Platelet activation by diacylglycerol or ionomycin is inhibited by nitroprusside.

Experiments were performed to elucidate the role of cyclic guanosine monophosphate (cGMP) on platelet activation induced by protein kinase C (PKC) activators and calcium ionophore. Human platelets were pretreated with acetylsalicylic acid and with hirudin and apyrase. Aggregation and ATP secretion in response to the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetylglycerol (OAG) were inhibited by the nitrovasodilator sodium nitroprusside (SNP), an activator of guanylate cyclase, and by 8-bromo-cyclic GMP (8-Br-cGMP). The experiments were performed in the presence of M&B 22948, an inhibitor of cGMP phosphodiesterase. SNP and 8-Br-cGMP also inhibited platelet aggregation and secretion evoked by the ionophore ionomycin. In fura-2 loaded platelets SNP did not affect basal cytosolic Ca2+ level nor the rise induced by low concentrations of ionomycin, both in the presence and absence of extracellular Ca2+. The phosphorylation of the 47 and 20 kDa protein induced by ionomycin or PMA were not significantly decreased by SNP or 8-Br-cGMP. The present results suggest that cGMP is able to inhibit both the PKC and the Ca(2+)-dependent pathways leading to platelet activation by interfering, similarly to cAMP, with processes following protein phosphorylation, close to the effector systems.     

The origin of the nascent blastocoele in preimplantation mouse embryos ultrastructural cytochemistry and effect of chloroquine

Summary Mouse morulae are known to undergo cavitation as soon as some external cells have entered the sixth cell cycle (Garbutt et al. 1987). Since the early cytological features of cavitation are still unclear, we undertook a careful ultrastructural analysis of late morulae-nascent blastocysts. In addition, since maturation of lysosomes might be involved in the first step of cavity formation, we focused our attention on these organelles by means of the cytochemical localization of trimetaphosphatase activity and by the study of the effects of chloroquine on precavitation embryos. Our results suggest that cavitation starts in a few external cells (presumably competent cells entering the sixth cell cycle), by the chloroquine-sensitive formation of degradative autophagic vacuoles engulfing lipid droplets and vacuoles containing osmiophilic material. These complex structures enlarge (as a result of lipid metabolism?) and so transform into intrablastomeric cavities which, by means of a membrane fusion process, very rapidly become extracellular cavities that coalesce. The abembryonic pole of the blastocyst is determined in this way. Moreover, we suggest that the juxtacoelic cytoplasmic processes covering the inner cell mass (ICM) cells, which are known to restrict the expression of their totipotency during early cavitation (Fleming et al. 1984), are the latest remnants of the walls of the growing intrablastomeric cavities.     

Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg⁶,Phe⁷] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized ¹²⁵I-labelled Met(O)-enkephalin[Arg⁶,Phe⁷]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg⁶,Phe⁷]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg⁶,Phe⁷]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.     

Transfer RNA and protein synthesis

The three-dimensional structure of yeast phenylalanine transfert RNA has been determined in orthorhombic crystals. The current status of this work is reviewed together with the relationship of the transfer RNA structure in the crystal to its biologically active form. In addition some speculations are put forward regarding the mode of interaction of tRNA molecules in the ribosome and the manner in which tRNA interacts with aminoacyl synthetase.     

Diet crude protein reduction on follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows

This study evaluated the effect of crude protein (CP) reduction in four diets (156, 139, 132, and 127 g Kg^-1 DM) maintaining constant metabolizable protein (188 g/day) on the follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows. Twenty-two Girolando cows with average of 21.55 ±3.19 L daily milk yield, 105.30 ±22.62 days in lactation and 3.22 ±0.03 body condition score were selected. To reduce CP in diets and maintain constant metabolizable protein, urea and soybean meal were gradually replaced by lignosulfonate-treated soybean meal (SoyPass^®, Cargill), resulting in an increase in rumen-undegradable protein and a reduction in rumen degradable protein. A linear and quadratic reduction was observed in the plasma and follicular fluid urea nitrogen concentration following CP reduction, with the most intense reduction occurring in the 127 g Kg^-1 DM group (p<0.001). As CP reduced, there was a tendency for a linear increase in the follicular growth rate (P=0.0696), on the number and proportion of viable oocytes (P<0.09), and also a linear increase for the number (P=0.0397) and proportion (P<0.09) of grade I viable oocytes. Plus, there was a linear effect for the number of cumulus oophorus cells. Cows fed with the lowest amount of CP had cumulus-oocyte complexes with higher numbers of cumulus oophorus cells (P=0.0238). Also, the reduction of diet crude protein was followed by a decrease in the probability of oocytes’ DNA degradation. In conclusion, the reduction of CP in the diet of mid-lactating Girolando cows, reduces urea nitrogen concentration in both blood plasma and follicular fluid, and, as a consequence, increases the viability of oocytes and the number of cumulus oophorus cells while reducing oocytes’ DNA degradation of follicular included cumulus-oocyte complex. The reduction on dietary CP may improve in vivo oocytes’ embryo development impacting fertility of lactating dairy cows.     

Co‐benefits,trade‐offs,barriers and policies for greenhouse gas mitigation in the agriculture,forestry and other land use (AFOLU) sector

The agriculture, forestry and other land use (AFOLU) sector is responsible for approximately 25% of anthropogenic GHG emissions mainly from deforestation and agricultural emissions from livestock, soil and nutrient management. Mitigation from the sector is thus extremely important in meeting emission reduction targets. The sector offers a variety of cost‐competitive mitigation options with most analyses indicating a decline in emissions largely due to decreasing deforestation rates. Sustainability criteria are needed to guide development and implementation of AFOLU mitigation measures with particular focus on multifunctional systems that allow the delivery of multiple services from land. It is striking that almost all of the positive and negative impacts, opportunities and barriers are context specific, precluding generic statements about which AFOLU mitigation measures have the greatest promise at a global scale. This finding underlines the importance of considering each mitigation strategy on a case‐by‐case basis, systemic effects when implementing mitigation options on the national scale, and suggests that policies need to be flexible enough to allow such assessments. National and international agricultural and forest (climate) policies have the potential to alter the opportunity costs of specific land uses in ways that increase opportunities or barriers for attaining climate change mitigation goals. Policies governing practices in agriculture and in forest conservation and management need to account for both effective mitigation and adaptation and can help to orient practices in agriculture and in forestry towards global sharing of innovative technologies for the efficient use of land resources. Different policy instruments, especially economic incentives and regulatory approaches, are currently being applied however, for its successful implementation it is critical to understand how land‐use decisions are made and how new social, political and economic forces in the future will influence this process.     

Evaluation of two new commercial tests for the diagnosis of acute dengue virus infection using NS1 antigen detection in human serum

Background

We compared the performance of two new commercial tests for the detection of dengue NS1 protein during the clinical phase of dengue virus (DENV) infection—an immunochromatographic test allowing rapid detection of the NS1 antigen, Dengue NS1 Ag STRIP (Bio-Rad Laboratories - Marnes La Coquette, France), and a two-step sandwich-format microplate enzyme-linked immunosorbent assay (ELISA), pan-E Dengue Early ELISA (Panbio - Brisbane, Australia)—with a one-step sandwich-format microplate ELISA, the Platelia Dengue NS1 Ag test (Bio-Rad).

Methods

We tested 272 serum samples from patients with dengue disease. Of these, 222 were from patients with acute infection of one of the four dengue serotypes, detected by RT-PCR and/or virus isolation. Forty-eight acute-phase serum samples from patients not infected with dengue virus were also included.

Results

The sensitivity of the Platelia Dengue NS1 Ag test on acute serum samples (n = 222) was 87.4% (95% confidence interval: 82.3% to 91.5%); that of Dengue NS1 Ag STRIP was 81.5% (95% CI: 75.8% to 86.4%) after 15 minutes and 82.4% (95% CI: 76.8% to 87.2%) after 30 minutes. Both tests had a specificity of 100% (97.5% CI, one-sided test: 92.6% to 100.0%). The pan-E Dengue Early ELISA had a sensitivity of 60.4% (95% CI: 53.4% to 66.8%) and a specificity of 97.9% (95% CI: 88.9% to 99.9%).

Conclusion

Our findings support the use of diagnostic tools based on the NS1 antigen detection for the diagnosis of acute DENV infection. The immunochromatographic test, Dengue NS1 Ag STRIP—the first rapid diagnostic test for DENV infection—was highly sensitive and specific, and would therefore be a suitable first-line test in the field. The pan-E Dengue Early ELISA was less sensitive than the Platelia test; this two-step ELISA should be combined with DENV IgM antibody detection for the diagnosis of DENV infection.