL) disease with pheochromocytoma in the Black Forest region of Germany: evidence for a founder effect.

We identified a germline missense mutation at nucleotide 505 (T to C) of the VHL tumor suppressor gene in 14, apparently unrelated, VHL type 2A families from the Black Forest region of Germany. This mutation was previously identified in two VHL 2A families living in Pennsylvania (USA). All affected individuals in the 16 families shared the same VHL haplotype indicating a founder effect. This missense mutation at codon 169 (Tyr to His) would probably cause an alteration in the structure of the putative VHL protein. The association of this distinct mutation with the pheochromocytoma phenotype in VHL may help to elucidate the genetic mechanism of carcinogenesis in this multi tumor cancer syndrome.

Adrenal Gland Neoplasms↗
PubMed1995

Differences in the growth pattern and clinical course of EBV-LMP1 expressing and non-expressing nasopharyngeal carcinomas.

All low differentiated or anaplastic forms of nasopharyngeal carcinoma (NPC) carry multiple copies of EBV-DNA and express EBNA1. The major membrane protein, LMP1, is only expressed in 65% of the tumours. The physiological function of LMP1 in the viral life cycle is unknown, but it has been shown to transform established rodent fibroblasts and immortalised human keratinocytes in vitro, and to increase the likelihood of a malignant transformation. We studied 74 cases collected from the Shanghai and Guanzhou areas in China. LMP1 expression was assessed in tumour biopsies by immunoblotting. Clinical and follow-up data were evaluated according to the classification of WHO. The laboratory and the clinical data were assembled in a mutually independent double blind fashion. Our findings indicate that the LMP1-positive tumours grew faster and more expansively than LMP1-negative tumours, but nevertheless had a better prognosis. LMP1-negative tumours recurred at a higher frequency, and showed an increased tendency to metastasise.

Carcinoma↗
PubMed1995

Infrequent MDM2 gene amplification and absence of gross WAF1 gene alterations in nasopharyngeal carcinoma.

We have investigated the possible involvement of MDM2 and WAF1 gene alterations in the development of nasopharyngeal carcinoma (NPC). MDM2 and WAF1 were analysed in 46 primary NPCs by Southern blot analysis. Forty-five tumours showed a normal EcoRI hybridisation pattern and hybridisation intensity with a human MDM2 cDNA probe. One tumour had more intense normal size MDM2 hybridising bands. Densitometric scanning revealed a 10-12-fold MDM2 gene amplification, as compared with human placenta DNA. All 46 tumours showed normal size WAF1 EcoRI bands that hybridised with normal intensity. This is the first demonstration of MDM2 gene amplification in NPC. Nonetheless, our analysis indicates that gross structural alterations of the MDM2 and WAF1 genes are infrequent events in the genesis of NPC.

Blotting, Southern↗
PubMed1995

Coupled transcription of Epstein-Barr virus latent membrane protein (LMP)-1 and LMP-2B genes in nasopharyngeal carcinomas.

The presence of transcripts of the Epstein-Barr virus genes for Epstein-Barr nuclear antigen (EBNA)-1 and EBNA-2 and for latent membrane protein (LMP)-1, LMP-2A and LMP-2B was investigated in 24 nasopharyngeal carcinoma (NPC) biopsies of Chinese origin and two NPC-derived solid tumour lines, CAO and C15, of Chinese and north African origin respectively, propagated by serial transplantation in nude mice. Transcripts were detected by PCR amplification of cDNA. EBNA-1 transcripts were present in all biopsies tested and originated exclusively from the FQ promoter while the C and W promoters were inactive. Using nested primers, LMP-1 and LMP-2B RNAs were found to be co-ordinately expressed in 22 of the 24 biopsies, while the two remaining tumours were negative for both. LMP-2A transcription was detected in 17 of the 22 LMP-1-positive biopsies. In summary, the following patterns of viral gene expression were observed in the tumour biopsies: (i) LMP-1-LMP-2B and LMP-2A positive (17 biopsies); (ii) LMP-1-LMP-2B positive but LMP-2A negative (five biopsies); (iii) no viral gene other than EBNA-1 expressed (two biopsies).

Animals↗
PubMed1995

Expression of protein kinase C gene in the brain and heart of spontaneously hypertensive rats.

1. The aim of this study was to investigate protein kinase C (PKC) gene expression in spontaneously hypertensive rats (SHR). 2. Using the PKC oligodeoxyribonucleotide probes (gamma, epsilon), we detected PKC isoforms gene expression in the heart and brain of 4 and 20 week old SHR with those of age-matched Wistar-Kyoto (WKY) rats by northern blot analysis. 3. In the cerebral cortex, there were significantly increased levels of expression of the Ca2+-dependent isoform PKC-gamma in 4 and 20 weeks SHR compared with that of WKY, while Ca2+ -independent isoform PKC-epsilon did not differ between SHR and WKY. 4. In ventricular myocytes, there was a significant expression of the Ca2+-independent isoform PKC-epsilon in 4 and 20 week old SHR compared with that of WKY, while Ca2+ -dependent isoform PKC-gamma could not be detected in the same extracts of SHR or WKY. 5. We conclude that both of the Ca2+ -dependent and Ca2+ -independent PKC could be involved in the pathogenesis of SHR. Ca2+ -dependent PKC-gamma may be mainly involved in the modulation of blood pressure in the level of the central nervous system, while Ca2+ -independent PKC-epsilon could be related to the genetic myocardial hypertrophy.

Animals↗
PubMed1995

Amplification of DNA polymerase gene fragments from viruses infecting microalgae.

Nested PCR with three highly degenerate primers was used for amplification and identification of DNA polymerase (pol) genes from viruses which infect three genera of microalgae. Group-specific primers (AVS1 and AVS2) were designed on the basis of inferred amino acid sequences unique to the DNA pol genes of viruses (PBCV-1 and NY-2A) that infect an endosymbiotic Chlorella-like alga (Chlorophyceae) and a virus (MpV-SP1) which infects the photosynthetic flagellate Micromonas pusilla (Prasinophyceae). In addition, a nested primer (POL) was designed on the basis of the highly conserved amino acid sequence YGDTDS found in most B-family (alpha-like) DNA pol genes. These primers were used to amplify DNA from the three viruses, PBCV-1, NY-2A, and MpV-SP1, for which the primers were designed, as well as eight clonal isolates of genetically distinct viruses which infect M. pusilla and others which infect Chrysochromulina spp. (Prymnesiophyceae), suggesting that these are a group of related viruses. In contrast, no product resulted from using DNA from viruses which infect the marine brown algae Ectocarpus siliculosis and Feldmannia sp. (Phaeophyceae), suggesting that these viruses may not be closely related to those that infect microalgae. These primers were also used to amplify DNA from natural virus communities. Our results indicate that nested PCR, even under low-stringency conditions, can be used as a rapid method to verify the presence in seawater of a group of related viruses which infect microalgae. Sequence analysis of these fragments should provide information on the genetic diversity and potentially the phyletic relationships among these viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Genetic relatedness of Cryptococcus neoformans clinical isolates grouped with the repetitive DNA probe CNRE-1.

Cryptococcus neoformans isolates from eight patients with cryptococcal infection were previously assigned into three groups on the basis of repetitive DNA probe (CNRE-1) restriction fragment length polymorphisms. These groups accounted for a disproportionate number of recent clinical isolates in New York City. To further examine the genetic relatedness of isolates within and across CNRE-1 groups, the DNA sequence of the 779-base URA5 gene from each strain was amplified and sequenced. The number of nucleotide differences occurred in the third codon position or in introns. Pairwise comparisons revealed average nucleotide differences within a CNRE-1 group of 4.8 +/- 2.6 (n = 8) and between CNRE-1 groups of 21.9 +/- 7.0 (n =20) (P <0.001) Analysis of URA5 sequences defined three groups that were congruent with those defined by CNRE-1 restriction fragment length polymorphisms. PCR amplification of an rDNA intergenic spacer revealed conservation of the intergenic spacer length within groups. Electrophoretic karyotyping did not distinguish between two isolates in each of two CNRE-1 groups. DNA from all isolates studied hybridized to an alpha mating type-specific probe. We interpret these results as suggesting a clonal population structure for some pathogenic isolates of C. neoformans in New York City.

Base Sequence↗
PubMed1995

A subpopulation of normal B cells latently infected with Epstein-Barr virus resembles Burkitt lymphoma cells in expressing EBNA-1 but not EBNA-2 or LMP1.

Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments mixing different proportions of Epstein-Barr virus (EBV)-carrying and EBV-negative cells. Based on the results, a method that detects viral transcripts for EBNA-1, EBNA-2, LMP1, and LMP2a from less than one positive cell among 10(5) negative cells was developed. With this method we have shown that the EBV DNA positive cells among small, high-density peripheral blood B-lymphocytes of normal healthy persons express EBNA-1-mRNA but not EBNA-2 or LMP1. A similar EBV expression pattern is found in type I Burkitt lymphoma cells. We suggest that the expression pattern in the lymphoma cells reflects the viral strategy in normal resting B cells and meets the requirements of latent persistence.

Animals↗
PubMed1995

Detection of germline mutations in the von Hippel-Lindau disease gene by the primer specified restriction map modification method.

Von Hippel-Lindau disease (VHL) is an inherited disorder characterised by a predisposition to develop tumours in the eyes, central nervous system, kidneys, and adrenal glands. Recently the VHL gene was cloned and shown to be mutated in 75% of US and Canadian VHL families. To develop simple, rapid methods for the detection of mutations found in large numbers of affected people, we designed based on the primer specified restriction site modification method. These tests have proved useful in identifying asymptomatic mutated VHL gene carriers who have the nt 505 T to C mutation or the nt 686 T to C mutation. Together with an MspI digestion test which can detect a mutation hot spot in codon 238, polymerase chain reaction/restriction endonuclease based tests can now detect VHL mutations in more than 50% of VHL type 2 families.

Base Sequence↗