Effects of prostaglandins of the E-series on pulmonary and systemic circulations of newborn goats during normoxia and hypoxia.

Effects of exogenous prostaglandins of the E-series on pulmonary and systemic circulations of newborn goats were investigated during normoxia and hypoxia. Pulmonary arterial infusion of prostaglandins E1 and E2 decreased pulmonary vascular resistance 20% and 14%, respectively, without systemic effects. Prostaglandin E1 abolished the pulmonary pressor response to hypoxia. Prostaglandin E2 was less effective in counteracting this hypoxic response. The increased pulmonary vascular resistance and augmented response to hypoxia following indomethacin administration was reversed by prostaglandin E1. Infusion of prostaglandin E1 directly into the pulmonary circulation may be of benefit to the distressed newborn with elevated pulmonary vascular resistance.     

Regulation of ET-1 biosynthesis in cerebral microvascular endothelial cells by vasoactive agents and PKC

Endothelin-1(ET-1) is the most potent vasoconstrictor agent known. ET-1 is elevatedin the cerebrospinal fluid following hemorrhage and brain injury andcan compromise cerebral microvascular homeostasis. The modulation ofET-1 production by cerebral microvascular endothelial cells and themechanism by which such changes take place are very important in ourunderstanding of the pathological roles of ET-1. In the present study,we investigated the effects of vasoconstrictor agents that can bereleased from hemolyzed blood, cAMP-dependent dilators, and the role ofprotein kinase C (PKC) in the regulation of ET-1 production by pigletcerebral microvascular endothelial cells in culture. ET-1 was measured by RIA. 1) Cerebral microvascularendothelial cells synthesize and release ET-1 into the media;2) 5-hydroxytryptamine (5-HT), lysophosphatidic acid (LPA), thromboxane analog U-46619, fetal bovineserum (20%), and phorbol 12-myristate 13-acetate significantly increase ET-1 production; 3) basaland vasoconstrictor agent-induced increases in ET-1 production byendothelial cells may be mediated via PKC;4) cAMP-dependent vasodilatorsattenuate the basal production of ET-1 by cerebral microvessels; and5) pretreatment of endothelial cellswith a higher concentration of LPA, U-46619, or 5-HT counterbalances the cAMP-dependent dilator agent-induced reduction in basal ET-1 production. Therefore, by-products of hemolyzed blood can stimulate theproduction of ET-1 by a PKC-mediated mechanism. cAMP-dependent dilatorscan attenuate the vasoconstrictor agent-induced elevation in ET-1production. These results suggest that cerebral microvascular homeostasis could be compromised by effects of interactions among vasoactive agents released during conditions injurious to the brain andthey may further the understanding of potential contributions ofhemolyzed blood clots to subarachnoid hemorrhage-induced vasospasm.     

Prostanoid synthesis and vascular responses to exogenous arachidonic acid following cerebral ischemia in piglets

In newborn pigs, cerebral ischemia abolishes both increased cerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuation of prostaglandin endoperoxide synthase activity could account for the failure to increase prostanoid synthesis and loss of responses to these stimuli. To test this possibility, arachidonic acid (3, 6, or 30 micrograms/ml) was placed under cranial windows in newborn pigs that had been exposed to 20 min of cerebral ischemia. The conversion to prostanoids and pial arteriolar responses to the arachidonic acid were measured. At all three concentrations, arachidonic acid caused similar increases in pial arteriolar diameter in sham control piglets and piglets 1 hr postischemia. Topical arachidonic acid caused dose-dependent increases of PGE2 in cortical periarachnoid cerebral spinal fluid. 6-keto-PGF1 alpha and TXB2 only increased at the highest concentration of arachidonic acid (30 micrograms/ml). Cerebral ischemia did not decrease the conversion of any concentration of arachidonic acid to PGE2, 6-keto-PGF1 alpha, or TXB2. We conclude that ischemia and subsequent reperfusion do not result in inhibition of prostaglandin endoperoxide synthase in the newborn pig brain. Therefore, the mechanism for the impaired prostanoid production in response to hypercapnia and hypotension following cerebral ischemia appears to involve reduction in release of free arachidonic acid.     

Mechanisms of stimulation of pulmonary prostacyclin synthesis at birth

In order to investigate the mechanism behind ventilation-induced pulmonary prostacyclin production at birth, chloralose anesthetized, exteriorized, fetal lambs were ventilated with a gas mixture that did not change blood gases (fetal gas) and unventilated fetal lungs were perfused with blood containing increased O₂ and decreased CO₂. Ventilation with fetal gas (3%O₂, 5%CO₂) increased net pulmonary prostacyclin (as 6-keto-PGF_1α production from −5.1 ± 4.4 to +12.6 ± 7.6 ng/kg·min. When ventilation was stopped, net pulmonary prostacyclin production returned to nondetectable levels. Ventilation with gas mixtures which increased pulmonary venous P_O2 and decreased P_O2 also stimulated pulmonary prostacyclin production, but did not have greater effects than did ventilation with fetal gas. In order to determine if increasing P_O2 or decreasing P_CO2 could stimulate pulmonary prostacyclin production independently from ventilation, unventilated fetal lamb lungs were perfused with blood that had P_O2 and P_CO2 similar to fetal blood, blood with elevated O₂, and blood that had P_O2 and P_CO2 values similar to arterial blood of newborn animals. Neither increased O₂ nor decreased CO₂ in the blood perfusing the lungs stimulated pulmonary prostacyclin synthesis. We conclude that the mechanism responsible for the stimulation of pulmonary prostacyclin with the onset of ventilation at birth is tissue stress during establishment of gaseous ventilation and rhythmic ventilation.     

Growth of Clostridium perfringens in cooked chili during cooling

U.S. Department of Agriculture regulations require that brick chili be cooled from 48.9 degrees C to 4.4 degrees C within 2 h of cooking, but processors may not always be able to comply. Studies were conducted to evaluate the extent of bacterial multiplication resulting from outgrowth of germinated Clostridium perfringens spores experimentally inoculated into chili and incubated at various temperatures. Inoculated samples were heated (75 degrees C for 20 min) to activate spores, quickly equilibrated, and held at one of five desired temperatures for 6 h. No growth was observed for C. perfringens in samples held at 26.7 degrees C and below for 6 h, but growth was observed by 6 h in samples held at 32.2 degrees C and after 2 h in samples held at temperatures between 37.8 degrees C and 48.9 degrees C. Using isothermal growth data, we developed a simple model for predicting the growth of bacteria with time under exponential cooling conditions. The model predicts both the lag phase and the numbers of bacteria at specific times during the growth phase. It was developed by using isothermal growth data and tested by using temperature-varying growth data from experiments with spores of C. perfringens in chili. Actual data agreed closely with predicted results. The results should be useful for evaluating the hazard potential for growth of C. perfringens in chili.     

High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies

Glycophorins are the most abundant sialoglycoproteins on the surface of human erythrocyte membranes. Genetic variation in glycophorin region of human chromosome 4 (containing GYPA, GYPB, and GYPE genes) is of interest because the gene products serve as receptors for pathogens of major public health interest, including Plasmodium sp., Babesia sp., Influenza virus, Vibrio cholerae El Tor Hemolysin, and Escherichia coli. A large structural rearrangement and hybrid glycophorin variant, known as Dantu, which was identified in East African populations, has been linked with a 40% reduction in risk for severe malaria. Apart from Dantu, other large structural variants exist, with the most common being deletion of the whole GYPB gene and its surrounding region, resulting in multiple different deletion forms. In West Africa particularly, these deletions are estimated to account for between 5 and 15% of the variation in different populations, mostly attributed to the forms known as DEL1 and DEL2. Due to the lack of specific variant assays, little is known of the distribution of these variants. Here, we report a modification of a previous GYPB DEL1 assay and the development of a novel GYPB DEL2 assay as high-throughput PCR-RFLP assays, as well as the identification of the crossover/breakpoint for GYPB DEL2. Using 393 samples from three study sites in Ghana as well as samples from HapMap and 1000 G projects for validation, we show that our assays are sensitive and reliable for genotyping GYPB DEL1 and DEL2. To the best of our knowledge, this is the first report of such high-throughput genotyping assays by PCR-RFLP for identifying specific GYPB deletion types in populations. These assays will enable better identification of GYPB deletions for large genetic association studies and functional experiments to understand the role of this gene cluster region in susceptibility to malaria and other diseases.     

Phosphorylation-dependent stimulation of prostanoid synthesis by nigericin in cerebral endothelial cells

Nigericin decreases intracellular pH(pH_i) and stimulates prostanoid(PG) synthesis in endothelial cells from cerebral microvessels ofnewborn pigs. Nigericin-induced PG production was abolished by proteintyrosine kinase (PTK) inhibitors and amplified by phorbol 12-myristate13-acetate (PMA) or protein tyrosine phosphatase (PTP) inhibitors.Nigericin-induced PG production in PMA-primed cells was potentiated byPTP inhibitors and abrogated by PTK inhibitors. PhospholipaseA₂(PLA₂) activity was stimulatedby nigericin in a phosphorylation-dependent manner. Nigericin'seffects on PG production and PLA₂activity were reproduced by ionomycin, which activates cytosolicPLA₂(cPLA₂).cPLA₂ was immunodetected in endothelial cell lysates. We found no evidence that nigericin's effects are mediated via mitogen-activated protein (MAP) kinase [extracellularly regulated kinase 1 (ERK1) and ERK2]activation: although nigericin stimulateddetergent-soluble MAP kinase, its effects were not amplified by PMA orPTP inhibitors. Phosphorylation-dependent stimulation of PG synthesiswas also observed when pH_i wasdecreased by sodium propionate or a high level ofCO₂. Altogether, our data indicatethat nigericin and decreased pH_istimulate PG synthesis by a protein phosphorylation-dependent mechanisminvolving cross talk between pathways mediated by PTK and PTP and byprotein kinase C; cPLA₂ appears tobe a key enzyme affected by nigericin and decreasedpH_i.