Multiple genotype analysis and sexing of IVF bovine embryos

Twenty-one in vitro-fertilized bovine blastocysts were quartered, lysed and subjected to primer elongation preamplification (PEP) procedure, allowing for the analysis of up to 40 genotypes per quarter embryo. The quarter-embryos were sexed by polymerase chain reaction (PCR) using BRY.1, Bov97M and ZFX/ZFY loci, and then genotyped for k-casein, bovine leukocyte adhesion deficiency (BLAD) and microsatellite D9S1. The mitochondrial cytochrome B locus was used as an internal control with a 95% success rate. The PEP procedure amplified genomic fragments in 93% of all cases. The embryos were identified to be 11 males and 10 females. Sexing accuracy was 87% for BRY.1, 97% for ZFX/ZFY and 100% for Bov97M. False genotyping was due mostly to amplification of BRY.1 in the female embryos and to the nonamplification of the ZFY locus in the male embryos. The results indicate that the combined use of Bov97M and ZFX/ZFY loci is a highly accurate procedure for sexing bovine embryos. Genotyping for kappa-casein, D9S1 and BLAD was successful in 94, 99 and 91% of assays, respectively. Sex ratios and allele frequencies of embryos for gk-casein, BLAD and D9S1 were all close to the observed frequencies in the Israeli Holstein population. These results support the conclusion that the genotyping of embryos is as accurate as that of mature animals. Thus, marker-assisted selection can be efficiently applied at the preimplantation embryo level for loci of economic importance.     

Does saline water consumption affect feeding and fuel deposition rate of a staging,long‐distance migrating passerine?

To accomplish their enduring journeys, migrating birds accumulate fuel consisting mainly of lipids in stopover sites located throughout their migration routes. Fuel deposition rate (FDR) is considered a key parameter determining the speed of migration and thereby bird fitness, and recent studies have demonstrated the positive effects of fresh water consumption on the FDR of migrating blackcaps Sylvia atricapilla. Sewage water reservoirs, characterized by higher water salinity than fresh water, were extensively built in different parts of the world and are used by birds during their travel, but their effects on wildlife and specifically on migrating birds have been largely overlooked thus far. We experimentally examined the effects of water salinity on blackcap FDR during migration. We captured birds in an autumn stopover site, transported them to the laboratory and provided them with fruits, mealworms and water of different salinity levels (0.3, 4.5 and 9‰ NaCl) for several days. We examined the effects of water salinity on the blackcaps’ diet, water consumption and FDR and found that FDR was mainly affected by fruit consumption rate and not by the water salinity levels. Water salinity nevertheless caused elevated water consumption as the birds consumed almost 3 times more saline water than fresh water per consumed fruit mass. Our work is the first to explore the consequences of saline water consumption on migrating passerines, specifically suggesting that anthropogenic alterations of habitats by sewage water treatment facilities may modulate bird nutrition and diet.     

Growth Rate of Escherichia coli at Elevated Temperatures: Reversible Inhibition of Homoserine Trans-Succinylase

The preceding paper (10) showed that the growth of Escherichia coli is slowed, without killing, at 40 to 45 C, and that in the several strains tested the cause is a decrease in the activity of homoserine trans-succinylase. These temperatures are now shown to inhibit the enzyme directly, in crude extracts and after partial purification. The effect is rapid and is immediately reversible, unlike the progressive and slowly reversible changes of conventional denaturation.     

Epigenetics and migraine; complex mitochondrial interactions contributing to disease susceptibility

Migraine is a common neurological disorder classified by the World Health Organisation (WHO) as one of the top twenty most debilitating diseases in the developed world. Current therapies are only effective for a proportion of sufferers and new therapeutic targets are desperately needed to alleviate this burden. Recently the role of epigenetics in the development of many complex diseases including migraine has become an emerging topic. By understanding the importance of acetylation, methylation and other epigenetic modifications, it then follows that this modification process is a potential target to manipulate epigenetic status with the goal of treating disease. Bisulphite sequencing and methylated DNA immunoprecipitation have been used to demonstrate the presence of methylated cytosines in the human D-loop of mitochondrial DNA (mtDNA), proving that the mitochondrial genome is methylated. For the first time, it has been shown that there is a difference in mtDNA epigenetic status between healthy controls and those with disease, especially for neurodegenerative and age related conditions. Given co-morbidities with migraine and the suggestive link between mitochondrial dysfunction and the lowered threshold for triggering a migraine attack, mitochondrial methylation may be a new avenue to pursue. Creative thinking and new approaches are needed to solve complex problems and a systems biology approach, where multiple layers of information are integrated is becoming more important in complex disease modelling.     

DAP-kinase and autophagy

DAP-kinase (DAPK) is a Ca²⁺-calmodulin regulated kinase with various, diverse cellular activities, including regulation of apoptosis and caspase-independent death programs, cytoskeletal dynamics, and immune functions. Recently, DAPK has also been shown to be a critical regulator of autophagy, a catabolic process whereby the cell consumes cytoplasmic contents and organelles within specialized vesicles, called autophagosomes. Here we present the latest findings demonstrating how DAPK modulates autophagy. DAPK positively contributes to the induction stage of autophagosome nucleation by modulating the Vps34 class III phosphatidyl inositol 3-kinase complex by two independent mechanisms. The first involves a kinase cascade in which DAPK phosphorylates protein kinase D, which then phosphorylates and activates Vps34. In the second mechanism, DAPK directly phosphorylates Beclin 1, a necessary component of the Vps34 complex, thereby releasing it from its inhibitor, Bcl-2. In addition to these established pathways, we will discuss additional connections between DAPK and autophagy and potential mechanisms that still remain to be fully validated. These include myosin-dependent trafficking of Atg9-containing vesicles to the sites of autophagosome formation, membrane fusion events that contribute to expansion of the autophagosome membrane and maturation through the endocytic pathway, and trafficking to the lysosome on microtubules. Finally, we discuss how DAPK's participation in the autophagic process may be related to its function as a tumor suppressor protein, and its role in neurodegenerative diseases.     

A Substrate Trapping Approach Identifies Proteins Regulated by Reversible S-nitrosylation

Protein S-nitrosylation, the nitric oxide-mediated posttranslational modification of cysteine residues, has emerged as an important regulatory mechanism in diverse cellular processes. Yet, knowledge about the S-nitrosoproteome in different cell types and cellular contexts is still limited and many questions remain regarding the precise roles of protein S-nitrosylation and denitrosylation. Here we present a novel strategy to identify reversibly nitrosylated proteins. Our approach is based on nitrosothiol capture and enrichment using a thioredoxin trap mutant, followed by protein identification by mass spectrometry. Employing this approach, we identified more than 400 putative nitroso-proteins in S-nitrosocysteine-treated human monocytes and about 200 nitrosylation substrates in endotoxin and cytokine-stimulated mouse macrophages. The large majority of these represent novel nitrosylation targets and they include many proteins with key functions in cellular homeostasis and signaling. Biochemical and functional experiments in vitro and in cells validated the proteomic results and further suggested a role for thioredoxin in the denitrosylation and activation of inducible nitric oxide synthase and the protein kinase MEK1. Our findings contribute to a better understanding of the macrophage S-nitrosoproteome and the role of thioredoxin-mediated denitrosylation in nitric oxide signaling. The approach described here may prove generally useful for the identification and exploration of nitroso-proteomes under various physiological and pathophysiological conditions.Protein S-nitrosylation, the covalent addition of a nitric oxide (NO)¹ group to a cysteine thiol, constitutes a widespread regulatory mechanism involved in various biological processes, such as control of cell growth, metabolism, differentiation, and apoptosis (1–4). S-nitrosylation is known to modulate the functional properties of a large number of proteins and thereby influence normal cell function and emerging evidence implicates aberrant protein S-nitrosylation in multiple pathological conditions, including cardiovascular disease, neurodegeneration, and cancer (5, 6). Although significant advances have been made in the field of S-nitrosylation, there is still limited knowledge regarding the constituents of the proteome that become nitrosylated (that is, the nitrosoproteome) across different cell types and conditions. Therefore, much remains unknown about the specific roles and functional significance of S-nitrosylation in cellular function and disease. In addition, there is a need to better understand the mechanisms and consequences of denitrosylation, both for individual proteins and on a systems level.Protein denitrosylation is substantially mediated by two cellular denitrosylating systems, namely the glutathione and S-nitrosoglutathione reductase (GSH/GSNOR) and the thioredoxin and thioredoxin reductase (Trx/TrxR) systems, with the latter representing a direct mechanism of protein denitrosylation (7–11). Trx/TrxR-mediated denitrosylation has been specifically linked to several cellular processes, including apoptosis (7), cell adhesion (12), exocytosis (13) and heme protein maturation (14). Despite recent progress in characterizing protein nitrosylation and denitrosylation, the dynamic cellular nitrosoproteome remains relatively unexplored, particularly under physiologically relevant conditions. This may be in part because of methodological challenges inherent to the proteomic analysis of S-nitrosylation and denitrosylation (See Discussion and (15–17)).The disulfide and nitrosothiol (SNO) reductase activities of Trx depend on a highly conserved Cys-Gly-Pro-Cys active site (8, 18). Recent evidence suggests that, similar to reduction of disulfides, SNO reduction occurs in a two-step mechanism (7, 8). First, the more N-terminal cysteine (Cys32 in human Trx1) attacks the sulfur atom on the SNO moiety of the substrate protein, thereby displacing NO (formally, NO⁻) and generating an intermolecular disulfide between Trx and the substrate. The second step entails an intramolecular attack by the second active site cysteine (Cys35, known as the “resolving cysteine”) on the mixed disulfide intermediate, thus releasing the reduced target protein and the oxidized Trx. The normally transient disulfide intermediate formed in the first step is stabilized in a reaction that involves a Trx mutant that lacks the resolving cysteine. This so-called “trap mutant” has been employed in the identification of disulfide targets of Trx in several cell systems (19–22). However, the utility and value of such a trapping approach in the context of nitrosylation proteomics has not been evaluated.In this study we adapted the Trx trapping strategy for global profiling of cellular nitrosylation and denitrosylation processes. Using this approach, we report the identification of hundreds of potential new nitrosylated targets in monoctyes and macrophages, followed by validation using biochemical and functional assays. The findings presented herein greatly expand our knowledge of the monocyte and macrophage nitrosoproteome and suggest multiple roles for nitrosylation and denitrosylation in macrophage activation and function. The approach employed in this study may be applied to exploring nitrosoproteomes in different cells and under various physiological and pathological conditions.