Biomedical subjects

A Weisz

Publications and source records attributed to A Weisz.

At least 37 records · Page 2Linked to original sources

PubMed1997

Angiogenesis by gene therapy: a new horizon for myocardial revascularization?

The concept of therapeutic angiogenesis is based on the premise that the potential for vascular growth inherent in vascular tissue can be utilized to promote the development of new blood vessels under the influence of the appropriate growth factors. Direct application of growth factors of the fibroblast (acidic, basic fibroblast growth factor, FGF-5), endothelial (vascular endothelial growth factor) and other series has been effective in preliminary studies. Angiogenesis by gene transfer provides an attractive alternative, with the advantage that the protein may continue to be secreted for a longer period of time and that the gene may be targeted to specific tissues to enhance efficacy and reduce systemic side effects. Angiogenesis by gene transfer is currently under investigation using a variety of growth factors and a wide array of potential delivery systems. These include application of the gene as naked DNA or by viral vector in the proximal vessel by direct intravascular injection, interventional cardiologic techniques (hydrogel coating on balloon, double balloon system, stent implantation) or by direct application to adventitia, pericardium or ischemic tissue distal to the site of arterial obstruction. As our understanding of the molecular and genetic processes underlying angiogenesis increases, and as we examine the results of preliminary animal and human protocols, we hope to develop the potential of angiogenesis by gene transfer for therapeutic use.

Animals↗

PubMed1997

Estrogen induces early and timed activation of cyclin-dependent kinases 4, 5, and 6 and increases cyclin messenger ribonucleic acid expression in rat uterus.

Cyclin-dependent kinases (cdks) are serine-threonine protein kinases that play a key role in the regulation of the mitotic cycle, in transcription initiation, and in the control of specific metabolic pathways in eukaryotic cells. cdk activity is controlled via phosphode-phosphorylation of the catalytic subunits of these enzymes and their physical association with cyclins and cdk inhibitors. In adult rats, estrogen stimulation results in massive proliferation of endometrial epithelial cells, accompanied by functional and structural modifications in all other tissue components of the uterus. We report here that administration of 17 beta-estradiol (E2) to adult ovariectomized rats induces within the first 25 h significant activation of cdk 4, 5, and 6, but not cdk 2, in the uterus, accompanied by increased expression of D-type (D1-3), A and E cyclin messenger RNAs (mRNAs). Furthermore, expression of the cdk inhibitor p27Kip1, a key regulator of uterine functions, is induced by E2 in this organ. Analysis of RNA extracted from E2-stimulated rat endometria shows early accumulation of D1 and D3, but not D2, cyclin mRNA, preceded by transient accumulation of c-fos mRNA. These results indicate an involvement of cdks and cyclins in estrogen actions in adult rat uterus and suggest that cyclins D1 and D3 are part of the molecular pathway that allows hormonal regulation of G1 progression in endometrial cells.

Animals↗

PubMed1996

17beta-Estradiol induces cyclin D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells.

MCF-7 human breast cancer cells express functional estrogen receptor and grow in response to estrogen stimulation. G(1)-synchronized MCF-7 cells, made quiescent by exposure to the HMG-CoA reductase inhibitor Simvastatin in estrogen-free medium, readily resume cell cycle progression upon stimulation with 17beta-estradiol (E(2)), even under conditions where polypeptide growth factor-triggered signal transduction pathways are inhibited by the continuous presence of Simvastatin in the culture medium. Under these conditions, cyclin D(1) gene transcription is transiently induced within the first 1-9 h of stimulation, as shown by the accumulation of cyclin D(1) mRNA and protein (p36(D(1))) in the cell and by enhanced expression of stably transfected D(1) promoter-luciferase hybrid genes. Estrogen-induced p36(D(1)) associates readily with p32(cdk2) and p34(cdk4), but not with p31(cdk5), which is however abundantly expressed in these cells. Only p36(D(1))-p34(cdk4) complexes are activated by E(2), as detected in cell extracts by immunoprecipitation with anti-D(1) antibodies followed by assessment of phosphotransferase activity toward the retinoblastoma (Rb) gene product and by analysis of p105(Rb) phosphorylation in vivo. An estrogen-responsive regulatory region has been mapped within the first 944 bp upstream of the transcriptional startsite of the human D(1) gene. Sequence analysis of this DNA region reveals that the cis-acting elements responsive to estrogen are likely to be different in this case from the canonical EREs.

Breast Neoplasms↗

PubMed1996

Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-gamma, bacterial lipopolysaccharide and NG-monomethyl-L-arginine.

Cytokines and bacterial lipopolysaccharides (LPSs) stimulate nitric oxide production in macrophages by inducing transcription of the gene coding for the inducible isoform of nitric oxide synthase (iNOS). We have cloned the mouse iNOS gene promoter and analysed its structural features and its response to interferon-gamma (IFN-gamma) and Escherichia coli LPS in RAW 264.7 mouse macrophage-like cells. Transcription of a recombinant reporter gene including the promoter and 4 kb of its 5'-flanking DNA, linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene, is stimulated by IFN-gamma and, more efficiently, by LPS upon transient transfection in RAW 264.7 cells. Two upstream DNA regions are required for maximal promoter activation of LPS: the first maps between positions -1541 and -775 and the other between -420 and -47, with respect to the major transcriptional start site of the iNOS gene. The upstream-most region also mediates promoter trans-activation by IFN-gamma. As reported earlier for transcription of the endogenous iNOS gene, combined stimulation of RAW 264.7 cells with IFN-gamma and LPS results in lower activation of the transfected promoter, when compared with LPS alone. NG-Monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase activity, enhances iNOS gene mRNA induction and promoter activation by IFN-gamma and LPS, indicating that nitric oxide can influence negatively the reponsiveness of this gene to inducers. These results suggest the possibility of a negative regulatory feedback exerted by iNOS on the transcriptional activation of its own gene.

Animals↗

PubMed1996

Isolation of 4'-bromo-4,5,6,7-tetrachlorofluorescein from a synthetic mixture by pH-zone-refining counter-current chromatography with continuous pH monitoring.

A synthetic mixture containing mainly 4'-bromo-4,5,6,7-tetrachlorofluorescein (4'BrTCF, ca. 25% by high-performance liquid chromatography) was prepared by direct bromination of tetrachlorofluorescein in ethanol. The 4'BrTCF was isolated and purified by pH-zone-refining counter-current chromatography (CCC), using two different two-phase solvent systems: diethyl ether-acetonitrile-water (4:1:5,v/v) and ter.-butyl methyl ether-water (1:1,v/v). For each system, the upper organic phase was acidified and used as the stationary phase and the lower aqueous phase was made basic and used as the mobile phase. pH-Zone-refining CCC of two 5-g batches of the crude mixture yielded 1.65 g of 4'BrTCF that was ca. 95% pure by HPLC. For these separations, a commercial counter-current chromatograph was fitted with a pH electrode in a flow cell to record the pH of the effluent "on line,' thereby replacing the tedious and time-consuming manual measurement of the pH of each fraction. Additionally, UV spectra of the effluent were continuously monitored and stored by using a computerized scanning UV-Vis detector equipped with an adjustable short-pathlength flow cell.

Chromatography, High Pressure Liquid↗

PubMed1996

Stimulation of human breast cancer MCF-7 cells with estrogen prevents cell cycle arrest by HMG-CoA reductase inhibitors.

Inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, such as Simvastatin and Lovastatin, reduce the rate of DNA synthesis and proliferation of a wide variety of cell types in vitro, by inducing a cell cycle arrest in G1. In estrogen-free medium, DNA synthesis is reduced by more that 90% following exposure of normal and transformed human breast epithelia] cells to 20 microM Simvastatin or Lovastatin for 24 to 42 hrs. We show here that stimulation of estrogen responsive MCF-7 cells with nanomolar concentrations of 17beta-estradiol (E2) prevents inhibition of DNA synthesis by these compounds. The effect of the hormone is antagonized by both steroidal and non steroidal antiestrogens, and it is not detectable in estrogen receptor-negative MCF-10a cells. Cell cycle analysis demonstrates that HMG-CoA reductase inhibitors are unable to induce G1 arrest of MCF-7 cells in the presence of E2.

Breast Neoplasms↗

PubMed1996

17 beta-Estradiol overcomes a G1 block induced by HMG-CoA reductase inhibitors and fosters cell cycle progression without inducing ERK-1 and -2 MAP kinases activation.

HMG-CoA reductase inhibitors, such as Lovastatin and Simvastatin, cause cell cycle arrest by interfering with the mitogenic activity of mitogens present in culture media. Cells are induced to pause in G1 and can readily resume growth upon removal of the enzymatic block. Estrogens, acting via their nuclear receptor, are mitogens for different normal and transformed cell types, where they foster cell cycle progression and cell division. In estrogen-responsive MCF-7 human breast cancer cells, but not in non responsive cells, 17 beta-estradiol (E2) induces cells arrested with Lovastatin or Simvastatin to proliferate in the presence of inhibitor, without restoring HMG-CoA reductase activity or affecting the protein prenylation pattern. Mitogenic stimulation of G1-arrested MCF-7 cells with E2 includes primary transcriptional activation of c-fos, accompanied by transient binding in vivo of the estrogen receptor and/or other factors to the ERE and the estrogen-responsive DNA region of this proto-oncogene, as detected by dimethylsulphate genomic footprinting analysis. Mitogenic stimulation of growth-arrested MCF-7 cells by E2 occurs, under these conditions, without evident activation of ERK-1 and -2 kinases, and thus independently from the mitogen-responsive signal transduction pathways that converge on these enzymes.

Base Sequence↗

PubMed1996

Fragmentation of isomeric N-quinolinylphthalimides on electron impact ionization.

The isomeric 2-, 3-, 5-, 6- and 8-quinolinylphthalimides give rise to different electron impact ionization mass spectra, which permit easy distinction. The specific fragmentation process are rationalized in terms of proximity effects and stabilization of cyclic ion structures. Collision-induced dissociation spectra were used to support the proposed ion structures of major fragment ions.

Electrons↗

PubMed1995

Inducible nitric oxide synthase in a human glioblastoma cell line.

Nitric oxide synthase (NOS) activity was induced in the cytosol of A-172 cells by treatment with lipopolysaccharide, tumor necrosis factor-alpha, and interferon-gamma. A 130-kDa human inducible NOS (iNOS) protein was detected with anti-rat iNOS antibody by western blot analysis. Northern blot analysis showed that the iNOS mRNA was approximately 4.5 kb, using a cDNA fragment for human iNOS, isolated from stimulated A-172 cells by reverse transcriptase-PCR, as a probe. The mRNA was induced by interferon-gamma at a trace level, and its expression was synergistically enhanced by simultaneous addition of lipopolysaccharide, tumor necrosis factor-alpha, and, to a larger extent, interleukin-1 beta. The mRNA expression was blocked by coincubation with actinomycin D or cycloheximide. Furthermore, by transfecting the mouse iNOS gene promoter into A-172 cells, transcriptional activation of the iNOS gene was detected in these cells upon stimulation with lipopolysaccharide and cytokines. The pattern of promoter activation correlated well with that of iNOS mRNA expression upon stimulation. These data indicate that expression of iNOS is transcriptionally regulated in A-172 cells. This process requires de novo protein synthesis with a mechanism similar to that in place in mouse macrophages.

Amino Acid Oxidoreductases↗

PubMed1995

Implication of nitric oxide synthase in carcinogenesis: analysis of the human inducible nitric oxide synthase gene.

Nitric oxide (NO) is a newly identified, multifunctional biological mediator. However, it also has deleterious effects on biological materials. For instance, nucleic acids, proteins, and some prosthetic groups of enzymes can be modified by NO or its reaction products with other reactive oxygen species. Endogenous nitrosamine formation through the reaction of NO or its oxidized products with amines might be involved in carcinogenesis. These deleterious effects of NO are often associated with inflammatory processes both in experimental animals and human. We analyzed the molecular mechanism of control of expression of the inducible nitric oxide synthase (NOS) gene in mouse cells by cloning its putative promoter region. This promoter responded to various cytokines and endotoxin similarly to the endogenous NOS gene in mouse cells. No appreciable induction of NOS was observed in human peripheral blood cells, but induction was detected in a human glioblastoma cell line A-172. Therefore, the human inducible NOS cDNA was cloned from A-172 cells and its cDNA-deduced amino acid sequence found to have about 80% similarity to those of both mouse and rat inducible NOSs. The effects of various cytokines on the induction of the gene were somewhat different from those observed in mouse cells, but the mouse promoter responded to these cytokines similarly to the endogenous NOS gene in human cells, indicating functional similarity of cis-elements of the genes encoding both human and mouse inducible NOS. Structural analysis of the human inducible NOS gene by Southern blot analysis revealed putative genetic restriction fragment length polymorphism in intron 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Preparative separation of stereoisomeric 1-methyl-4-methoxymethylcyclohexanecarboxylic acids by pH-zone-refining counter-current chromatography.

The application of pH-zone-refining counter-current chromatography (CCC) to the preparative separation of stereoisomeric acids is described. The separation was accomplished on the basis of the difference in acidity of the two stereoisomers. pH-Zone-refining CCC of 400 mg of a crude synthetic mixture of stereoisomeric 1-methyl-4-methoxymethylcyclohexanecarboxylic acids yielded 49.5 and 40 mg of the pure Z- and E-stereoisomers respectively. The two-phase solvent system consisted of hexane-ethyl acetate-methanol-water (1:1:1:1). Trifluoro acetic and octanoic acids were used as retainer acids. The eluent base was aqueous ammonia. The eluted fraction were monitored by gas chromatography-mass spectrometry.

Carboxylic Acids↗

PubMed1994

Preparative separation of components of the color additive D&C Red No. 28 (phloxine B) by pH-zone-refining counter-current chromatography.

A pH-zone-refining counter-current chromatographic method was developed for the preparative (multigram) separation and purification of components of the commercial color additive D&C Red No. 28 (phloxine B). The chromatography of 3 and 6 g of color additive yielded 1.07 and 4.06 g, respectively, of pure 2',4',5',7'-tetrabromo-4,5,6,7-tetrachlorofluorescein, the principal component of D&C Red No. 28. The importance of the quantity of retainer acid (trifluoroacetic acid) relative to the amount of salt in the color additive is discussed.

Chromatography, High Pressure Liquid↗

PubMed1994

Dual mechanism for the control of inducible-type NO synthase gene expression in macrophages during activation by interferon-gamma and bacterial lipopolysaccharide. Transcriptional and post-transcriptional regulation.

Production of nitric oxide (NO) by macrophages is enhanced upon activation by bacterial endotoxins and cytokines mainly via an increase of the intracellular content of the inducible isoform of nitric oxide synthase (i-NOS). We have studied in detail the effect of several modulators of macrophage activity on steady state levels of i-NOS mRNA in the mouse macrophage-like cell line RAW 264.7. Bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) were found to be effective inducers of i-NOS mRNA, in accordance with their known ability to stimulate both i-NOS activity and NO production in macrophages from different sources, while TNF-alpha, IL-1, or IL-6 was ineffective in this regard. Accumulation of i-NOS mRNA in response to either LPS or IFN-gamma stimulation was accompanied by increased i-NOS gene transcription, as detected both by using a nuclear "run-on" transcription assay and by transient transfection of the cloned gene promoter in RAW 264.7 cells. Co-stimulation of the cells with both inducers resulted in higher steady state levels of i-NOS mRNA in the absence, however, of a corresponding potentiation of the rate of gene transcription. This was due primarily to a considerable effect of LPS on i-NOS mRNA stability, with prolongation of its half-life from 1-1.5 h, in the presence of IFN-gamma alone, to 4-6 h in the presence of both LPS and IFN-gamma.

Amino Acid Oxidoreductases↗

PubMed1994

Enhancement of inducible-type NO synthase gene transcription by protein synthesis inhibitors. Activation of an intracellular signal transduction pathway by low concentrations of cycloheximide.

Treatment of mouse macrophage-like RAW 264.7 cells with certain protein synthesis inhibitors is followed by accumulation of the mRNA for the inducible isoform of nitric oxide synthase (i-NOS). The activity of these compounds on the i-NOS gene in RAW 264.7 cells was analyzed here in detail. Results show that both cycloheximide and anisomycin can efficiently induce i-NOS mRNA, even when used at concentrations so low (0.25 microgram/ml) to have only negligible effects on protein synthesis; puromycin, on the other hand, shows only a limited effect on i-NOS mRNA expression, detectable only when cells are treated with higher concentrations of inhibitor (25 micrograms/ml). In RAW 264.7 cells, low concentrations of cycloheximide trigger an immediate-early gene response, as indicated by induction of c-fos and JE mRNAs, and can efficiently activate transcription of transiently transfected recombinant reporter genes including either the i-NOS or the c-fos gene promoters.

Amino Acid Oxidoreductases↗

PubMed1994

IFN consensus sequence binding protein (ICSBP) is a conditional repressor of IFN inducible promoters.

IFN stimulated genes (ISGs) contain common DNA motif termed IFN consensus sequence (ICS) at their promoters that enable IFN responsiveness. Different transcription factors capable of interacting with the ICS have been described. Previously, we reported the cloning of a factor capable of binding to the ICS (ICSBP) that demonstrates similarity at DNA the binding domain with three other ICS binding factor, i.e. IRF-1, IRF-2 and ISGF3 gamma. ICSBP is expressed constitutively in hematopoietic cells and its expression is further induced by IFN-gamma. This is a negative trans-acting regulator of ISGs; however, its effect is attenuated following prolonged exposures of cells to both types of IFNs. In this communication, we show that short exposures of cells to IFNs (priming) are sufficient to alleviate ICSBP mediated repression. Further, exposure of primed cells to the synthetic dsRNA (polyl-polyC) results in total abrogation of ICSBP repression. In an attempt to unravel the molecular mechanism governing this conditional repression of ICSBP, the direct involvement of transcriptional activator IRF-1 is demonstrated. We postulate that constitutive expression of ICSBP in hematopoietic cells is mediating submaximal expression of ISGs such as MHC class I. Our data demonstrate that IRF-1 competes with ICSBP for the binding to the ISRE element, resulting in the alleviation of ICSBP repression. Thus, the magnitude of ISGs expression is a result of a fine balance between positive and negative regulators.

Antisense Elements (Genetics)↗

PubMed1993

Functional antagonism between the estrogen receptor and Fos in the regulation of c-fos protooncogene transcription.

Estrogen hormones induce transient transcriptional activation of c-fos during the early phases of mitogenic stimulation of target cells. This is mediated by a functional estrogen response element (ERE) that in the human c-fos gene is localized 1kb up-stream of the transcription start site. This is the first known example of transient transcriptional activation induced by a steroid hormone acting via its nuclear receptor. Starting with the hypothesis that the product of c-fos (Fos) interferes with estrogen receptor (ER) activity on this gene promoter, generating in this way a feedback inhibition mechanism responsible for the rapid transcriptional down-regulation detected in vivo, we tested the effects of Fos overexpression on ER-mediated activation of the c-fos promoter in transfected HeLa cells. Transient transfection of an ER expression vector is followed by hormone-dependent trans-activation of reporter genes comprising the c-fos ERE linked to its own promoter. Coexpression of Fos in the cell induces a significant reduction in the activity of ER on the reporter genes. Fos antagonism is effective on both transcription activation functions of the receptor molecule and is independent of the nature of the target promoter. Furthermore, under the same experimental conditions, the estrogen-receptor complex antagonizes activation of an AP-1-responsive test gene by Fos. ER mutants deprived of the DNA-binding domain are efficient inhibitors of Fos activity, indicating that reciprocal antagonism is likely to be mediated by the formation of inactive complexes between the two factors. These results reveal the existence of a functional interference between the ER and Fos for regulation of c-fos protooncogene transcription. It is the first case in which the product of an estrogen-induced growth-related gene is shown to exert a negative feedback control on ER regulation of its own promoter.

Animals↗

PubMed1993

Nasal distribution of budesonide inhaled via a powder inhaler.

The distribution pattern of budesonide in the nasal passages and lungs was investigated in 10 healthy subjects after nasal inhalation. The subjects inhaled drug powder, radiolabelled with 99mTc, at maximum flow rate (46.3 +/- 6.8 l/min) and at 29.9 +/- 2.5 l/min via Turbuhaler. At both flows, the majority of the dose was deposited in the anterior part of the nasal cavity on a single, rather localized area, but some particles also penetrated more posteriorly into the main nasal passages and to the lungs. At maximum flow rate the nasal deposition was 65.2% (range 39.5-84.1%) and the lung deposition 4.7% (range 1.4-9.3%) of the metered dose, and at 30 l/min, the nasal deposition was 67.6% (range 49.7-81.6%) and the lung deposition was 4.2% (range 1.7-7.9%). A large fraction of the metered dose was deposited in the nasal adaptor of the inhaler during the administration (mean values 29 and 28%, for the two inhalation flows). Of the dose actually reaching the subject, 91 and 93% (mean values) was deposited in the nose. There were no statistically significant differences in distribution pattern between the two inhalation flows.

Administration, Inhalation↗

PubMed1993

Identification of a specific pattern of "immediate-early" gene activation induced by estrogen during mitogenic stimulation of rat uterine cells.

Estrogen hormones are potent mitogens for certain target tissues, where they stimulate cell growth by inducing recruitment of quiescent cells in cycle and by fostering cell cycle progression. To define the molecular bases of the mitogenic action of these steroid hormones, the pattern of "immediate-early" gene expression was monitored during the early phases of estrogen stimulation of rat uterine cells in vivo. Nuclear run-on transcription and/or Northern blot RNA analysis indicate that c-jun, junB, jun-D, c-fos, TIS 1 (also called NGFI-B or nur/77) and TIS 8 (zif-268, krox24, egr-1, or NGFI-A) genes are all transiently activated in the uterus (up to 20-fold) within 30-120 min after treatment of adult ovariectomized rats with a mitogenic dose of 17b-estradiol. Conversely, JE gene mRNA accumulates progressively in estrogen-stimulated uterine cells, whereas TIS 11 and 21 genes are only slightly responsive to the hormone (less than twofold induction) and fos B,fra-1,fra-2,krox20 (egr-2), TIS 7 and 10, KC, and c-rel mRNAs are undetectable in rat uterus either before or after estrogen treatment. Stimulation in the presence of cycloheximide shows that only c-jun, jun-D, c-fos, and JE gene activations are primary responses to the hormone in rat uterine cells. These findings establish the direct mitogenic role of estrogen and identify for the first time a specific genetic program activated by these steroid hormones during stimulation of target cell proliferation. Furthermore, since most of the activated genes encode for transcription factors, these results enable us to envision how the mitogenic signal transmitted by the hormone can be elaborated and amplified within target cells by the products of estrogen-responsive genes, leading to a cascade of growth-dependent gene regulation, cell cycle progression, and, ultimately, cell division.

Animals↗