Block RNA Synthesis Research Articles

Actin is closely associated with RNA polymerase II and involved in activation of gene transcription

Biochemical and morphological studies have demonstrated the presence of actin in the nucleus of different eukaryotic cells, whereas its role remains unclear. In this work, we studied the interaction and the functional relationship between nuclear actin and RNA polymerase II (RNAP II). The immunofluorescence study demonstrated a clear co-localization of nuclear actin with RNAP II in HeLa cells. Meanwhile, actin can be immunoprecipitated by anti-RNAP II antibody, indicating that they could interact with each other. Treatment of cells with α-amanitin induced the formation of actin bundle network in the nucleoplasm. Blocking of the formation of filamentous actin (F-actin) by cytochalasin B modified the distribution of actin. Although the actin content remained unchanged in resting and concanavalinA stimulated mouse lymphocytes, the actin content in the nuclei showed a progressive increase after stimulation. Furthermore, the antibody against actin blocked RNA synthesis in a eukaryotic in vitro transcription system. These observations implicate that nuclear actin interacts with RNAP II and may have function on the RNAP II-mediated transcription.

DNA damage responses triggered by a highly cytotoxic monofunctional DNA alkylator, hedamycin, a pluramycin antitumor antibiotic

Long-term exposure (72 h) to hedamycin, a monofunctional DNA alkylator of the pluramycin class of antitumor antibiotics, decreased growth of mammalian cells by 50% at subnanomolar concentrations. Short-term treatment (4 h) rapidly reduced DNA synthesis by 50% also at subnanomolar concentrations, but substantially higher levels were needed to block RNA synthesis while protein synthesis even at very high hedamycin concentrations remained unaffected. Hedamycin treatment at concentrations below its growth IC(50) induced only a transient and temporary accumulation of cells in G(2). Somewhat higher concentrations resulted in substantial S-phase arrest, and at increasing concentrations, complete cell cycle arrest in G(1) was observed without the appearance of a sub-G(1) cell population. Neither inhibition of cell growth nor cell cycle arrest appeared to be dependent on ataxia and Rad-related kinase expression. DNA damage checkpoint proteins including p53, chk1, and chk2 were differentially activated by hedamycin depending on the concentration and duration of treatment. The level of downstream cell cycle regulators such as cdc25A, E2F1, cyclin E, and p21 were also altered under conditions that induced cell cycle arrest, but atypically, p21 overexpression was observed only in S-phase-arrested cells. Apoptotic indicators were only observed at moderate hedamycin concentrations associated with S-phase arrest, while increasing concentrations, when cells were arrested in G(1), resulted in a reduction of these signals. Taken together, the responses of cells to hedamycin are distinct with regard to its effect on cell cycle but also in the unusual concentration-dependent manner of activation of DNA damage and cell cycle checkpoint proteins as well as the induction of apoptotic-associated events.

Photorepair of RNA polymerase arrest and apoptosis after ultraviolet irradiation in normal and XPB deficient rodent cells.

Cyclobutane pyrimidine dimers (CPDs) are directly involved in signaling for UV-induced apoptosis in mammalian cells. Failure to remove these lesions, specially those located at actively expressing genes, is critical, as cells defective in transcription coupled repair have increased apoptotic levels. Thus, the blockage of RNA synthesis by lesions is an important candidate event triggering off active cell death. In this work, wild-type and XPB mutated Chinese hamster ovary (CHO) cells expressing a marsupial photolyase, that removes specifically CPDs from the damaged DNA, were generated, in order to investigate the importance of this lesion in both RNA transcription blockage and apoptotic induction. Photorepair strongly recovers RNA synthesis in wild-type CHO cell line, although the resumption of transcription is decreased in XPB deficient cells. This recovery is accompanied by the prevention of cells entering into apoptosis. These results demonstrate that marsupial photolyase has access to CPDs blocking RNA synthesis in vivo, and this may be affected by the presence of a mutated XPB protein.

Suppression of programmed cell death by intracellular cAMP is not mediated by expression of genes encoding an inhibitor of apoptosis.

The elevation of intracellular cAMP content is accompanied by expression of genes whose promoter contains a Ca(2+)-cAMP responsive element. In vascular smooth muscle cells (VSMC), activation of cAMP signaling blocks apoptosis triggered by serum deprivation. In the present study we investigated the role of gene expression in the inhibition of apoptosis by cAMP. In VSMC transfected with E1A adenovirus, incubation in the absence of serum for 6 h led to 20-fold elevation of chromatin fragmentation and 10-fold activation of caspase-3 activity, these being employed as markers of apoptosis. Forskolin-induced activation of cAMP signaling was accompanied by 50% elevation of RNA synthesis and completely abolished the development of apoptosis during the initial 6 h incubation in growth factor-free medium. In 12 h apoptosis in forskolin-treated VSMC was slowly developed and after 24 h the content of chromatin fragments was 2-fold less than in control cells. Addition of actinomycin D and cycloheximide completely blocked RNA synthesis and decreased protein synthesis by 80%, respectively. Neither compound affected baseline apoptosis or its inhibition by forskolin. More than 70 newly phosphorylated proteins were observed by 2D-electrophoresis of VSMC after incubation with forskolin for 3 h; in 24 h the number of phosphoproteins triggered by forskolin was decreased by 2-3-fold. These results show that suppression of VSMC apoptosis under activation of cAMP signaling is mediated via posttranslational modification of pre-existing intermediates of the apoptotic machinery rather than by de novo synthesis of inhibitors of programmed cell death.

Bacteriophage T7 RNA polymerase transcription elongation is inhibited by site-specific, stereospecific benzo[c]phenanthrene diol epoxide DNA lesions.

Benzo[c]phenanthrene diol epoxide (B[c]PhDE), the ultimate carcinogenic metabolite of the environmental pollutant benzo[c]phenanthrene, reacts with DNA primarily at the exocyclic amino groups of purines, forming B[c]PhDE-DNA adducts that differ in their stereochemical configurations and their effect on biological processes such as transcription. To determine the effect of these stereoisomers on RNA synthesis, in vitro T7 RNA polymerase transcription assays were performed using DNA templates modified on the transcribed strand by either a site-specific (+)-trans- or (-)-trans-anti-B[c]PhDE-N(6)-dA lesion located within the sequence 5'-CTCTCACTTCC-3'. The results show that both (-)-trans-anti-B[c]PhDE-N(6)-dA and (+)-trans-anti-B[c]PhDE-N(6)-dA block RNA synthesis. Furthermore, both B[c]PhDE-dA stereoisomeric adducts lead to lower levels of initiation of transcription relative to that observed using an unmodified DNA template. In contrast to these results, placement of the adduct on the nontranscribed strand within the template does not impede transcription elongation. In addition to the assessment of the effect of the lesions on transcription elongation, the resulting transcripts were characterized in terms of their base composition. A high level of base misincorporation is detected at the 3'-ends of truncated transcripts, with guanosine being most frequently incorporated opposite the modified nucleotide rather than the expected uridine. This result supports the notion that translocation past a modified base in a DNA template relies in part on correct base incorporation, and suggests that stalling of RNA polymerases at damaged sites in DNA may well be dependent on both the presence of the lesion and the base which is incorporated opposite the modified nucleotide.

Regulation of HGF/SF Gene Expression in MRC-5 Cells by N-Acetylcysteine

The effect of N-acetylcysteine (NAC) on levels of hepatocyte growth factor/scatter factor (HGF/SF) gene transcripts was investigated in the human lung embryonic fibroblast cell line, MRC-5. NAC increased expression of HGF/SF mRNA, in a dose- and time-dependent fashion, by a mechanism independent of glutathione synthesis but sensitive to oxidant stress induced by H2O2. Using actinomycin D to block RNA synthesis, it was observed that NAC had no effect on the stability of the HGF/SF mRNA transcripts. NAC increased HGF/SF promoter activity in cells transiently transfected with chloramphenicol acetyltransferase (CAT) reporter genes driven by HGF/SF gene 5′-flanking sequences. Primer extension analysis demonstrated that NAC enhanced the expression of HGF/SF mRNA transcribed from the main transcription initiation site. Although the 5′ flanking region of the HGF/SF gene contains a sequence at −1019 to −1011 with homology to the NF-κB response element, electrophoretic mobility shift assay demonstrated that this site did not bind nuclear factors in MRC-5 cells in the presence or absence of NAC. In contrast to the effect on HGF/SF mRNA, NAC did not increase HGF/SF protein production by MRC-5 cells.

Mechanism of Regulation of HGF/SF Gene Expression in Fibroblasts by TGF-β1

The effect of transforming growth factor β 1 (TGF-β1) on levels of hepatocyte growth factor/scatter factor (HGF/SF) gene transcripts was investigated in the human lung embryonic fibroblast cell line, MRC-5. TGF-β1 markedly reduced the expression of the 6.0-kb and 3.0-kb HGF/SF mRNA, which encode full-length HGF/SF, but it had little effect on the expression of the alternatively spliced 1.5-kb mRNA, which encodes NK2, a competitive HGF/SF antagonist. Using actinomycin D to block RNA synthesis, it was observed that TGF-β1 had little effect on the stability of the 1.5-kb NK2 mRNA but increased the rate of degradation of the 6.0- and 3.0-kb HGF/SF mRNA transcripts by a mechanism that was dependent on new protein synthesis. TGF-β1 minimally increased rather than reduced HGF/SF promoter activity in cells transiently transfected with chloramphenicol acetyltransferase (CAT) reporter genes driven by HGF/SF gene 5′-flanking sequences. In MRC-5 cells, TGF-β1 modulates HGF/SF gene transcripts at the posttranscriptional level in order to favour expression of the 1.5-kb mRNA that encodes the truncated protein NK2.

Calcium and phosphate supplementation promotes bone cell mineralization: implications for hydroxyapatite (HA)-enhanced bone formation.

Organic phosphate, in particular beta-glycerophosphate (beta-GP), has been used to induce mineralization in cell culture systems. It serves as a source of inorganic phosphate when hydrolyzed by alkaline phosphatase. This study examined the effect of supplemental calcium and phosphate as well as the influence of various metabolic inhibitors on mineralization in a rat osteoblast-like cell-culture system. Mineralization was induced by supplementation of 1.8 mM of Ca(+2) and 5 mM of beta-GP or Pi. Mineral deposits associated with in vitro mineralization were revealed under SEM and TEM. Levamisole (10-100 microM) inhibited alkaline phosphatase activity and effectively reduced mineral formation. Actinomycin (500 ng/mL) and cycloheximide (50 microg/mL) also reduced mineral depositions by blocking RNA synthesis and protein synthesis, respectively. Levamisole and beta-GP did not appear to influence DNA synthesis. Spontaneous precipitation of calcium phosphate mineral was not detected in the culture medium with calcium and phosphate supplements in the absence of cell culture. The findings suggest that an elevated concentration of calcium and phosphate is crucial for in vitro mineralization. Furthermore, the mineralization process is associated with biologic events rather than with a spontaneous precipitation of calcium phosphate mineral. In view of the degradation potential of hydroxyapatite (HA)-coated implants, these results may be a viable indication that HA enhances bone formation through a similar mechanism.

Targeted gene repair in mammalian cells using chimeric oligonucleotides.

In the past several years, significant advances in the area of genome modification have taken place. In some cases, the objective has been to block RNA synthesis and change developmental progression (1), while in other cases more permanent alteration at the chromosomal level has been attempted (2). These types of approaches seek not to replace traditional protocols of gene knock-out, gene knock-in or gene replacement, but rather augment them, adding to the arsenal of techniques available to workers who wish to study functional aspects of gene expression. In fact, since the strategies for creating mouse knock-outs first became available, relatively little improvement in the rate of success has occurred.

Signaling pathways for tumor necrosis factor-alpha and interleukin-6 expression in human macrophages exposed to titanium-alloy particulate debris in vitro.

Loosening of the implant after total joint arthroplasty remains a serious problem. The activation of macrophages by wear debris from implants, mediated by the release of cytokines that elicit bone resorption, may lead to loosening. The purpose of the present study was to elucidate the mechanisms of macrophage activation by titanium particles from the components of implants and to identify the signaling pathways involved in particle-mediated release of cytokines. Macrophages were isolated from mononuclear leukocytes obtained from healthy human donors and were exposed to titanium-alloy particles that had been obtained from periprosthetic membranes collected at revision total joint arthroplasties and then enzymatically prepared. The experimental protocols included examination of the effects of the inhibition of phagocytosis and the binding of antibodies to macrophage complement receptors on particle-induced macrophage activation. The release of the proinflammatory cytokines TNF-alpha (tumor necrosis factor-alpha) and IL-6 (interleukin-6) was used to assess macrophage activation. The signaling pathways involved in the induction of cytokine release were analyzed by identification of phosphorylated proteins with use of the Western blot technique and by translocation of the transcription factors nuclear factor-kappa B (NF-kappaB) and nuclear factor-interleukin-6 (NF-IL-6) into the nuclear protein fraction with use of electrophoretic mobility shift assays. The role of serine/threonine and tyrosine kinase pathways in the activation of nuclear factors and the release of cytokines was examined with use of selective pharmacological agents. Exposure of macrophages to titanium-alloy particles in vitro for forty-eight hours resulted in a fortyfold increase in the release of TNF-alpha and a sevenfold increase in the release of IL-6 (p<0.01). Phagocytosis of particles occurred in approximately 73 percent of the macrophages within one hour of exposure. Pretreatment of the macrophages with cytochalasin B reduced phagocytosis by 95 percent but did not reduce the release of TNF-alpha or IL-6. Thus, phagocytosis of particles was not necessary for induction of the release of TNF-alpha or IL-6 in the cultured macrophages. Ligation of the macrophage CD11b/CD18 receptors by integrin-specific antibodies also increased the release of TNF-alpha and IL-6. Antibodies to CD11b/ CD18 receptors (macrophage Mac-1 receptors) reduced phagocytosis of particles by 50 percent (p<0.05). (The CD11b/CD18 macrophage receptor is the macrophage receptor for the complement component CR3bi. The CD11b/CD18 macrophage receptor can also bind to ICAM-1 and ICAM-2. CD is the abbreviation for cluster of differentiation, and ICAM is the abbreviation for intercellular adhesion molecule.) Inhibition of phagocytosis was not accompanied by a decrease in the release of TNF-alpha and IL-6. Blocking RNA synthesis with actinomycin D or preventing protein synthesis with cycloheximide abolished or decreased particle-induced release of TNF-alpha and IL-6 from the macrophages. Macrophage release of TNF-alpha and IL-6 in response to particles coincided with increased tyrosine phosphorylation and mitogen-activated protein kinase activation. Inhibition of tyrosine and serine/threonine kinase activity decreased the particle-induced release of cytokines. Exposure of macrophages to either titanium-alloy particles or to antibodies to the receptor proteins CD11b and CD18 for thirty minutes activated the transcription factors NF-kappaB and NF-IL-6. Inhibition of particle phagocytosis did not block activation of the transcription factors. However, inhibition of tyrosine and serine/threonine kinase activity decreased the activation of NF-kappaB and NF-IL-6. These data suggest that particle induced macrophage release of TNF-alpha and IL-6 does not require phagocytosis but is dependent on tyrosine and serine/threonine kinase activity culminating in activation of

Effects of ethanol on GLUT1 protein and gene expression in rat astrocytes.

Effects of ethanol on glucose transporter gene expression were examined in cultured rat astrocytes. Exposure to 50 or 100 mM ethanol for 18 hours significantly inhibited hexose uptake and reduced the number of glucose transporters, as indicated by binding studies with cytochalasin B. Indirect immunofluorescence and immunoperoxidase staining showed marked reduction of the GLUT1 glucose transporter by exposure to 100 mM ethanol for 5 or 18 hours, but no obvious change in response to 50 mM ethanol. Western blot analysis showed GLUT1 protein levels to be decreased by 52 +/- 12% (p < 0.05) after exposure to 100 mM ethanol for 18 hours. In situ hybridization histochemistry indicated an increase in steady-state GLUT1 mRNA in astrocytes exposed to 50 or 100 mM ethanol for 5 or 18 hours. Quantitation of GLUT1 mRNA levels by northern blot analysis showed that GLUT1 mRNA levels were increased by 59 and 112% in cells treated for 5 h with 50 and 100 mM ethanol, respectively. A similar effect was observed after treatment for 18 hours, but ethanol did not alter actin gene expression. Experiments using actinomycin D to block RNA synthesis suggest that this increase in steady-state mRNA level results from increased message stability. These results suggest that ethanol acts on GLUT1 gene expression at the post-transcriptional level.

Induction of DNA replication by transcription in the region upstream of the human c-myc gene in a model replication system.

An important relationship between transcription and initiation of DNA replication in both eukaryotes and prokaryotes has been suggested. In an attempt to understand the molecular mechanism of this interaction, we examined whether transcription can induce DNA replication in vitro by constructing a system in which both replication and transcription were combined. Relaxed circular DNA possessing a replication initiation zone located upstream of the human c-myc gene and a T7 promoter near the P1 promoter of the gene was replicated in the presence of T7 RNA polymerase. In our model system, replication was carried out with the proteins required for simian virus 40 DNA replication. DNA synthesis, which was dependent on both T7 RNA polymerase and the replication proteins, was detected mainly in the promoter and upstream regions of the c-myc gene. Blocking RNA synthesis at the initial stage of the reaction severely reduced DNA synthesis, suggesting that RNA chain elongation is required to induce DNA synthesis. The results indicated that transcription can induce DNA replication in the upstream region of the transcribed gene, most likely by introducing negative supercoiling into the region, which results in unwinding of the DNA duplex.

Amanitin Greatly Reduces the Rate of Transcription by RNA Polymerase II Ternary Complexes but Fails to Inhibit Some Transcript Cleavage Modes

The toxin alpha-amanitin is frequently employed to completely block RNA synthesis by RNA polymerase II. However, we find that polymerase II ternary transcription complexes stalled by the absence of NTPs resume RNA synthesis when NTPs and amanitin are added. Chain elongation with amanitin can continue for hours at approximately 1% of the normal rate. Amanitin also greatly slows pyrophosphorolysis by elongation-competent complexes. Complexes which are arrested (that is, which have paused in transcription for long periods in the presence of excess NTPs) are essentially incapable of resuming transcription in the presence of alpha-amanitin. Complexes traversing sequences that can provoke arrest are much more likely to stop transcription in the presence of the toxin. The substitution of IMP for GMP at the 3' end of the nascent RNA greatly increases the sensitivity of stalled transcription complexes to amanitin. Neither arrested nor stalled complexes display detectable SII-mediated transcript cleavage following amanitin treatment. However, arrested complexes possess a low level, intrinsic transcript cleavage activity which is completely amanitin-resistant; furthermore, pyrophosphorolytic transcript cleavage in arrested complexes is not affected by amanitin.

8-Methoxypsoralen DNA interstrand cross-linking of the ribosomal RNA genes in Tetrahymena thermophila. Distribution, repair and effect on rRNA synthesis.

The distribution and repair of 8-methoxypsoralen-DNA interstrand cross-links in the ribosomal RNA genes (rDNA) in Tetrahymena thermophila have been studied in vivo by Southern blot analysis. It is found that the cross-links at a density of < or = 1/2 x 10(4) base pairs (bp) are distributed equally between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis (BamHI and ClaI). It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is sufficient to block RNA synthesis. Finally, it is shown that the cross-links in the rDNA molecules are repaired at equal rate in all three domains within 24 h and that RNA synthesis is partly restored during this repair period. The majority of the cells also go through one to two cell divisions in this period but do not survive.

Specific Inhibitors of Protein Synthesis Do Not Block RNA Synthesis or Settlement in Larvae of a Marine Gastropod Mollusk (Haliotis rufescens).

Antibiotic inhibitors of protein synthesis were tested for their effectiveness in larvae of the red abalone, Haliotis rufescens (gastropod mollusk). Emetine and anisomycin proved highly effective in this system, while cycloheximide, fusidic acid, puromycin, and tetracycline were less effective. Emetine and anisomycin specifically inhibited protein synthesis but not RNA synthesis. The contribution to protein synthesis by chloramphenicolsensitive prokaryotic contaminants was found to be undetectable, except following the onset of symptoms of toxicity resulting from prolonged exposure to emetine or anisomycin. The induction of larval settlement and plantigrade attachment by γ-aminobutyric acid (GABA), a functional analog of the natural inducer of settlement, occurred even under conditions in which most protein synthesis was inhibited, as expected for a chemosensory system response, whereas subsequent developmental metamorphosis was completely blocked. Because emetine and anisomycin block protein synthesis--including the synthesis of new transcription factors--but do not block early transcription, treatment of marine invertebrate embryos and larvae with these inhibitors can be used to obtain a selective enrichment in the mRNA population of "early gene" transcripts induced directly by GABA and other morphogenetic signals, without dilution by new mRNAs, the appearance of which is dependent on the synthesis of new protein transcription factors.

Nicotinamide-induced activity of alkaline phosphodiesterase I toward tumor-derived cultured cells from neurofibromatosis patients.

Membrane-bound alkaline phosphodiesterase I was investigated in control fibroblasts and tumor-derived fibroblasts from patients with neurofibromatosis. Alkaline phosphodiesterase I activity of tumor-derived cells increased in a dose response to nicotinamide (0-9 mM) in culture; 5'-thymidine p-nitrophenyl phosphate was used as substrate. The enzyme activity increased 1.7-fold after 30 h of incubation with 9 mM nicotinamide, and after 3 weeks increased 5-fold. The nicotinamide-dependent enhancement of alkaline phosphodiesterase I activity was inhibited by actinomycin D, which specifically blocks RNA synthesis, but not by cycloheximide. These results suggest that the increase in the enzyme activity caused by nicotinamide was due to induction of alkaline phosphodiesterase I at the transcriptional level in tumor-derived cells. The metabolic effect of nicotinamide on alkaline phosphodiesterase I may be related to tumorigenicity in neurofibromatosis.

Yeast nucleosomal particles: structural and transcriptional properties

Yeast nucleosomal core particles have been characterized by thermal denaturation, circular dichroism, and digestion with DNase I and with trypsin. Practically all nucleosomal DNA melts in one transition centered at 70 degrees C, and the circular dichroism spectrum is displaced to lower wavelengths as compared to that corresponding to chicken nucleosomal cores. The susceptibility of yeast nucleosomal particles to dissociation by salt is significantly higher than that of chicken nucleosomal cores, a substantial dissociation being observed at 0.5 M NaCl. Treatment of yeast nucleosomal particles with the amino group reagent dimethylmaleic anhydride is accompanied by selective release of histones H2A and H2B. The results indicate not large but significant structural differences between yeast and chicken nucleosomal cores. However, the in vitro transcription properties of complete and H2A.H2B-deficient nucleosomal cores are similar in the two kinds of particles: the histone octamer blocks RNA synthesis, this block being eliminated in part by the partial loss of histones H2A and H2B.

Hydrostatic pressure influences histone mRNA

Exposure of HeLa S3 cells to high hydrostatic pressure (6.89 x 10(3) to 6.89 x 10(4) kPa: 1000 to 10,000 lbfin-2) reduced core and H1 histone mRNA levels as determined by hybridization to specific histone DNA probes. At 4.14 x 10(4) kPa for 10 min core histone and H1 histone mRNA levels were reduced 32-38% and 56%, respectively. At 30 min postdecompression core mRNA levels returned to atmospheric control levels while H1 histone mRNA levels continued to be suppressed. Levels of macromolecular synthesis were monitored under hydrostatic pressure with radioactive precursors of RNA, DNA and protein. Macromolecular synthesis was shown to be suppressed in a dose-dependent manner with increasing magnitude and duration of pressure. To determine the influence of pressure on histone mRNA stability, actinomycin D (10 micrograms ml-1) was used to block RNA synthesis. Relative amounts of H4 and H1 mRNA were determined at atmospheric pressure and following treatment with actinomycin D (10 micrograms ml-1), pressure (4.14 x 10(4) kPa) and a combination of pressure and actinomycin D. This study shows that a synthesis component and a stability component are involved in the pressure-induced reduction of core histone mRNA. At 4.14 x 10(4) kPa for 15 min, there was a 42% reduction in core histone mRNA of which approximately one third was due a suppression of transcription and two thirds to a loss of mRNA stability. The pressure-induced reduction in histone mRNA is attributed to the instability of endogenous histone mRNA and a reduction in transcription/processing of new histone mRNA.

The Isoquinoline Sulfonamide Inhibitors of Protein Phosphorylation, H-7, H-8, and HA-1004, Also Inhibit RNA Synthesis: Studies on Responses of Adipose Tissue to Growth Hormone*

To evaluate the possibility that some of the metabolic effects of GH in rat adipose tissue depend upon phosphorylation-dephosphorylation reactions, we examined the effects of the isoquinoline sulfonamide family (H-7, H-8, and HA-1004) of protein kinase inhibitors on the actions of GH. In the course of these studies it became clear that these compounds may also block RNA synthesis. In the concentration range of 50-200 microM, H-7, H-8, and HA-1004 completely blocked lipolysis in response to the combination of 100 ng/ml dexamethasone and 30 ng/ml human GH in segments of epididymal fat from normal rats, but were less effective in blocking lipolysis in response to either 1 mM (Bu)2cAMP or 1 ng/ml isoproterenol, which are known to depend upon activation of protein kinase-A. Activation of protein kinase-C with phorbol myristate nearly doubled the rate of glucose oxidation in segments of normal adipose tissue, and this insulin-like response was completely inhibited with 200 microM H-7. At concentrations as high as 500 microM, H-7, H-8, and HA-1004 failed to inhibit the insulin-like response to GH in tissue segments of either normal or hypophysectomized rats. However, when 200 microM H-7 or H-8, but not HA-1004, was present during the first 3 h of treatment with GH, it prolonged the duration of the insulin-like response (acceleration of glucose oxidation) from its normal termination within 2-3 h to more than 4 h. Identical results were obtained with 5 micrograms/ml actinomycin-D. The effect of H-7 or H-8 was reversible and required the continuous presence of these agents, whereas actinomycin-D was required only during the first 60 min after GH. Termination of the insulin-like response normally is followed by a period of several hours in which the tissues are refractory to further insulin-like stimulation by GH. When actinomycin-D, H-7, H-8, or HA-1004 was added to tissues of hypophysectomized rats 60 min after GH, the insulin-like response terminated at its normal time, but the tissues were not refractory to insulin-like stimulation upon reexposure to GH. These agents also prevented GH from sustaining refractoriness in normal adipose tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular Mechanism of Action of the Antibiotic Rifampicin

AbstractThe lipophilic antibiotic rifampicin is successfully used in the treatment of tuberculosis. On the molecular level it interferes with the metabolism of Eubacteria by blocking RNA synthesis. This effect is the consequence of the tight binding of the drug to a single and highly specific binding site on the DNA‐dependent RNA polymerase. The enzyme‐bound drug strongly inhibits RNA chain initiation and chain elongation. This inhibition can be explained by the influence of enzyme‐bound rifampicin on binding sites for the reaction products diphosphate and RNA. In order to reach its target the antibiotic must penetrate into the cytoplasm of the bacteria. Mutants have been discovered which are resistant to rifampicin because its uptake from the medium is significantly reduced. The gene responsible for this effect has been cloned. It confers on bacterial cells with highly sensitive RNA polymerases a remarkable resistance to the drug.