Thromboxane and Prostacyclin Levels in Fetal Rabbit Brain and Placenta After Intrauterine Partial Ischemic Episodes

The appearance of arachidonic acid (AA) oxidation products in fetal rabbit brain and placenta under normal or partial short-term ischemic episodes induced by placental blood vessel restriction was examined. Intracerebral administration of [3H]AA into close-to-term rabbit fetuses gave rise to radioactively labeled prostaglandin (PG) E2, thromboxane B2, and 6-keto-PGF1 alpha metabolites as detected by HPLC analysis. A significant increase of 20-30% of [3H]AA precursor into eicosanoids was detected in brain of fetuses after 2-h restriction. The thromboxane B2 and 6-keto-PGF1 alpha levels were determined by radioimmunoassay technique over a period of 48 h following ischemic episodes. Thromboxane B2 content in affected animals was higher by five- and twofold at 3 h over control fetal brain and placental tissue values, respectively, and remained significantly higher for 24 h. 6-Keto-PGF1 alpha levels reached a peak value that was greater by 2.5- and 1.5-fold at 6 h for the ischemic brain and placental tissue, respectively, compared with control fetuses. PGE2 levels were less affected, attaining a maximum of 1.9- and 1.1-fold in brain and placenta correspondingly. The thromboxane/prostacyclin ratio reached a maximum in the brain after approximately 3 h, while that in the placenta continued to rise even after 20 h. Persisting high levels of thromboxane are indicative of cerebral vasoconstriction and may suggest possible damaging effects.     

Specific localization and quantification of biotin transport components in yeast by use of a biotin-conjugated,impermeant, electron-dense label

Summary Two approaches are described for the localization and quantification of biotin transport components in yeast cells. One approach is based on tracing the fate of a radioactive affinity label for the biotin transport system, [¹⁴C]-biotinyl-p-nitrophenyl ester (pBNP), through various stages of subcellular fractionations. A complementary method involves the use of a biotinderivatized, impermeant, electron-dense, affinity-cytochemical label (ferritin-biotin conjugates) for subsequent visualization by electron microscopy. Values of approximately 8,000 and 4,000 sites/cell, respectively, were achieved by the two methods. Complicating factors, future perspectives and the relevance of the two methods to the isolation of transport components are discussed.     

Cell lineage analysis of the mammalian female germline

Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.     

The Use of Kosher Phenotyping for Mapping QTL Affecting Susceptibility to Bovine Respiratory Disease

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in feedlot cattle, caused by multiple pathogens that become more virulent in response to stress. As clinical signs often go undetected and various preventive strategies failed, identification of genes affecting BRD is essential for selection for resistance. Selective DNA pooling (SDP) was applied in a genome wide association study (GWAS) to map BRD QTLs in Israeli Holstein male calves. Kosher scoring of lung adhesions was used to allocate 122 and 62 animals to High (Glatt Kosher) and Low (Non-Kosher) resistant groups, respectively. Genotyping was performed using the Illumina BovineHD BeadChip according to the Infinium protocol. Moving average of -logP was used to map QTLs and Log drop was used to define their boundaries (QTLRs). The combined procedure was efficient for high resolution mapping. Nineteen QTLRs distributed over 13 autosomes were found, some overlapping previous studies. The QTLRs contain polymorphic functional and expression candidate genes to affect kosher status, with putative immunological and wound healing activities. Kosher phenotyping was shown to be a reliable means to map QTLs affecting BRD morbidity.     

Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.     

The insulin paradox: aging, proteotoxicity and neurodegeneration

Distinct human neurodegenerative diseases share remarkably similar temporal emergence patterns, even though different toxic proteins are involved in their onset. Typically, familial neurodegenerative diseases emerge during the fifth decade of life, whereas sporadic cases do not exhibit symptoms earlier than the seventh decade. Recently, mechanistic links between the aging process and toxic protein aggregation, a common hallmark of neurodegenerative diseases, have been revealed. The insulin/insulin-like growth factor 1 (IGF1) signalling pathway - a lifespan, metabolism and stress-resistance regulator - links neurodegeneration to the aging process. Thus, although a reduction of insulin signalling can result in diabetes, its reduction can also increase longevity and delay the onset of protein-aggregation-mediated toxicity. Here we review this apparent paradox and delineate the therapeutic potential of manipulating the insulin/IGF1 signalling pathway for the treatment of neurodegenerative diseases.     

The Effect of Chemical Chaperones on the Assembly and Stability of HIV-1 Capsid Protein

Chemical chaperones are small organic molecules which accumulate in a broad range of organisms in various tissues under different stress conditions and assist in the maintenance of a correct proteostasis under denaturating environments. The effect of chemical chaperones on protein folding and aggregation has been extensively studied and is generally considered to be mediated through non-specific interactions. However, the precise mechanism of action remains elusive. Protein self-assembly is a key event in both native and pathological states, ranging from microtubules and actin filaments formation to toxic amyloids appearance in degenerative disorders, such as Alzheimer''s and Parkinson''s diseases. Another pathological event, in which protein assembly cascade is a fundamental process, is the formation of virus particles. In the late stage of the virus life cycle, capsid proteins self-assemble into highly-ordered cores, which encapsulate the viral genome, consequently protect genome integrity and mediate infectivity. In this study, we examined the effect of different groups of chemical chaperones on viral capsid assembly in vitro, focusing on HIV-1 capsid protein as a system model. We found that while polyols and sugars markedly inhibited capsid assembly, methylamines dramatically enhanced the assembly rate. Moreover, chemical chaperones that inhibited capsid core formation, also stabilized capsid structure under thermal denaturation. Correspondingly, trimethylamine N-oxide, which facilitated formation of high-order assemblies, clearly destabilized capsid structure under similar conditions. In contrast to the prevailing hypothesis suggesting that chemical chaperones affect proteins through preferential exclusion, the observed dual effects imply that different chaperones modify capsid assembly and stability through different mechanisms. Furthermore, our results indicate a correlation between the folding state of capsid to its tendency to assemble into highly-ordered structures.