Chronic renal failure and anterior hypophysial hormones.

Kidneys are not only organs with an excretory function but they produce their own endocrine factors which are involved in supporting homeostasis in the organism. The kidneys are the organs in which metabolism and biodegradation of many hormones take place. Together with the liver, the kidneys actively take part in the catabolism of hormones.

Influence of dopamine energic pharmacology drugs on secretion of the adrenocorticotropic hormone from the hypophysis.

To elucidate the role of dopamine as a neuromediator in the adrenocorticotropic hormone (ACTH) secretion, investigations were carried out with dopaminergic pharmacology drugs on male white Wistar rats. In the first series of experiments, the effects of 200 mg/kg body wt L-DOPA, of the combination of 200 mg/kg L-DOPA and 50 mg/kg body wt carbidopa, and of 2.5 mg/kg body wt bromocriptine, after a single intraperitoneal injection of ACTH in the serum of rats after 30, 90 and 120 min, following the injection, were studied. In the second series of experiments, the effect of 200 mg/kg body wt L-DOPA, of the combination of 200 mg/kg body wt L-DOPA and 50 mg/kg body wt carbidopa, of 1 mg/kg body wt bromocriptine, after intraperitoneal injection, on the concentration of ACTH in the serum within 7 days, were assessed. The inhibition of agonists of dopamine after ACTH secretion with repeated application has been shown. Using a radioimmunology assay with test kits, the amount of ACTH in the serum was determined.

Effect of cryopreservation on lipids and some physiological features of spermatozoa from rams pastured in highlands and in valleys.

The effect of low temperature preservation on the motility and morphology of acrosomes, acrosomal proteolytic activity, phospholipid and fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE), and the cholesterol/phospholipid molar ratio in sperm from rams housed in the highlands or in the valleys, were studied. The indices of motility and morphological integrity of sperm from highland rams were much greater compared with those of valley rams. Phosphatidyl choline (PC) of the highland rams was more unsaturated, while PE was more saturated compared with those of valley rams. Cryopreservation of the sperm from highland rams significantly increased the content of choline plasmalogen, accompanied by a slight rise in the levels of lysophosphatidyl choline (LPC) and phosphatidyl inositol (PI) in their sperm. The fatty acid composition altered following cryopreservation. These variations were mainly due to a decrease in the amount of docosahexaenic acid and an increase in the amounts of linoleic and palmitic fatty acids. The results may be indicative of the fact that the alterations in the sperm of the valley rams were more pronounced and they may be attributed to the structural features of the sperm, as well as a reduced concentration of oxygen in the organs and tissues of the highland rams.

The effect of prostaglandin E1 on in vitro transcription of sperm chromatin, isolated from patients with azoospermia, teratospermia and chronic prostatitis.

We have investigated the influence of Prostaglandin E1 on the in vitro transcription of chromatin, isolated from spermatozoa of patients suffering from different pathologies, leading to infertility, namely, azoospermia, teratospermia and chronic prostatitis. Our studies indicate that prostaglandin E1 has a stimulatory effect both on in vitro transcription, on the number of RNA polymerase molecules and the polyribonucleotide elongation rates as compared to sperm chromatin from healthy patients. The results on the incorporation of alpha-32P-ATP in to RNA in the presence and absence of Prostaglandin E1 correlate well with the data on the number of actively transcribing RNA polymerase molecules and the rate of RNA elongation, which might be due to low levels of prostaglandin E1 in human semen.

The effect of magnesium ions during beer fermentation.

When cells of Saccharomyces cerevisiae ale strain C1028 were grown aerobically, ethanol production displayed a hyperbolic increase over a limited range of magnesium concentrations up to approximately 0.7 mM. Entry of cells into the stationary growth phase and the time of maximum ethanol and minimum sugar concentrations correlated with a period of maximum Mg2+ concentration in the growing media. It is suggested that magnesium accumulation by yeast cells may be usefully exploited in biotechnology concerned with the production of beer.

Isolation of an osmotolerant ale strain of Saccharomyces cerevisiae.

Saccharomyces cerevisiae (ale strain) grown in batch culture to stationary phase was tested for its tolerance to heat (50 degrees C for 5 min), hydrogen peroxide (0.3 M) and salt (growth in 1.5 M sodium chloride/YPD medium). Yeast cells which have been exposed previously to heat shock are more tolerant to hydrogen peroxide and high salt concentrations (1.5 M NaCl) than the controls. Their fermentative activity as judged by glucose consumption and their viability, as judged by cell number and density have higher levels when compared with cells not previously exposed to heat shock. Experimental conditions facilitated the isolation of S. cerevisiae ale strain, which was tolerant to heat, and other agents such as hydrogen peroxide and sodium chloride.

Alternative splicing of the rabbit beta-globin pre-mRNA in vivo after transient transfection of myoblast and myotube cells.

The relationship between preferences among alternative 5' splice sites and their sequences was investigated using as model mouse myoblasts and myotubes after transient transfection with the rabbit beta-globin gene. The preferences for the use of two different 5' splice sites, acting with different efficiencies to direct splicing in vivo are reported. The predominant selection of the upstream splice site has been shown in normal mouse myoblasts. In the case of differentiated myotubes the downstream splice was 1.4 times better used. The results indicate that there were differences in the preferences for the use of the two alternative splice sites between non-differentiated and terminally differentiated cells, within the same-cell line.

Isolation of a heat-labile protein factor involved in the 3'-processing of H4 pre-mRNA.

It is well documented that the snRNP particles play a role in the processing of histone pre-mRNAs. There is also evidence that in addition to the snRNP particles, at least two more proteins are involved in the vitro 3'-processing of histone pre-mRNAs. The aim of the present work was the isolation and purification of one of these PF250 factors. Processing factor PF250 was isolated from HeLa nuclear extracts using ion exchange chromatography. Its ability to partially restore the processing activity of heat inactivated nuclear extract was demonstrated in vitro using labelled H4- pre-mRNA. Our results indicate that nuclear extracts exposed to 50 degrees C for 15 min lacked any processing activity. Using a series of column chromatography purification steps we isolated a thermolabile protein factor that partially restored the activity of heat inactivated HeLa cell nuclear extracts. It can be concluded that besides U7 snRNP particles at least one more thermolabile protein factor is required for the maturation of H4 pre-mRNA in vitro. It has a molecular mass of 30 kDa and is inactivated by heating at 50 degrees C. These characteristics make it a likely candidate to be a regulatory factor involved in pre-mRNA processing.

Immunofluorescent study after transient transfection of myoblast cells with the plasmid PSV40-beta-globin.

The level of transfection of myoblast C-2/C-12 cells with the plasmid PSV40-beta-globin, using the immunofluorescence assay, was investigated. Myoblasts, which are non-terminally differentiated cells, were chosen since it is known that terminally differentiated myotubes are very poorly transfected. The results showed that myoblast cells of the C-2/C-12 cell line were readily transfected within 24 h with the PSV40-beta-globin gene. The immunofluorescence assay showed the nucleoplasmic localization of the SV40 large T antigen, and indicated the possibility that some fraction of it might be localized in the cytoplasm.

The influence of prostaglandin E1 on in vitro transcription of sperm chromatin from healthy males.

Run-on transcription experiments with sonicated chromatin from healthy individuals in the presence and absence of prostaglandin E1 showed different levels of transcription. Thus the level of incorporation of 14C-UTP into pre-mRNA proved to be about twice as high in the presence of postaglandin E1 as in the controls. The kinetic data of the in vitro transcription indicated that there were two peaks of incorporation of 14C-UTP into RNA both in the controls and in prostaglandin E1 treated probes but at different time intervals. The results show that prostaglandin E1 has a stimulatory effect on in vitro transcription of chromatin, isolated from normal patients.

Quantification of actively transcribing RNA polymerase B molecules in human sperm chromatin and the effect of prostaglandin E1.

Run-on transcription experiments with sonicated chromatin from spermatozoa of healthy men revealed the presence of in vitro transcription. The number of actively transcribing RNA polymerase B molecules in sperm chromatin in vitro, isolated from healthy individuals, and the effect of prostaglandin El, were investigated. The results indicate that the number of RNA polymerase B molecules actively engaged in transcription increased one and a half times when 5 units of prostaglandin E1 was added to the reaction mixtures. These results correlate well with the authors' previous findings that the incorporation of labelled uridine triphosphate into pre-mRNA was higher when prostaglandin E1 was added to the reaction mixtures in run-on transcription experiments, and when sperm chromatin from healthy individuals was used as a template.

Electron microscopic data for the presence of post-meiotic gene expression in isolated ram sperm chromatin.

A whole mount electron microscopic technique facilitated a direct visualization of transcripts in ram spermatozoa in run-on experiments. The localization of transcripts in sperm chromatin by spreading enabled identification of regions where transcription complexes, presumably pre-mRNA species, were seen related to chromatin. In another series of experiments the localization of polymerase II was demonstrated using a specific antibody against the conservative tail domain of the polymerase II molecule, followed by protein A-gold visualization on spread chromatin and on thin sections from mature spermatozoa. Incorporation of bio-UTP in transcripts was visualized by streptavidin-gold on spreading and on thin sections. The data suggest that transcription occurs at the periphery of the mature spermatozoa.

Electron microscopic demonstration of transcription of ram sperm chromatin.

Transcription of ram sperm chromatin was examined by two electron microscopic techniques, namely spreading and thin sections. Labelling by Streptavidin-gold particles permits identification of transcription complexes, that have previously incorporated biotinylated uridine triphosphate. This supports previous electron microscopic data for randomly distributed transcription complexes and the presence of polymerase II molecules, documented by means of specific antibody using immunoelectron microscopy. Labelling of transcripts with biotin permits exact visualization of the transcription process, as well as identification of regions, where transcription occurs.

Influence of freezing at different temperatures on the transcription activity of buffalo sperm chromatin.

The effect of freezing at different temperatures, -20 degrees C, -70 degrees C and -196 degrees C, on the transcription activity of buffalo sperm chromatin was investigated. The kinetic data of incorporation of 3H-UTP in pre-mRNA indicate that temperatures of -70 degrees C and -196 degrees C are the most favourable for preservation, since transcription activity gradually increases with time, reaching maximum values within 30 min. Freezing at -20 degrees C or preservation at 4 degrees C leads to a rapid decrease of transcriptional activity within 10 and 20 min, respectively. It is suggested that the optimal temperature for storage of buffalo spermatozoa is below -20 degrees C as judged by the transcription assay.

Isolation and purification of U7 snRNP particles.

1. A procedure has been developed for the isolation of U7 small nuclear ribo-protein particles involved in the processing of histone pre-mRNAs. They were fractionated from the rest of the snRNPs through a series of gel filtration and affinity chromatography steps. 2. The isolated assembly of U7 snRNP particles appeared to be intact and pure as judged by the gel electrophoresis of their RNA. No detectable RNA species were monitored other than U7 RNA.

Isolation and purification of U7 and snRNP particles.

A procedure was developed for the isolation of U7 small nuclear ribonucleoprotein particles involved in the processing of histone pre-mRNAs. U7 snRNP particles were fractionated from the rest of the snRNPs by means of a series of gel filtration and affinity chromatography steps. The isolated assembly of U7 snRNP particles appeared to be intact and pure as judged by gel electrophoresis of their RNA. No detectable RNA species were monitored other than U7 RNA.

The effect of three cryoprotective diluents on the in vitro transcription of ram sperm cells.

The effect of three different solvents was investigated in run-on experiments, using purified ram sperm nuclei. The best cryoprotective diluent proved to be Nagase-Niwa, which had the most positive effect on transcription. The kinetic data of incorporation of 32P-ATP in pre-mRNA indicated that there was a stepwise increase in the level of transcription during the first 20 min of incubation, being lowest in the control and highest in the presence of the diluent Nagase-Niwa.

Characterization of the protein moiety of U7 small nuclear RNP particles.

U7 snRNP particles contain a set of seven proteins with molecular weights in the range of 13.5 to 50 kD. One protein with a molecular weight of 18 kD carried the antigenic determinant responsible for the interaction of U7 snRNPs with autoantibodies from patients with connective tissue diseases. It is suggested that U7 RNA is associated with proteins representing a ribonucleoprotein particle analogous to U1, U2, U4, U5 and U6 snRNPs.

3' processing of histone H4 precursor mRNA requires the presence of a small nuclear RNP particle.

1. Incubation of in vitro synthesized mouse histone H4 mRNA precursors in nuclear extracts of mouse 3T6 fibroblasts, rat L6-5 myoblasts and myotubes yields processed mRNA species. A processing activity was identified in all three kinds of extracts that cleaves the precursor transcripts, generating mRNA species with mature 3' termini. 2. The processing activity is present both in proliferating (mouse 3T6 fibroblast, rat L6-5 myoblast) and terminally differentiated (rat L6 myotube) cells. 3. The efficiency of the endonucleolytic cleavage reactions was higher in a homologous system, i.e. in the presence of nuclear extracts from mouse 3T6 fibroblasts, than in a heterologous system, i.e. in the presence of rat myoblast or myotube extracts. 4. The in vitro processing activity is specifically inhibited by anti-Sm antibodies, which suggests the requirement of an snRNP particle for H4 pre-mRNA maturation.

U7 snRNP particles bind H4 pre-mRNA in vitro.

1. U7 snRNP particles were bound in vitro to H4 pre-mRNA using immunoassay experiments. 2. The processing of in vitro H4 pre-mRNAs is an SmRNP-dependent reaction. 3. U7 RNA was isolated as a unique species from the complex H4-pre-mRNA-snRNPs, which indicates its involvement in the processing of histone pre-mRNAs.