Thrombosis prophylaxis using plasmin and its combination with alpha-adrenoreceptor antagonists]

Using two models of experimental thrombosis (arterio-venous shunt and Wessler's model) the effect of plasmin and its combination with alpha-adrenoreceptor antagonists on the formation of thrombus was studied on white rats. It was established that the efficacy of prophylactic of thrombosis by plasmin only was low: middle ball of thrombosis was 2.5-3.0. The combination of plasmin with alpha-adrenoblockers dihydroergotoxine or prazosin under these conditions is most efficient against the formation of thrombus (middle ball of thrombus in this case was 0.8-1.1). Prazosin under certain conditions have some advantages.     

Electrophysiological changes induced by lysophosphatidylcholine, an ischaemic phospholipid catabolite, in rabbit atrial and ventricular cardiac cells.

The effects of palmitoyl-lysophosphatidylcholine (LPC) were studied on the cellular electrical activity of rabbit heart preparations. LPC (100 mumol/l) caused a considerable enhancement of the automaticity of the SA nodal and Purkinje fibers and frequently induced irregular firing in both supraventricular (SA node, atrium, AV junction) and ventricular (Purkinje fibers, papillary muscle) myocardial regions. The 'automatotropic' and arrhythmogenic effects of LPC were accompanied by a lengthening of the atrioventricular conduction time. In ventricular muscle fibers LPC (100 mumol/l) decreased the resting potential (RP), the maximum rate of depolarization (Vmax) and the amplitude (APA) and duration (APD) of the action potential, and often evoked action potentials of 'slow response' type. In atrial muscle cells, 100 mumol/l LPC was capable of inducing hyperpolarization, with concomitant increases in RP, Vmax, APA and APD; higher concentrations (300 and 600 mumol/l) of LPC resulted in decreases in RP, Vmax, APA and APD, i.e. phenomena similar to those observed with 100 mumol/l LPC in the ventricular myocardium. The results seem to support the assumption that lysolipids accumulating in the ischaemic myocardium may play a pathogenetic role in the development of both supraventricular and ventricular dysrhythmias accompanying coronary artery occlusion.     

Impact of single neonatal allylestrenol treatment on the estrus cycle of rats treated with FSH+LH or TSH.

Neonatal allylestrenol treatment administered to female rats significantly increases the duration of estrus phase in the sexual cycle. Treatment with follicle stimulating hormone (FSH) + luteinizing hormone (LH) in adulthood prolongs the duration of estrus even on its own; the effect, however, is more pronounced in those animals who were treated (imprinted) with allylestrenol neonatally. When administered to the control animals, the chemically related thyreotrop hormone (TSH) is either indifferent or it even decreases the estrus index. In animals having received neonatal allylestrenol treatment, however, TSH administration increases significantly the duration of the estrus phase. Either with or without FSH+LH treatment, the ratio of estrogenic to gestagenic phase increases following neonatal allylestrenol treatment. The experiments call attention to the potential functional risks inherent in neonatal allylestrenol treatment. The actual risks, however, seem to be smaller than the effects seen at the receptor level.     

Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases.

Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli. Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta-lactamase precursor. The SipS protein consisted of 184 amino acids (mol. wt 21 kDa). The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae. Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex. Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases. The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases.     

A novel and conserved salt-induced protein is an important determinant of salt tolerance in yeast.

We have isolated a novel yeast gene, HAL1, which upon overexpression improves growth under salt stress. In addition, disruption of this gene decreases salt tolerance. Therefore HAL1 constitutes a rate-limiting determinant for halotolerance. It encodes a polar protein of 32 kDa located in the yeast cytoplasm and unrelated to sequences in data banks. The expression of this gene is increased by high concentrations of either NaCl, KCl or sorbitol. On the other hand, the growth advantage obtained by overexpression of HAL1 is specific for NaCl stress. In cells overexpressing HAL1, sodium toxicity seems to be counteracted by an increased accumulation of potassium. The HAL1 protein could interact with the transport systems which determine intracellular K+ homeostasis. The HAL1 gene and encoded protein are conserved in plants, being induced in these organisms by salt stress and abscisic acid. These results suggest that yeast serves as a convenient model system for the molecular biology of plant salt tolerance.     

GTPase-activating protein SH2-SH3 domains induce gene expression in a Ras-dependent fashion.

The p21ras GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p21ras. Here, we have investigated the role of GAP by using a transient expression assay with a fos luciferase reporter plasmid. We used GAP deletion mutants that lack the domain involved in interaction with p21ras and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction of fos promoter activity similar to induction by activated p21ras. Expression of a full-length GAP construct had no effect on the activity of the fos promoter. Activation of the fos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21ras, Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21ras activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p21ras, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p21ras. The combined effect of Ras(Leu-61) and the GAP deletion mutants was not inhibited by Ras(Asn-17), indicating that GAP SH2-SH3 domains do not function to activate endogenous p21ras but cooperate with another signal coming from active p21ras. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21ras but that additional signals coming from p21ras are required for them to function.     

NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.     

Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.

A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways.     

Comparative analysis of the intracellular localization of c-Myc, c-Fos, and replicative proteins during cell cycle progression.

In eukaryotic cells, nucleus-cytoplasm exchanges play an important role in genomic regulation. We have analyzed the localization of four nuclear antigens in different growth conditions: two replicative proteins, DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), and two oncogenic regulatory proteins, c-Myc and c-Fos. A kinetic study of subcellular localization of these proteins has been done. In cultures in which cells were sparse, these proteins were detected in the nucleus. When proliferation was stopped by the high density of culture cells or by serum starvation, these proteins left the nucleus for the cytoplasm with different kinetics. DNA polymerase alpha is the first protein to leave the nucleus, with the PCNA protein, c-Fos, and c-Myc leaving the nucleus later. In contrast, during serum stimulation c-Fos and c-Myc relocalize into the nucleus before the replicative proteins. We also noticed that in sparse cell cultures, 10% of the cells exhibit a perinuclear staining for the DNA polymerase alpha, PCNA, and c-Myc proteins but not for c-Fos. This peculiar staining was also observed as an initial step to nuclear localization after serum stimulation and in vivo in Xenopus embryos when the G1 phase is reintroduced in the embryonic cell cycle at the mid-blastula stage. We suggest that such staining could reflect specific structures involved in the initiation of the S phase.