Inhibition of DNA polymerization and DNA transcription to RNA by seminal plasma peptides.

An oligopeptide fraction purified from the extracellular compartment of bull semen and strongly interacting with DNA was shown to hinder mononucleotide polymerizations to DNA and RNA in vitro. The fraction, collectively called seminal plasma inhibitor, was active in the endogenous DNA and RNA polymerase reactions of the nuclei from rat hepatocytes and in the analogous nucleotide polymerizations catalyzed by purified enzymes of bacterial origin. The type of the induced inhibition was studied using the RNA polymerase from Escherichia coli as a representative nucleotidyl transferase. In the enzymatic polycondensation of mononucleotides, the seminal plasma inhibitor appeared to exert its effect mainly by a competitive inhibition for the utilization of DNA templates without specificity with respect to the source and the base sequence of DNA. Concavities of the plots of V0/Vi versus the amounts of inhibitor in the nucleotide polymerizing reactions and of the Dixon plots in the assays of RNA polymerase from E. coli suggested that the isolated oligopeptide fraction contained more than one active molecular species with differential effects at low and high doses. Preliminary results on the microheterogeneity of the seminal plasma inhibitor supported this contention.

Acidic pentapeptide phosphorylated in vitro by calf thymus protein kinase NII binds to DNA in the presence of Mg2+ cations.

The pentapeptide pyroGlu-Ala-Glu-Ser-Asn has been synthetized and phosphorylated in vitro at level of serine by protein kinase NII isolated from calf thymus chromatin. It is noteworthy that the calf thymus kinase NII shows a remarkable affinity for this peptide. The [32P]peptide is able to bind to several DNAs in the presence of Mg2+ (lambda phage, calf thymus, pBR540 plasmid). This binding appears not specific with regard to the type of DNA and its base sequence. These data support the hypothesis that phosphorylated acidic domains of nuclear nonhistone proteins could bind directly to DNA in the presence of Mg2+ cations.

A non-steroidal gametic factor linked to DNA modulates delta-aminolevulinic acid synthase inducibility acting on liver transcriptional and translational processes.

The effect of an acidic factor of low molecular weight (about 1,000 daltons), extracted from bovine spermatozoan DNA, on the inducibility of delta-aminolevulinic acid synthase by ethanol during aging in rat has been examined. The increased enzyme inducibility in 600-day old rats is supported by stimulation of transcriptional and translational processes; on the contrary, in 30-day old rats, the higher enzyme values induced by ethanol are significantly decreased after factor treatment. The active factor is strongly DNA-bound in the native spermatozoan DNA. This would imply a possible role in regulating gene expression in vivo.

Effect of adrenergic and Ca2+ antagonists on increased ornithine decarboxylase expression in regenerating rat liver.

Partial hepatectomy (PH) (70% resection) causes within 4 hr an accumulation of ornithine decarboxylase (EC 4.1.1.17, ODC) mRNAs concomitant with an increase in ODC activity, maximum values being observed at 8 and 16 hr, respectively. In the early hours of hepatic regeneration, enhancement of transcriptional-rate of ODC gene, demonstrated by nuclear run-on analysis, can account for the accumulation of ODC mRNAs. The involvement of catecholamines in these processes is demonstrated by using prazosin and propranolol, specific antagonists of alpha 1 and beta adrenoceptors, respectively. Prazosin reduces almost completely the rise of ODC activity at 4 hr, without affecting mRNA levels. At 16 hr, enzyme activity and mRNAs increase, however, over the values observed in regenerating liver of prazosin-untreated animals. These findings suggest that alpha 1-receptor activation triggers positive control signals for ODC gene expression at the early time of liver regeneration and, on the contrary, negative signals at later times by mainly post-transcriptional and transcriptional mechanisms, respectively. Propranolol reduces similarly the initial 4 hr-rise of ODC activity. These results indicate that activation of both alpha 1- and beta-adrenoceptors causes the large increase in ODC activity. Pharmacological manipulation of intracellular Ca2+ levels by verapamil, a Ca2(+)-channel blocker, or neomycin, an inhibitor of Ca2+ release from endogenous stores, diminishes ODC activity at 4 and 16 hr after PH. ODC mRNA levels, which are not modified at 4 hr, increase over the values of partially hepatectomized rat liver at 16 hr. Trifluoperazine inhibits both ODC activity and mRNA accumulation at the times studied. As a working hypothesis it is proposed that Ca2(+)-mediated processes induced by catecholamines are involved in ODC gene expression during the prereplicative phase of liver regeneration.

Inhibition of gastric acid secretion by a glycoprotein isolated from human urine (human urinary gastric inhibitor).

The gastric antisecretory activity of an inhibitor newly isolated from human urine (Human Urinary Gastric Inhibitor or HUGI) has been studied. HUGI was given intravenously and its activity determined in the following test systems: gastric secretion in the rat with pyloric ligation; gastric secretion in the dog with a Heidenhain pouch stimulated with pentagastrin, histamine and a protein meal; acid secretion by the isolated gastric mucosa of the rat; gastrointestinal motility; bile flow and gall-bladder tone and arterial and venous blood pressure and heart rate. HUGI was found to have marked activity only in the pyloric-ligated rats and in the dogs with Heidenhain pouches stimulated by a protein meal. Particularly in the dog, HUGI (0.1 to 6.4 micrograms/kg, i.v) markedly inhibited gastric secretion, dose-dependently and without changing the plasma gastrin concentration. Negative results were obtained in the other tests, but these results serve to demonstrate that HUGI is an inhibitor well-differentiated from other glycoproteins or peptides with gastric antisecretory activity, such as urogastrone and GIP. The results obtained to date are not sufficient to allow the mechanism of action of HUGI to be defined.

Comparison of human urine gastric inhibitor (HUGI) and bacterial endotoxin as inhibitors of acid secretion.

In conscious cats with gastric fistulas, 10 micrograms . kg-1 of human urinary gastric inhibitor (HUGI) given as an intravenous bolus injection increased mean rectal temperature 1.4 degree C and inhibited mean gastrin-stimulated acid secretion by 64%. The sample of HUGI contained an amount of beta-hydroxymyristic acid corresponding to a 5% contamination of the HUGI with bacterial endotoxin. Injection of bacterial endotoxin in an amount corresponding to the beta-hydroxymyritic acid content of HUGI mimicked, both in magnitude and time course, the increase in body temperature and the inhibition of acid secretion produced by HUGI. We conclude that inhibition of acid secretion by HUGI may be due to the presence of an endotoxin-like contaminant.

Effect of a nonsteroidal gametic factor on senile cataract in the dog.

We have studied the clarifying activity of a purified peptide fraction of gametic origin in senile cataract of the dog. The active substance, administered intramuscularly, had significant and durable clarifying effects on the cortical area of the lens.

Seminal plasma peptides inhibiting RNA synthesis.

A peptide fraction, called seminal plasma inhibitor (SPI), present in mammalian semen, was shown to block DNA transcription in vitro (Lugaro et al., 1984). Peptides responsible for this effect were partially purified from bovine seminal plasma. This report outlines the preparative procedure for the isolation of SPI and provides preliminary information on the action mechanism of the RNA synthesis inhibition.

Bovine seminal plasma contains factors that enhance lymphocyte transformation in vitro.

The important immunosuppressive properties of seminal plasma have significant functions in the processes of reproduction. They mask the presence of an immunostimulating activity. From bovine seminal plasma two active factors have been isolated and characterized with marked enhancing activity for in vitro PHA-dependent lymphocyte transformation. They have inosine and hypoxanthine structures, as confirmed by UV absorption profiles, TLC, mass spectrometry, HPLC patterns, behavior to enzymatic treatments, and breaking of purine ring after acid treatment. Nevertheless, their biological activities are about two orders of magnitude higher than those of commercially available inosine and hypoxanthine standards. Biological activities became practically identical when these were processed (HPLC) in the same way as native molecules. A study to explain such a discrepancy is in progress.

Activity of a purified nonsteroidal gametic factor on the inducibility of hepatic delta-aminolevulinic acid synthetase, NADPH-oxidase and tyrosine aminotransferase in senescent rats.

The effect of a water soluble nonsteroidal factor extracted from the male gamete on the activity of certain liver inducible enzymes during aging has been examined. Three enzymes have been studied: delta-aminolevulinic acid synthetase, NADPH-oxidase and tyrosine aminotransferase whose inducibility by ethanol, phenobarbital and ACTH, respectively, show age dependent alterations. The results here reported show that this factor is able to restore the enzyme inducibility in the liver of aging (600-day-old) rats without affecting the response of young (40-day-old) rats. Since the enzyme inducibility is altered during aging, and in the majority of rat hepatomas this factor might enter, possibly, in the regulation of enzyme activity also of neoplastic cells.