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N Wang

Publications and source records attributed to N Wang.

At least 343 records · Page 19Linked to original sources
PubMed1994

Synthesis, processing, and localization of human Lon protease.

The synthesis, maturation, and localization of the human homolog of the bacterial ATP-dependent Lon protease have been studied in cultured cells and in a cell-free system. Immunofluorescence microscopy of cells transfected with a recombinant human Lon protease-FLAG epitope chimeric protein confirmed the mitochondrial location of human Lon protease. The primary product of transcription and translation directed by a human LON cDNA in vitro had an apparent mass of approximately 107 kDa, and incubation of the translation product with isolated rat liver mitochondria converted the precursor into the approximately 100-kDa mature form. The latter pelleted with mitochondria and was resistant to trypsin digestion. Maturation of human Lon protease in vitro was dependent on mitochondrial inner membrane potential, as the uncoupling agent 2,4-dinitrophenol (DNP) completely blocked this process. In intact cultured cells treated with DNP, newly synthesized Lon protease also accumulated as the precursor form, and subsequent removal of DNP allowed the precursor to be translocated into mitochondria and cleaved to its mature form. A pulse-chase experiment in the absence of DNP showed that the Lon protease precursor is converted to the mature form with a half-time of < 2 min and that the mature human Lon protease is indefinitely stable in intact cells. Submitochondrial fractionation and immunoblot analysis of rat liver mitochondrial proteins using the polyclonal anti-human Lon protease antiserum indicated that the protease is located exclusively in the matrix. These data demonstrate that the maturation of human Lon protease is characterized by steps similar to those reported for other mitochondrial matrix proteins.

ATP-Dependent Proteases↗
PubMed1994

Role of gamma-glutamyltranspeptidase-mediated glutathione transport on the radiosensitivity of B16 melanoma variant cell lines.

PURPOSE: To elucidate the role of gamma-glutamyltranspeptidase-mediated glutathione transport on the radiosensitivity of B16 melanoma variant cell lines. METHODS AND MATERIALS: B16 melanoma variant cell lines were examined for their levels of gamma-glutamyltranspeptidase (GGTP; E.C. 2.3.2.2), a plasma membrane-associated ectoenzyme that is involved in the transport of extracellular glutathione, by flow cytometric and biochemical analysis. B16 cell lines were examined for rates of de novo glutathione synthesis from extracellular glutathione and for their sensitivity to gamma-irradiation and glutathione synthesis inhibition. The GGTP inhibitors were examined for their effect on the radiosensitivity of B16 melanoma cells. RESULTS: B16-F10-BL6 (BL6) melanoma cells were shown to express a 20-fold higher level of GGTP than the B16-F1 melanoma variant cells. Cultures of BL6 and B16-F1 cells depleted extracellular glutathione at rates of 2.4 and < 0.1 nmol glutathione/10(6) cells/h, respectively, and BL6 cells exported glutathione at a rate 7.2-fold higher than B16-F1 cells (710 and 98 pmol glutathione/10(6) cells/h, respectively). BL6 melanoma cells replenished exhausted intracellular glutathione levels from an extracellular glutathione source at a rate of 1.21 nmol glutathione/h (18% basal glutathione/h); however, B16-F1 cells lacked the capacity to replenish intracellular glutathione despite the presence of exogenous glutathione in the culture medium. BL6 melanoma cells were radioresistant compared to the B16-F1 cell line, exhibiting extrapolation numbers (ñ) of 14.9 and 1.0, respectively, and a lower surviving fraction to a wide range of radiation doses. The GGTP inhibitor combination of L-serine and sodium borate blocked the repletion of intracellular glutathione and in the presence or absence of buthionine sulfoximine-mediated depletion of glutathione reverses the radiation resistance in BL6 melanoma cells to near baseline levels observed with the B16-F1 parent clone. Serine-borate treatment of low-GGTP expressing B16-F1 cells had no effect on the ñ value or the surviving fraction of cells to a range of ionizing irradiation doses. CONCLUSIONS: These results suggest that GGTP plays an important role in the extracellular metabolism and transport of glutathione, which also provides radioresistance to BL6 melanoma cells in vitro.

Animals↗
PubMed1994

Codon reiteration and the evolution of proteins.

Sequence data banks have been searched for proteins possessing uninterrupted reiterations of any amino acid. Hydrophilic amino acids, and particularly glutamine, account for a large proportion of the longer reiterants. In the genes for these proteins, the most common reiterants are those that contain poly(CAG), even out-of-frame or, to a lesser degree, those that contain repeated doublets of CA, AG, or GC. The preferential generation of such reiterants requires that DNA strand-specific signals predispose to reiteration and thus to the extension of coding regions.

Amino Acid Sequence↗
PubMed1994

Delineation of three different thyroid hormone-response elements in promoter of rat sarcoplasmic reticulum Ca2+ATPase gene. Demonstration that retinoid X receptor binds 5' to thyroid hormone receptor in response element 1.

Thyroid hormone (3,5,3'-triiodothyronine) positively regulates transcription of the sarcoplasmic reticulum Ca2+ATPase gene in rat heart, and sequences within 559 nucleotides upstream from the transcription start site confer thyroid hormone responsiveness upon a reporter gene. In the present study, three thyroid hormone-response elements (TREs) are identified between nucleotides -485 and -190. Each TRE is active in transient transfection assays and specifically binds 3,5,3'-triiodothyronine receptors (TRs) alpha 1 and beta 1 alone and in combination with retinoid X receptors (RXRs) alpha and beta. TRE 1 is a direct repeat of two half-sites separated by four nucleotides; TREs 2 and 3 are inverted palindromes of two half-sites separated by four and six nucleotides, respectively. Methylation interference analysis of TRE 1 showed binding of a TR alpha 1 monomer to the 3' half-site, whereas the heterodimer contacts both half-sites. Subsequent studies employed TR beta and RXR alpha mutants in which their P-boxes were replaced with the P-box of the glucocorticoid receptor. Bandshifts of wild type and mutant proteins with either wild type TRE 1 or a mutant version, in which the 5' half-site was converted to a glucocorticoid response element half-site, demonstrated preferential binding of RXR to the 5' half-site and of TR to the 3' half-site of TRE 1.

Animals↗
PubMed1994

Pre- and post-synaptic modulators of excitatory neurotransmission: comparative effects on hypoxia/hypoglycemia in cortical cultures.

Neuron-enriched cultures from embryonic rat cerebral cortex were exposed to hypoxia and hypoglycemia, and the resulting cellular injury was quantified by measuring lactate dehydrogenase (LDH) release, which was maximal after 20-24 h. The increase in LDH release produced by hypoxia/hypoglycemia was prevented by N-methyl-D-aspartate (NMDA) antagonists, but not by three classes of drugs thought to modulate glutamate release: Ca2+ channel antagonists (nimodipine, omega-conotoxin GVIA, omega-agatoxin-IVA), KATP channel activators (cromakalim, diazoxide), and glutamate transport inhibitors (dihydrokainate, DL-threo-beta-hydroxyaspartate).

Animals↗
PubMed1994

Requirement for the yeast gene LON in intramitochondrial proteolysis and maintenance of respiration.

The role of protein degradation in mitochondrial homeostasis was explored by cloning of a gene from Saccharomyces cerevisiae that encodes a protein resembling the adenosine triphosphate (ATP)-dependent bacterial protease Lon. The predicted yeast protein has a typical mitochondrial matrix-targeting sequence at its amino terminus. Yeast cells lacking a functional LON gene contained a nonfunctional mitochondrial genome, were respiratory-deficient, and lacked an ATP-dependent proteolytic activity present in the mitochondria of Lon+ cells. Lon- cells were also impaired in their ability to catalyze the energy-dependent degradation of several mitochondrial matrix proteins and they accumulated electron-dense inclusions in their mitochondrial matrix.

ATP-Dependent Proteases↗
PubMed1994

Gamma-glutamyltranspeptidase expression regulates the growth-inhibitory activity of the anti-tumor prodrug gamma-L-glutaminyl-4-hydroxy-3-iodobenzene.

gamma-L-glutaminyl-4-hydroxy-3-iodobenzene (I-GHB), a novel iodinated analog of gamma-L-glutaminyl-4-hydroxybenzene (GHB), demonstrates greater anti-tumor activity in human and in murine melanoma cell lines. These phenolic amides are substrates for gamma-glutamyltranspeptidase (GGTP; E.C. 2.3.2.2), a cell-membrane-associated ecto-enzyme which is elevated in a number of tumor systems. We now present data to show that the growth-inhibitory activity of I-GHB and GHB may be mediated via GGTP-catalyzed reactions. The growth-inhibitory activity of I-GHB and GHB in pigmented B16-BL6 melanoma cells was blocked significantly by rabbit anti-rat GGTP polyclonal antibodies. The combination of L-serine and sodium borate, a specific transition-state inhibitor of GGTP, as well as acivicin, a glutamine antagonist and irreversible GGTP inhibitor, inhibited the killing of BL6 cells by GHB and I-GHB. To further define the role of GGTP expression in the regulation of phenolic amide cytotoxicity, GGTP-negative Chinese hamster ovary cells (CHO-K1) were transfected with a functional rat renal cDNA representing the full-length GGTP transcript. I-GHB and GHB were significantly more cytotoxic in GGTP cDNA transfected Chinese hamster ovary (CHO-K1-GGTP) cells than in non-transfected CHO-K1 cells. The combination of L-serine and sodium borate blocked the cytotoxic activity of these pro-drugs and also inhibited GGTP-catalyzed formation of polymerized products from these phenolic amides in intact BL6 melanoma and CHO-K1-GGTP cells. Furthermore, melanin formation from GHB was not observed in non-transfected CHO-K1 cells lacking GGTP expression. The combined data strongly suggest that GGTP-catalyzed hydrolysis of the anti-tumor pro-drugs I-GHB and GHB to 4-aminophenols mediates the expression of antitumor activity.

Animals↗
PubMed1994

Identification and sequence of an accessory factor required for activation of the human interferon gamma receptor.

Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by induction of class I human leukocyte antigen (HLA) and protection against encephalomyocarditis virus (EMCV) infection. Whereas human chromosome 6 encodes the Hu-IFN-gamma receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. Probes from a genomic clone were used to identity cDNA clones expressing a species-specific accessory factor. These cDNA clones are able to substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer full antiviral protection against EMCV, confirming that an additional factor encoded on human chromosome 21 is required for reconstitution of antiviral activity against EMCV. We conclude that this accessory factor belongs to a family of such accessory factors responsible for different actions of IFN-gamma.

Amino Acid Sequence↗
PubMed1994

Ultraviolet radiation induces phosphorylation of the epidermal growth factor receptor.

Ultraviolet light in solar radiation is responsible for more than 600,000 malignancies each year in the United States alone, making it the most efficient environmental carcinogen known. Ultraviolet radiation-induced direct DNA damage is thought to be responsible for its initiating properties, while the promotional aspects of such radiation are poorly defined and only recently gaining attention. We show here for the first time that physiologically relevant doses of ultraviolet radiation induce phosphorylation of the epidermal growth factor receptor in A431 keratinocytes at tyrosine sites within 30 min. Such alteration of this major signal transduction system is probably an important step in the ultraviolet radiation-induced, epidermal cell-signalling cascade.

Carcinoma, Squamous Cell↗
PubMed1994

Bacterial expression of Chinese hamster regulatory type-I and catalytic subunits of cyclic AMP-dependent protein kinase and mutational analysis of the type-I regulatory subunit.

The type-I regulatory subunit (RI) of the cyclic AMP-dependent protein kinase (PKA) from Chinese hamster ovary (CHO) cells has been cloned and expressed in a strain of BL21(DE3) Escherichia coli lacking adenylate cyclase [BL21(DE3)/delta cya]. RI expressed in this bacterial system free of cyclic AMP is soluble and can reconstitute functional PKA. Recombinant CHO C alpha is predominantly insoluble with some active soluble protein. C beta is entirely insoluble and inactive. Soluble recombinant RI and soluble recombinant C alpha can associate in vitro and be activated by cyclic AMP. Six site-directed mutations of RI were generated to study the interaction of cyclic AMP with RI and RI-C alpha subunit interactions. Four cyclic AMP-binding-site point mutants were generated [W261R (tryptophan to arginine at position 261), a novel mutation in site A; V376G, a novel mutation in site B; G200E (site A), and Y370F (site B), previously described in bovine RI were introduced into the CHO RI for comparison purposes]. Mutants W261R, Y370F, and G200E demonstrated decreased 8-N3-[3H]cyclic AMP binding as well as 5-fold reduced affinity for [3H]cyclic AMP, with threefold increased EC50 values for cyclic AMP activation of kinase activity from reconstituted mutant holoenzymes. The mutation at V376G did not alter cyclic AMP binding or activation by cyclic AMP of mutant holoenzyme. A truncation mutant, G200Stop, which lacks both cyclic AMP-binding sites, did not bind cyclic AMP but can inhibit C alpha subunit activity. A novel mutation outside the cyclic AMP-binding regions of RI (V89A) weakened the interaction with C alpha indicated by a 7-fold lower EC50 for mutant holoenzyme activation by cyclic AMP.

Amino Acid Sequence↗
PubMed1994

Modulation of human fibroblast activity by selected angiogenesis inhibitors.

Tenon's fibroblast (TF) bleb encapsulation is a common cause of filtering surgery failure. Various pharmacologic agents have been used in an attempt to inhibit this response. The effects of three angiogenesis inhibitors were studied. AGM-1470, a fumagillin analog and hydrocortisone 21-phosphate (HC21P) complexed with heparin or with the heparin analog beta-cyclodextrin tetradecasulfate (BCD-TDS) in modulating human tenon's fibroblasts in cell culture. It has been clinically demonstrated that well vascularized blebs are associated with a poorer prognosis. In addition to reducing bleb neovascularization, angiogenesis inhibitors may have an additional therapeutic role in decreasing fibroblast activity. Drug effects were assessed by studying cell growth rates by cell Coulter counting and rate of wound closure. TF's were grown in DMEM with 10% FBS and 1% PCN-strep with fungizone. Angiogenesis inhibitors were added to growth medium in varying concentrations: AGM-1470 alone, HC21P complexed with heparin and HC21P complexed with BCD-TDS. Controls were grown in control medium alone, medium with added ethanol, or with added BCD-TDS, heparin or HC21P. We found a dose related inhibition of cell growth with all of the angiogenesis inhibitor combinations, which was not seen in the control groups. Tenon's fibroblast proliferation was significantly inhibited by AGM-1470 as seen in the four higher concentrations (P < 0.05) with insignificant inhibition at the lowest concentration of AGM-1470 (P = 0.38). The heparin:HC21P and heparin:BCD-TDS combinations demonstrated significant inhibition at all concentrations (P < 0.05). Wound closure was significantly inhibited by all the added agents, except AGM-1470 and the controls. The rate of wound closure was significantly reduced by the highest concentrations of the heparin:HC21P and heparin:BCD-TDS combinations (P < 0.05), although it was not significantly affected by lower concentrations (P > 0.05). Rank order of potencies of these agents for inhibition of TF proliferation was AGM-1470, BCD-TDS:HC21P, heparin:HC21P, HC21P, while the rank order of potencies for these agents for inhibition of wound closure was HC21P, heparin:HC21P, BCD-TDS:HC21P, AGM-1470. These selected angiogenesis inhibitors appear to have marked inhibitory effects on TF proliferation and migration, which may have a potential clinical role in modulating wound healing associated with glaucoma filtering surgery.

Cell Division↗
PubMed1994

Preperitoneal insertion of the HeartMate 1000 IP implantable left ventricular assist device.

During a 16-month period, 12 consecutive patients underwent insertion of a HeartMate 1000 IP left ventricular assist device in a preperitoneal pocket that separated the device from the abdomen. All patients were in cardiogenic shock awaiting heart transplantation. Preoperatively, the mean cardiac index was 1.6 L.min-1.m-2, with 11 patients on intraaortic balloon pump support and 2 on extracorporeal membrane oxygenation. The pocket was formed below the rectus abdominis and internal oblique muscles and above the posterior rectus sheath. The pump fit easily in all patients. One patient died of progressive multiorgan failure. Four patients are still on support. Seven patients underwent successful transplantation after a mean duration of 55 days of support (mean pump index was 3.1 L.min-1.m-2 during support). One patient had driveline revision because of an exit site infection but had successful transplantation. The pump was explanted without difficulty at each transplant operation. All patients having transplantation are alive and well. Preperitoneal insertion of the HeartMate left ventricular assist device can be safely performed and may avoid problems posed by intraabdominal left ventricular assist device insertion.

Abdominal Muscles↗
PubMed1994

Pericardioperitoneal shunt: an alternative treatment for malignant pericardial effusion.

The treatment of 37 consecutive cases of symptomatic malignant pericardial effusion over a period of 13 years was retrospectively analyzed. The most common diagnoses were lung cancer (59%) and breast cancer (11%). In the most recent 4 patients, the Denver pleuroperitoneal shunt was used to drain the pericardial effusion into the peritoneal cavity. In each case, the procedure was performed under local anesthesia, and the patient was discharged 2 to 4 days later without complications. Three of the patients subsequently died of the disease process without evidence of cardiac failure or tamponade during 6-month follow-up. The more traditional means of pericardial drainage, the subxiphoid approach (14 patients) and the anterior thoracotomy approach (19 patients), were associated with higher postoperative morbidity (21% and 53%, respectively) and mortality (7% and 42%, respectively). Because of the small number of patients treated by pericardioperitoneal shunting, a significant difference was demonstrated only in the length of hospital stay (shunt, 2.8 +/- 0.5 days; subxiphoid, 11.2 +/- 4.6 days; thoracotomy, 14.9 +/- 6.1 days). Median survivals were essentially the same (shunt, 3.5 months; subxiphoid, 2.7 months; thoracotomy, 1.2 months). It is apparent that the pericardioperitoneal shunt, although a much simpler procedure, can accomplish similar palliation effectively in the treatment of malignant pericardial effusion.

Breast Neoplasms↗
PubMed1994

Repair of insufficient bicuspid aortic valves.

A technique for the repair of bicuspid aortic valves that includes resection of the flail segment of the prolapsing leaflet, annuloplasty, and resection of the raphe, when present, has been reported. To assess the efficacy of this technique in the repair of insufficient bicuspid aortic valves, the results in 72 consecutive patients were assessed. The mean age of the patients was 39 +/- 11 years; 94% were male. Fifty-six patients (78%) underwent isolated aortic valve repair, 9 (12.5%) underwent aortic and mitral valve repair, and 7 (9.7%) had other associated procedures. All patients underwent leaflet resection, including 35 (48%) at the raphe. The mean aortic occlusion time was 39 +/- 12 minutes. There were no operative deaths. The severity of aortic insufficiency, as assessed by Doppler echocardiography (graded from 0 to 4) preoperatively and intraoperatively and at late follow-up, was 3.6 +/- 0.6, 0.4 +/- 0.4, and 0.9 +/- 0.8, respectively, with a p value of < 0.0001 for the latter two values versus the preoperative one. There have been no postoperative deaths. Patients did not receive anticoagulation treatment and there were no strokes or episodes of endocarditis. Six patients have required reoperation; 3 underwent repeat repair. The Kaplan-Meier freedom from aortic valve reoperation probabilities at 12 and 24 months were 94% and 89.5%, respectively. We conclude that valvuloplasty for insufficient bicuspid aortic valves is technically safe, is associated with a low incidence of recurrent insufficiency, and has been associated with no other valve-related complications.

Actuarial Analysis↗