Endogenous protein C is essential for the functional integrity of human endothelial cells

Circulating protein C (PC) plays a vital role as an anti-coagulant and anti-inflammatory mediator. We show here that human endothelial cells produce PC that acts through novel mediators to enhance their own functional integrity. When endogenous PC or its receptor, endothelial protein C receptor (EPCR), was suppressed by small interfering (si) RNA, human umbilical cord endothelial cell (HUVEC) proliferation was decreased and apoptosis elevated. Interestingly, PC or EPCR siRNA significantly increased HUVEC permeability, which is likely via reduction of the angiopoietin (Ang)1/Ang2 ratio and inhibition of the peripheral localization of the tight junction protein, zona occludins-1. In addition, PC or EPCR siRNA inhibited type IV collagen and matrix metalloproteinase-2, providing the first evidence that PC contributes to vascular basement membrane formation. These newly described actions of endogenous PC act to stabilize endothelial cells and enhance barrier function, to potentially promote the functional integrity of blood vessels.     

Evidence for the presence of viable endothelial cells in cultures derived from dissociated rat brain

Summary The morphology and histochemistry of dissociated newborn rat brain was studied in tissue culture. Direct microscopy of developing cells, electron microscopy and the alkaline phosphatase activity were used to identify the capillary endothelial cells.Acknowledgments. We thank Dr J. Gripenberg for technical assistance. This research was supported by Finnish Cultural Foundation and carried out during the tenure of a fellowship provided for F. J. from the Finnish-Hungarian Cultural Exchange Program.     

Diurnal fluctuation in the rate of synthesis of a specific protein fraction in the rat brain

Summary A protein fraction has been identified in microsomes prepared from the rat hypothalamus whose rate of synthesis fluctuates diurnally in ovariectomized animals.     

Requirement for protein synthesis for the transfer of meiotic induction in a multicellular complex ofBlepharisma

Summary In the ciliateBlepharisma japonicum it is possible to induce meiosis in multicellular homotypic chains. In this work we demonstrate that protein synthesis is required to transfer meiotic activation from one cell to another in a chain.This work was supported by CNR, Programma finalizzato Biologia della Riproduzione.     

Serotonin is localized in endothelial cells of coronary arteries and released during hypoxia: a possible new mechanism for hypoxia-induced vasodilatation of the rat heart

In this report we demonstrate the immunocytochemical localization of serotonin in endothelial cells of rat coronary vessels and a significant increase in the release of serotonin into the perfusate of Langendorff rat heart preparations during hypoxia. It is suggested that serotonin, localized in endothelial cells, is released during hypoxia and could provide part of a pathophysiological mechanism for vasodilatation to protect the heart from damage due to hypoxia.     

Cell penetrating peptides: overview and applications to the delivery of oligonucleotides

Crossing biological barriers represents a major limitation for clinical applications of biomolecules such as nucleic acids, peptides or proteins. Cell penetrating peptides (CPP), also named protein transduction domains, comprise short and usually basic amino acids-rich peptides originating from proteins able to cross biological barriers, such as the viral Tat protein, or are rationally designed. They have emerged as a new class of non-viral vectors allowing the delivery of various biomolecules across biological barriers from low molecular weight drugs to nanosized particles. Encouraging data with CPP-conjugated oligonucleotides have been obtained both in vitro and in vivo in animal models of diseases such as Duchenne muscular dystrophy. Whether CPP-cargo conjugates enter cells by direct translocation across the plasma membrane or by endocytosis remains controversial. In many instances, however, endosomal escape appears as a major limitation of this new delivery strategy.     

Possibility of using131I-albumin as a marker for the estimations of microbial protein synthesis rates in the rumen

Summary Total microbial protein synthesis rates in the rumen of buffaloes were estimated by isotope dilution technique, using¹³¹I-albumin treated with tannic acid as a marker. The animals were fed groundnut cake treated with formaldehyde to meet 50% of their digestible crude protein (DCP) requirement and 2.5% urea molasses mixture was given to meet the remaining requirement of DCP. Wheat straw was fed as the basal roughage. The total average microbial protein synthesis was 58.14 g/day.     

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.     

Migration of lymphoid cells to the bone marrow of rat following eradication of cells in DNA synthesis and in mitosis

Summary Eradication of replicating bone marrow cells of rat by means of combined administration of single doses of hydroxyurea and vinblastin is followed within 9–10 h by an inflow of lymphoid cells of extramedullary origin in the range of 13,200,000/femur. The rat bone marrow with a high content of lymphoid cells was previously shown to be concentrated in stem cells. The factor(s) which convey the information of decrease of replicating marrow cells to extramedullary sites is at present unknown.Acknowledgment. This study was supported by a grant from the Chief Scientist Office, Ministry of Health, Israel.Hydroxyurea for this investigation was given as a gift by the Squibb Institute of Medical Research, Princeton, N.J. USA, to which the authors are indebted.     

Nucleic acids and protein synthesis in mice tumor and normal cells after irradiation at 365 nm in the presence of psoralen

Riassunto L'irradiazione a 365 nm in presenza di psoralene, furocumarina fotosensibilizzatrice cutanea non sostituita, influisce in modo diverso sulla sintesi macromoleculare in alcune cellule tumorali e normali del topo.     

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: effect of nafenopin and electroporation

Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive beta-oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes. In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. 'Whorl formation' by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.     

Action of juvenile hormone on the follicle cells ofRhodnius prolixus: Evidence for a novel regulatory mechanism involving protein kinase C

Summary Juvenile hormone (JH) is known to act on the membranes of the follicle cells ofRhodnius, activating a specific Na⁺, K⁺-ATPase. This leads to a decrease in volume of the cells and the appearance of spaces between them (patency). The addition of an inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), to the medium in vitro inhibits the action of JH on the follicle cells. PDBU (phorbol-12,13-dibutyrate) mimics the action of JH in vitro and the response of the follicle cells to, PDBU is blocked by ouabain. It is concluded that the activation of protein kinase C is a required step in the chain of events leading to activation of the JH-dependent ATPase and set in train by the binding of JH to the membrane.     

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: Effect of nafenopin and electroporation

Summary Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive -oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes.In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. Whorl formation by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.