Biomedical subjects

D Chen

Publications and source records attributed to D Chen.

At least 397 records · Page 22Linked to original sources

PubMed1998

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) stimulation of GLUT4 translocation is tyrosine kinase-dependent.

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) treatment of permeabilized adipocytes results in GLUT4 translocation similar to that elicited by insulin treatment. However, although the selective phosphatidylinositol 3-kinase inhibitor, wortmannin, completely prevented insulin-stimulated GLUT4 translocation, it was without effect on GTPgammaS-stimulated GLUT4 translocation. In addition, insulin was an effective stimulant, whereas GTPgammaS was a very weak activator of the downstream Akt serine/threonine kinase. Consistent with an Akt-independent mechanism, guanosine 5'-O-2-(thio)diphosphate inhibited insulin-stimulated GLUT4 translocation without any effect on the Akt kinase. Surprisingly, two functionally distinct tyrosine kinase inhibitors, genistein and herbimycin A, as well as microinjection of a monoclonal phosphotyrosine specific antibody, inhibited both GTPgammaS- and insulin-stimulated GLUT4 translocation. Phosphotyrosine immunoblotting and specific immunoprecipitation demonstrated that GTPgammaS did not elicit tyrosine phosphorylation of insulin receptor or insulin receptor substrate-1. In contrast to insulin, proteins in the 120-130-kDa and 55-75-kDa range were tyrosine-phosphorylated following GTPgammaS stimulation. Several of these proteins were identified and include protein-tyrosine kinase 2 (also known as CAKbeta, RAFTK, and CADTK), pp125 focal adhesion tyrosine kinase, pp130 Crk-associated substrate, paxillin, and Cbl. These data demonstrate that the GTPgammaS-stimulated GLUT4 translocation utilizes a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.

3T3 Cells↗

PubMed1998

Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state.

Human P-glycoprotein (Pgp), a plasma membrane protein that confers multidrug resistance, functions as an ATP-dependent drug efflux pump. Pgp contains two ATP binding/utilization sites and exhibits ATPase activity that is stimulated in the presence of substrates and modulating agents. The mechanism of coupling of ATP hydrolysis to drug transport is not known. To understand the role of ATP hydrolysis in drug binding, it is necessary to develop methods for purifying and reconstituting Pgp that retains properties including stimulation of ATPase activity by known substrates to an extent similar to that in the native membrane. In this study, (His)6-tagged Pgp was expressed in Trichoplusia ni (High Five) cells using the recombinant baculovirus system and purified by metal affinity chromatography. Upon reconstitution into phospholipid vesicles, purified Pgp exhibited specific binding to analogues of substrates and ATP in affinity labeling experiments and displayed a high level of drug-stimulated ATPase activity (specific activity ranging from 4.5 to 6.5 micromol min-1 mg-1). The ATPase activity was inhibited by ADP in a competitive manner, and by vanadate and N-ethylmaleimide at low concentrations. Vanadate which is known to inhibit ATPase activity by trapping MgADP at the catalytic site inhibited photoaffinity labeling of Pgp with substrate analogues, [125I]iodoarylazidoprazosin and [3H]azidopine, only under ATP hydrolysis conditions. Because vanadate-trapped Pgp is known to resemble the ADP and phosphate-bound catalytic transition state, our findings indicate that ATP hydrolysis results in a conformation with reduced affinity for substrates. A catalytic transition conformation with reduced affinity would essentially result in substrate dissociation and supports a model for drug transport in which an ATP hydrolysis-induced conformational change leads to drug release toward the extracellular medium.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Evidence for the separate regulation of the human papillomavirus type 11 E7 and E6 promoters by Viral cis sequences near the E6 promoter.

The human papillomavirus type 11 (HPV-11) E7 protein can modulate host cell functions and is required for papilloma formation, but little is known concerning the regulation of its expression. This study was designed to determine whether the viral upstream regulatory region controlled expression from the E7 promoter and whether cis sequences differentially regulated E6 and E7 expression in laryngeal mucosal keratinocytes, the natural target cells for this virus. Reporter constructs were designed to study expression of the luciferase gene from the HPV-11 E7 promoter in its natural position downstream of a functional E6 promoter. E7 expression, like E6 expression, required upstream regulatory sequences. However, E7 expression was less sensitive to repression by viral E2 protein and to mutation of the Spl binding site adjacent to the E2 binding site. Moreover, there was differential sensitivity of the two promoters to mutation of the E6 TATA box, with E7 expression more affected than E6 expression. These findings show that, in the normal host cells for this virus, the E6 and E7 promoters can be independently regulated by the cis regulatory region adjacent to the E6 promoter.

DNA Transposable Elements↗

PubMed1998

Role of c-fos and E2F in the induction of cyclin A transcription and vascular smooth muscle cell proliferation.

Excessive proliferation of vascular smooth muscle cells (VSMCs) contributes to vessel renarrowing after angioplasty. Here we investigated the transcriptional regulation of the cyclin A gene, a key positive regulator of S phase that is induced after angioplasty. We show that Ras-dependent mitogenic signaling is essential for the normal stimulation of cyclin A promoter activity and DNA synthesis in VSMCs. Overexpression of the AP-1 transcription factor c-fos can circumvent this requirement via interaction with the cAMP-responsive element (CRE) in the cyclin A promoter. Moreover, c-fos overexpression in serum-starved VSMCs results in the induction of cyclin A promoter activity in a CRE-dependent manner, and increased binding of endogenous c-fos protein to the cyclin A CRE precedes the onset of DNA replication in VSMCs induced by serum in vitro and by angioplasty in vivo. We also show that E2F function is essential for both serum- and c-fos-dependent induction of cyclin A expression. Taken together, these findings suggest that c-fos and E2F are important components of the signaling cascade that link Ras activity to cyclin A transcription in VSMCs. These studies illustrate a novel link between the transcriptional and cell cycle machinery that may be relevant to the pathogenesis of vascular proliferative disorders.

Activating Transcription Factor 2↗

PubMed1998

Potentiated angiogenic effect of scatter factor/hepatocyte growth factor via induction of vascular endothelial growth factor: the case for paracrine amplification of angiogenesis.

BACKGROUND: Scatter factor/hepatocyte growth factor (SF/HGF) is a pleiotropic growth factor that stimulates proliferation and migration of endothelial cells (ECs) via the c-Met receptor, present on ECs as well as other cell types, including smooth muscle cells (SMCs). We studied the effects of recombinant human (rh) SF/HGF in vitro and in vivo in a rabbit model of hindlimb ischemia. We further compared these effects with those of recombinant human vascular endothelial growth factor (rhVEGF165), an EC-specific mitogen. METHODS AND RESULTS: In vitro, rhSF/HGF and rhVEGF165 exhibited similar effects on proliferation and migration of ECs. When both cytokines were administered together, the result was an additive effect on EC proliferation and a synergistic effect on EC migration. Application of rhSF/HGF to cultures of human SMCs resulted in the induction of VEGF mRNA and protein. In vivo, administration of rhSF/HGF (500 microg x 3) was associated with significant improvements in collateral formation (P<.001) and regional blood flow (P<.0005) and with a significant reduction in muscle atrophy (P<.0001). These effects were significantly more pronounced than those of rhVEGF165 administered according to the same protocol (P<.05). Neither remote angiogenesis nor other pathological sequelae were observed with either rhSF/HGF or rhVEGF165. CONCLUSIONS: The pleiotropic effects of certain growth factors may potentiate angiogenesis via a combination of direct effects on EC proliferation and migration and indirect effects that result in the generation of other potent EC mitogens from non-EC populations. The synergistic effects demonstrated when SF/HGF and VEGF are administered together in vitro may be reproduced in vivo by SF/HGF-induced upregulation of VEGF in vascular SMCs.

Animals↗

PubMed1998

Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12.

Since the bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that induce the differentiation of mesenchymal precursor cells into the osteogenic cells, we identified the relevant signaling molecules responsible for mediating BMP-2 effects on mesenchymal precursor cells. BMP-2 induces osteoblastic differentiation of the pluripotent mesenchymal cell line C2C12 by increasing alkaline phosphatase activity and osteocalcin production. As recent studies have demonstrated that cytoplasmic Smad proteins are involved in TGF-beta superfamily signaling, we plan to isolate the relevant Smad family members involved in osteoblastic differentiation. We identified human Smad5, which is highly homologous to Smad1. BMP-2 caused serine phosphorylation of Smad5 as well as Smad1. In contrast, TGF-beta failed to cause serine phosphorylation of Smad1 and Smad5. We found Smad5 is directly activated by BMP type Ia or Ib receptors through physical association with these receptors. Following phosphorylation, Smad5 bound to DPC4, another Smad family member, and the complex was translocated to the nucleus. Overexpression of point-mutated Smad5 (G419S) or a C-terminal deletion mutant DPC4 (DPC4 delta C) blocked the induction of alkaline phosphatase activity, osteocalcin production, and Smad5-DPC4 signaling cascades upon BMP-2 treatment in C2C12 cells. These data suggest that activation of Smad5 and subsequent Smad5-DPC4 complex formation are key steps in the BMP signaling pathway, which mediates BMP-2-induced osteoblastic differentiation of the C2C12 mesenchymal cells.

Amino Acid Sequence↗

PubMed1998

Activation of the leu-500 promoter by a reversed polarity tetA gene. Response to global plasmid supercoiling.

The leu-500 promoter is inactivated by a mutation in the -10 region but can be activated in topA Escherichia coli and Salmonella strains. We have found that the tetA gene plays a vital role in the topA-dependent activation of a plasmid-borne leu-500 promoter. In previous studies, the leu-500 promoter and tetA gene have been arranged divergently. In this study we have reversed the polarity of the tetA gene, thus locating the leu-500 promoter at the 3' end of tetA. Despite being formally located in the downstream region of tetA, the leu-500 promoter is equally well activated in a topA strain in this environment, even though it is 1.6 kilobase pairs away from the promoter of the reversed tetA gene. Activation of the leu-500 promoter depends on transcription and translation of tetA but is largely insensitive to the function of other transcription units on the plasmid. These results require a change in viewpoint of the role of tetA, from local to global supercoiling. We conclude that transcription of the tetA gene is the main generator of transcription-induced supercoiling that activates the leu-500 promoter. Unbalanced relaxation of this supercoiling leads to a net increase in the negative linking difference of the plasmid globally, and there is a linear correlation between the change in global plasmid topology and the activation of the leu-500 promoter. Thus the leu-500 promoter appears to respond to the negative supercoiling of the plasmid overall.

Antiporters↗

PubMed1998

The 4(3)Pig and 6(3)Pig States of Na2: Observation and Assignment.

Two 3Pig states of Na2 have been observed in the 35 000-38 000 cm-1 energy region by pulsed perturbation facilitated optical-optical double resonance (PFOODR) fluorescence excitation spectroscopy and assigned to the 5dpi and 6dpi (the fourth and sixth according to the energy rank at Re) 3Pig Rydberg states, respectively. Molecular constants and RKR potential curves are reported. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

In vitro bone resorption is dependent on physiological concentrations of zinc.

The addition of physiological concentrations of zinc (25-200 (microg/dL) to Dulbecco's Modified Eagle's Medium containing tibiae from 19-d chick embryos resulted in a concentration-dependent increase in tibial content of tartrate-resistant acid phosphatase (TRAP) and an increase in bone resorption, as measured by tibial calcium release. This increase in bone resorption was additive to the resorptive effect resulting from the addition of 10(-9)-10(-7) M parathyroid hormone (PTH), but was not additive to similar effects produced by the addition of 10(-9)-10(-7) M prostaglandin E2 (PGE2). An inhibitor of prostaglandin synthesis, flurbiprofen (10[-6] M), did not influence the effect of zinc on bone resorption. However, the addition of 2,6-pyridinedicarboxylic acid (10[-3] M, 2,6-PDCA), a chelator of zinc, did attenuate the effects of zinc, as did the addition of an inhibitor of DNA replication (hydroxyurea, 10[-3] M). Hydroxyurea also attenuated the bone resorptive response to PGE2, but had no influence on the effects of PTH. These results indicate that physiological concentrations of zinc alter bone resorptive rates in vitro by a mechanism that is dependent on DNA replication.

Acid Phosphatase↗

PubMed1998

Glioma prostaglandin levels correlate with brain edema.

The present study was designed to prospectively investigated the prostaglandin (PG) levels and extent of peritumoral edema in 30 cases of glioma by using methods of radioimmunoassay and imaging. Both TXB2 and 6-keto-PGF1 alpha levels in all glioma groups went up over that in the control group. TXB2 level and ratio of TXB2/6-keto-PGF1 alpha were markedly increased with the extent of tumor malignancy. Water concentration in anaplastic astrocytoma and glioblastoma were significantly elevated. Difference in TXB2 level and TXB2/6-keto-PGF1 alpha ratio among three edema grades were statistically significant. TXB2 level and ratio of TXB2/6-keto-PGF1 alpha were closely correlated with water concentration (r1 = 0.53, r2 = 0.72, P < 0.01). Our findings suggested that the metabolism of PG in glioma were in the state of disorder, and that the imbalance between PGI2 and TXA2 may be one of factors which affect the formation of peritumoral edema.

6-Ketoprostaglandin F1 alpha↗

PubMed1998

Formation of 8-oxodeoxyguanosine in brain DNA of rats exposed to acrylonitrile.

Acrylonitrile (ACN) produces tumors in rats, particularly gliomas of the brain, but tests for genotoxicity have yielded mixed results and no ACN-DNA adducts have been identified in the brain. To examine the possibility that ACN-related brain tumors were not a consequence of binding of ACN to brain DNA, experiments were conducted to investigate possible epigenetic mechanisms. Male Sprague-Dawley rats were exposed to 0, 3, 30, and 300 ppm ACN in drinking water for 21 days, a range that includes doses associated with brain tumorigenesis. In the 30 and 300 ppm ACN groups, 8-oxodeoxyguanosine (8-oxodG) levels were two fold greater than in the controls. Measures of glutathione levels, glutathione peroxidase and catalase were not significantly changed, but cyst(e)ine was somewhat increased. No changes were found in brain cytochrome oxidase activity, which indicates a lack of metabolic hypoxia. Also, no effects on thiobarbituric acid reactive substances were found, indicating a lack of lipid peroxidation. In an additional experiment, male Sprague-Dawley rats were exposed to 0 or 100 ppm ACN in drinking water for 94 days; interim sacrifices were conducted at 3, 10, and 31 days. Levels of brain nuclear DNA 8-oxodG were significantly increased in ACN-exposed rats compared with controls. Another group of animals were given weekly i.v. injections of 5 mg/kg methylnitrosurea and no increases in 8-oxodG were found. These studies suggest the possibility that ACN-induced tumors may be produced by a mode of action involving 8-oxodG. The formation of 8-oxodG is not understood, but does not appear to involve lipid peroxidation or disruption of antioxidant defenses.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1998

Morphometric analysis of gastrectomy-evoked osteopenia.

Gastrectomy leads to osteopenia in the rat. The present study describes the effects of gastrectomy on bone morphology. Rats were subjected to gastrectomy or sham operation. Four weeks after the operation the rats were killed and both tibiae were removed. Bone morphology of the left tibia was analyzed with quantitative computer tomography, the right tibia with histomorphometry. Bone length, bone mineral content, as well as indices of bone resorption and formation were measured in the metaphysis and the diaphysis. Gastrectomy had no effect on longitudinal bone growth but it led to a low bone mineral content at both sites. Bone resorption was increased by gastrectomy, as shown by an increase in the medullary cavity area in the diaphysis. Gastrectomy also reduced bone formation, as shown by a decreased periosteal circumference and a decrease in the mean periosteal bone apposition in the diaphysis. In conclusion, gastrectomy-evoked osteopenia reflects impaired formation and increased resorption of bone.

Animals↗

PubMed1998

Gastrin stimulates epithelial cell proliferation in the oesophagus of rats.

Gastrin can induce mitotic stimulation in the oxyntic mucosa of the stomach, sometimes leading to abnormal growth. We examined whether gastrin was able to influence cell proliferation in the oesophageal epithelium. Rats were treated with gastrin, omeprazole or saline for 3 days, or were subjected to fundectomy or sham operation. Bromodeoxyuridine labelling (LI) and mitotic (MI) indices were counted in the proliferative zone of the squamous epithelium. Infusion of exogenous gastrin, treatment with omeprazole or fundectomy raised the LI and the MI values in the oesophageal epithelium, indicating that gastrin stimulates cell proliferation in the oesophageal mucosa in the rat.

Animals↗

PubMed1998

Reversibility of omeprazole-evoked changes in the ultrastructure of ECL cells in the rat stomach.

The ECL cells are histamine- and peptide hormone-producing endocrine cells in the rat oxyntic mucosa. They are rich in secretory vesicles and also contain microvesicles and electron-dense granules. They operate under the control of circulating gastrin. In the present study, we examined the ECL-cell ultrastructure after long term treatment with omeprazole, which is known to induce hypergastrinemia, and after withdrawal of the drug. Rats received omeprazole (400 micromol/kg per day, orally) for 16 days and were killed 1, 5, 20, or 40 days after the last dose of the drug. Oxyntic mucosal specimens were processed for electron microscopy. Electron micrographs of ECL-cell profiles were analyzed planimetrically. The ECL-cell profile area increased promptly in response to omeprazole, the secretory vesicles and granules were reduced in number and volume density, the microvesicles were unchanged in number but reduced in volume density, and vacuoles appeared. Within a week after stopping the omeprazole treatment, the numbers and volume densities of secretory vesicles and microvesicles returned to pre-stimulation values. Also, the vacuoles disappeared promptly. The ECL-cell profile area decreased below the pre-stimulation level within five days after stopping treatment, while, in contrast, the granules increased in number and volume density. Somewhat surprisingly, the cell size and the granule compartment did not return to normal until 40 days after stopping treatment.

Animals↗

PubMed1998

ECL cells of the rat stomach: development of lipofuscin in response to sustained gastrin stimulation.

Ageing cells, especially post-mitotic cells, are known to accumulate pigments, i.e. highly electron-dense material, referred to as ceroid or lipofuscin. This material is formed as a consequence of autophagocytosis and peroxidation of the products undergoing degradation. The present study describes the development of lipofuscin in the ECL cells of the rat stomach. These cells produce and secrete histamine in response to gastrin. They are rich in secretory vesicles, which fuse to form vacuoles in hypergastrinaemic rats. Hypergastrinaemia was induced by continuous infusion of human Leu15-gastrin-17 for 6 days or by daily treatment with omeprazole for 10 weeks. Either treatment caused both vacuoles and lipofuscin bodies to appear in large numbers; the vacuoles disappeared promptly after interruption of the hypergastrinaemia, whereas the lipofuscin bodies remained. Antrectomy-evoked hypogastrinaemia was associated with a reduced number and volume density of lipofuscin bodies. Treatment with alpha-fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, resulted in depletion of ECL-cell histamine and was found to prevent the omeprazole-evoked formation of vacuoles and lipofuscin. The numbers of both vacuoles and lipofuscin bodies were well-correlated with the serum gastrin concentration, suggesting that gastrin stimulates the development not only of vacuoles but also of lipofuscin, perhaps through enhanced autophagocytosis and/or oxidative stress. Thus, lipofuscin bodies may develop from vacuoles, and both vacuoles and lipofuscin bodies may reflect the efforts of overstimulated ECL cells to cope with the excessive formation of secretory products.

Animals↗

PubMed1998

Eicosanoids in sickle cell disease: potential relevance of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid to the pathophysiology of vaso-occlusion.

The monohydroxyeicosanoid 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), which is derived from oxygenation of arachidonic acid by 12-lipoxygenase, is one of the major metabolites in platelets. In a recent study, we have showed that this eicosanoid stimulated basal sickle-red-cell-endothelial-cell adhesion. To understand the pathophysiologic significance of 12-HETE, we measured the levels of this eicosanoid in plasma and urine from children with sickle cell disease. We found that as compared with controls, plasma 12-HETE levels are increased in patients with sickle-cell disease in the steady state, and are increased further during vaso-occlusive crises. Urinary 12-HETE levels were also increased during the steady state. We also assessed plasma levels of soluble P-selectin (another potential marker for platelet activation), and found changes in the levels of this marker similar to those seen with plasma 12-HETE. In additional studies, we found that 12-HETE enhanced hypoxia-induced sickle-red-cell-endothelial adherence, and that this effect was mediated by potentiation of agonist-induced upregulation of the expression of the mRNA for vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. Because 12-HETE appears to enhance both basal and agonist-induced sickle-red-cell adhesion, this metabolite could potentially play a role in the pathogenesis of the vaso-occlusive crisis (VOC) in sickle-cell disease.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1998

Role of free thiol groups in the biological activities of stonustoxin, a lethal factor from stonefish (Synanceja horrida) venom.

Stonustoxin (SNTX) is a two-subunit protein purified from the venom of a stonefish, Synanceia horrida. It has potent lethal activity and is also a membrane pore-forming cytolysin. The role of thiol groups in the biological activities of SNTX was investigated. Both the hemolytic and lethal activities of SNTX were potentiated by the reducing agent, dithiothreitol (DTT). The hemolytic activity of SNTX was sensitive to the modification of thiol groups by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The loss of haemolytic activity correlated with the number of thiol groups that were titrated with DTNB. Thiol modification of SNTX with DTNB also inhibited its lethality. These inhibitory effects of thiol modification could be reversed by reduction with DTT. It was also found that the haemolytic activity of SNTX could not be inhibited by cholesterol. These observations indicate that free thiol groups play an important role in the haemolytic activity and lethality of SNTX but unlike other thiol-activated cytolysins, SNTX was not inhibited by cholesterol. Thus, SNTX may represent a new class of cytolytic toxin.

Animals↗

PubMed1998

Short-wave cone signal in the red-green detection mechanism.

Previous work shows that the red-green (RG) detection mechanism is highly sensitive, responding to equal and opposite long-wave (L) and middle-wave (M) cone contrast signals. This mechanism mediates red-green hue judgements under many conditions. We show that the RG detection mechanism also receives a weak input from the short-wave (S) cones that supports the L signal and equally opposes M. This was demonstrated with a pedestal paradigm, in which weak S cone flicker facilitates discrimination and detection of red-green flicker. Also, a near-threshold +S cone flash facilitates detection of red flashes and inhibits green flashes, and a near-threshold -S cone flash facilitates detection of green flashes and inhibits red flashes. The S contrast weight in RG is small relative to the L and M contrast weights. However, a comparison of our results with other studies suggests that the strength of the absolute S cone contrast contribution to the RG detection mechanism is 1/4 to 1/3 the strength of the S contribution to the blue-yellow (BY) detection mechanism. Thus, the S weight in RG is a significant fraction of the S weight in BY. This has important implications for the 'cardinal' color mechanisms, for it predicts that for detection or discrimination, the mechanisms limiting performance do not lie on orthogonal M-L and S axes within the equiluminant color plane.

Color Perception↗