Seroprevalence of Leptospira spp. in clinically healthy horses in Switzerland.

A retrospective, cross-sectional study was conducted to determine the leptospiral seroprevalence in clinically healthy horses in Switzerland. A representative sample of 615 horse sera was examined by microscopic agglutination test for the presence of antibodies against 15 Leptospira spp. serovars. In total, 58.5 % (n = 360) of the horses were positive for one or more of the antigens analysed, with 20.3 % of them showing titres >= 400. The most prevalent serovar was Pyrogenes (22.6 %), followed by serovars Canicola (22.1 %) and Australis (19.2 %). Older horses, mares, ponies and animals spending increased time on pasture exhibited significantly higher prevalence rates (p < 0.05). Moreover, the prevalence was higher in summer and autumn (p = 0.003). The high seroprevalence in healthy horses indicates that they are often exposed to or infected with Leptospira spp. without developing signs of disease. Therefore, other laboratory and clinical data should always be taken into consideration when interpreting serological test results for Leptospira spp.

Regulation of differentiation, proliferation and cancer suppressor activity.

This review suggests that carcinogenesis is linked to defects in linkages between cellular differentiation and regulation of cell proliferation. These linkages are discussed in terms of terminal and nonterminal states of differentiation and their relationship to the control of proliferation. The ability of differentiation to regulate proliferation potential proteins and cancer suppressor genes is also discussed, because these mechanisms may be important for cancer prevention and therapy.

Stable induction of c-jun mRNA expression in normal human keratinocytes by agents that induce predifferentiation growth arrest.

A variety of agents can induce predifferentiation growth arrest (PGA) in human keratinocytes; these include transforming growth factor beta 1 (TGF-beta 1) and razoxane. We evaluated the ability of these and other agents to induce the expression of a variety of transcription factor genes including c-fos, c-myc, junB, and c-jun. The results show that both TGF-beta 1 and razoxane induce maximal c-jun mRNA expression 4 days after initiation of treatment concurrent with the development of PGA. In contrast, no detectable induction of c-fos, c-myc, or junB was observed. Keratinocytes maintained in the presence of TGF-beta 1 for an additional 3 days continued to show high levels of c-jun mRNA, indicating stable induction. Razoxane treatment also induces PGA and high c-jun mRNA levels for 4 days, but thereafter a decay of c-jun expression occurs. Run-off transcription experiments comparing rapidly growing cells with cells treated with TGF-beta 1 for 4 days demonstrated a significant increase in transcriptional activity of the c-jun gene. This result indicates that the increase in c-jun gene expression is due in part to a change in transcriptional regulation of c-jun. The stable induction of c-jun mRNA in keratinocytes at the PGA state is unique because the induction of this gene is usually transient. The finding that c-fos is not coinduced suggests that c-Jun homodimers or other AP-1 heterodimers may be formed at the PGA state to facilitate the stable induction of c-jun mRNA. This experimental system should therefore serve as a model system to study the molecular mechanisms for the stable control of c-jun gene expression and the control of AP-1-dependent gene expression during the process of keratinocyte differentiation.

Cloning of murine tissue factor and regulation of gene expression by transforming growth factor type beta 1.

We have cloned a serum- and cycloheximide-inducible mRNA from AKR-2B murine fibroblasts which encodes a protein with significant sequence similarity to human tissue factor, a cellular initiator of the blood coagulation cascade. Information derived from this clone was used to establish the presence of a virtually identical sequence in mouse brain. Most importantly, cDNA-directed expression in a quail fibroblast cell line produced high levels of tissue factor procoagulant activity, confirming the identity of this protein as murine tissue factor. Additional studies demonstrate that transforming growth factor type beta 1 stimulates tissue factor gene transcription and is a potent inducer of tissue factor procoagulant activity in fibroblasts. Other tested mitogens such as platelet-derived growth factor, epidermal growth factor, and insulin were weak inducers. These results may reflect a role for transforming growth factor beta 1 in the maintenance of hemostasis or, alternatively, a role for tissue factor in cellular functions unrelated to blood coagulation.

Induction of fibronectin gene transcription and mRNA is a primary response to growth-factor stimulation of AKR-2B cells.

A cDNA library, prepared from poly(A)+ RNA isolated from quiescent AKR-2B cells 4 hr after stimulation with epidermal growth factor in the presence of cycloheximide, was screened to identify RNA transcripts whose abundance is specifically increased as a primary response to growth stimulation. Approximately 40% of the inducible clones detected by this procedure corresponded to either cytoskeletal beta- or gamma-actin genes. One nonactin clone, designated c99, was found to be derived from an 8.5-kilobase RNA whose abundance began to increase as early as 30 min after stimulation. DNA sequencing established the identity of this RNA as fibronectin. Several additional mitogens were then tested and found to efficiently induce fibronectin mRNA. These included fetal calf serum, platelet-derived growth factor, and transforming growth factor type beta. For at least one inducer, fetal calf serum, the increase in mRNA was preceded by an increase in fibronectin gene transcription. This increase was rapid, reaching maximal levels within 10 min, and was accompanied by near-coordinate increases in both c-fos and beta-actin transcription. These results indicate that fibronectin is a member of a class of "early-response" genes, typified by c-fos and including beta-actin, whose rapid expression may be important in mediating cellular responses to peptide growth factors.

Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II.

DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.

Hormonal control of transcription in the rat uterus. Stimulation of deoxyribonucleic acid-dependent RNA polymerase III by estradiol.

DNA-dependent RNA polymerases were extracted from rat uterine tissue, partially purified and resolved by DEAE-Sephadex chromatography. RNA polymerases I, II, IIIA, and IIIB eluted at the characteristic ammonium sulfate concentrations of 0.15, 0.28, 0.34, and 0.42 M, respectively. The sensitivity of each peak of polymerase to alpha-amanitin was examined and was shown to be essentially identical to the three classes of RNA polymerases in other mammalian systems. RNA polymerase I was insensitive to high levels of alpha-amanitin, RNA polymerase II was sensitive to low concentrations of alpha-amanitin (50% inhibition at 0.006 mug/ml) and RNA polymerases IIIA and IIIB were sensitive to high concentrations of alpha-amanitin (50% inhibition at 18 mug/ml). The alpha-amanitin sensitivity curve of total RNA synthesis measured in isolated nucleo demonstrated that the activity of each class of RNA polymerase could be quantitated in uterine nuclei. Thus the initial decrease in activity at low concentrations of alpha-amanitin (50% inhibition at 0.005 mug/ml) was attributed to the inhibition of RNA polymerase II activity, the second decrease in activity at higher concentrations of alpha-amanitin (50% inhibition at 15 mug/ml) was attributed to the inhibition of RNA polymerase III activity, and the activity which was resistant to the highest alpha-amanitin concentration tested was attributed to RNA polymerase I activity. When estradiol was given to immature rats 6 h before killing both RNA polymerases I and III levels in nuclei were increased significantly over the control values. The time course of these changes demonstrated that the increases in RNA polymerases I and III were first evident between 1.5 and 3 h following hormone treatment. Significantly, these increases in polymerase I and III in nuclei parallel the published increases for rRNA and tRNA synthesis following hormone treatment. However, the amount of RNA polymerase I and III was not altered upon extraction, suggesting that these changes are due to the alteration in chromatin template activity. Both estradiol and estriol produced identical increases in uterine RNA polymerase I and III 6 h after treatment.

HeLa cell deoxyribonucleic acid dependent RNA polymerases: function and properties of the class III enzymes.

The class III DNA dependent RNA polymerases (nucleoside triphosphate:RNA nucleotidyltransferase EC 2.7.7.6 from HeLa cells have been solubilized and characterized as to function and properties. Two chromatographically distinct forms of enzyme III, designated polymerases IIIA and IIIB, can be resolved when cell extracts are chromatographed on DEAE-Sephadex columns. Enzymes IIIA and IIIB exhibit nearly identical catalytic properties such as divalent cation stimulation, broad biphasic ammonium sulfate optima, and characteristic alpha-amanitin sensitivities which clearly distinguish them from the homologous enzymes, forms I and II. Polymerases IIIA and IIIB are both primarily localized in the nucleus (greater than 60%). The most notable characteristic of the class III enzymes is a unique sensitivity to inhibition by alpha-amanitin (50% inhibition at 15 mug/ml). HeLa cell enzyme I is not inhibited by the mushroom toxin even at very high concentrations (greater than 400 mug/ml), while HeLa cell polymerase II is inhibited by very low concentrations of amanitin (50% inhibition at 0.003 mug/ml). The three major classes of enzyme (I, II, III) exhibit characteristic sensitivities to alpha-amanitin whether assayed in nuclei, crude homogenates, or in a chromatographically purified state. Using a nuclear in vitro RNA synthesizing system to investigate the alpha-amanitin sensitivities of the synthesis of tRNA precursor (4.5S pre-tRNA) and 5S ribosomal RNA, it was found that the synthesis of these RNA species was inhibited 50% at 15 mug/ml of alpha-amanitin. The alpha-amanitin inhibition curves for the synthesis of pre-tRNA-5S ribosomal RNA in nuclei and the alpha-amanitin titration curves for the partially purified class III enzymes (IIIA and IIIB) are identical. These data, therefore, show that the in vivo functional role of the class III RNA polymerases (IIIA-IIIB) is the transcription of the genes coding for transfer RNA and 5S ribosomal RNA.

Partial purification and properties of calf thymus deoxyribonucleic acid dependent RNA polymerase III.

DNA-dependent RNA polymerase III (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.-7.6) has been isolated and partially purified from calf thymus tissue. Significant amounts of enzyme III are present in this tissue (up to 15% of the total activity of thymus homogenates). This enzyme has been characterized with respect to its chromatographic properties, broad ammonium sulfate optimum (0.04-0.2 M), template requirements, divalent metal optima, and its unique alpha-amanitin sensitivity (50% inhibition of activity occurring at an alpha-amanitin concentration of 10 mug/ml).

Molecular structures of DNA-dependent RNA polymerases (II) from calf thymus and rat liver.

DNA-dependent RNA polymerase II has been purified to high specific activity and apparent homogeneity from both calf thymus and rat liver. Two form II enzymes are present in rat-liver preparations, one with the molecular structure [(190,000)(1)(150,000)(1)(35,000)(1)(25,000)(1)], the other with a molecular structure of [(170,000)(1)(150,000)(1)(35,000)(1)(25,000)(1)] (molecular weights are within +/-5% but the absolute values are approximate). Inclusion of a proteolytic inhibitor during the isolation procedure decreases the proportion of the molecule containing the 170,000 subunit. Calf-thymus RNA polymerase preparations typically exhibit four components on polyacrylamide gels that contain sodium dodecyl sulfate, with an apparent molecular structure of [(190,000)(1)(150,000)(1)(35,000)(1)(25,000)(1)]. In addition, some calf-thymus polymerase II preparations contain small quantities of the [(170,000)(1)(150,000)(1)(35,000)(1)(25,000)(1)] species; the quantity of this species may also be increased from less than 5% in the normal preparation to at least 40% in an "aged" preparation. Thus, the 170,000 subunit may be derived from the 190,000 subunit in both tissues. Until unequivocal evidence is obtained on this point, however, the possibility that the large subunits are unique species should not be eliminated. The general structural similarity of the eukaryotic RNA polymerase II with that of the prokaryotic polymerase suggests that the modes of action and regulation may be analogous.

RNA-dependent RNA polymerase activity in influenza virions.

An RNA-dependent RNA polymerase activity has been detected in purified preparations of influenza virus. In contrast to the replicase activity induced in influenza-infected cells, the virion-associated enzyme has an absolute requirement for Mn(++). Most of the RNA synthesized in vitro is complementary to virion RNA.