Influence of predation risk and plant structure on vigilance and intermittent locomotion in <Emphasis Type="Italic">Microcavia australis</Emphasis> (Rodentia,Caviidae)

The aim of this study was to analyze and compare vigilance behavior and intermittent locomotion at two sites (El Leoncito and Ñacuñán, Argentina) that differ in predation risk, plant structure, and plant resource availability. Subjects were lesser cavies (Microcavia australis), a social species that is semi-fossorial, diurnal, and native to South America. Continuous focal sampling was conducted during the day, at times of food shortage, food abundance, and reproduction from 2003 to 2005. The proportion of time spent vigilance was significantly higher at Ñacuñán, where vigilance peaked at midday and reached a minimum in the evening. This midday peak of vigilance at Ñacuñán was associated with a midday peak of danger from raptors as indicated by a raptor activity peak at that time. In contrast, both vigilance and predator activity at El Leoncito were constant through the day. Records of intermittent locomotion and number and duration of pauses in locomotion were significantly higher at El Leoncito, a difference that may have been due to the need for greater vigilance while moving across areas of less protective cover at this site.     

Potential CRF1R PET imaging agents: N-fluoroalkyl-8-(6-methoxy-2-methylpyridin-3-yl)-2,7-dimethyl-N-alkylpyrazolo[1,5-a][1,3,5]triazin-4-amines

A series of N-fluoroalkyl-8-(6-methoxy-2-methylpyridin-3-yl)-2,7-dimethyl-N-alkylpyrazolo[1,5-a][1,3,5]triazin-4-amines were prepared and evaluated as potential CRF₁R PET imaging agents. Optimization of their CRF₁R binding potencies and octanol-phosphate buffer phase distribution coefficients resulted in discovery of analog 7e (IC₅₀ = 6.5 nM, log D = 3.5).     

Differential effects of proinflammatory cytokines on cell death and ER stress in insulin-secreting INS1E cells and the involvement of nitric oxide

Proinflammatory cytokines produced by immune cells destroy pancreatic beta cells in type 1 diabetes. The aim of this study was to investigate the cytokine network and its effects in insulin-secreting cells. INS1E cells were exposed to different combinations of proinflammatory cytokines. Cytokine toxicity was estimated by MTT assay and caspase activation measurements. The NFκB-iNOS pathway was analyzed by a SEAP reporter gene assay, Western-blotting and nitrite measurements. Gene expression analyses of ER stress markers, Chop and Bip, were performed by real-time RT-PCR. Cytokines tested in this study, namely IL-1β, TNFα and IFNγ, had deleterious effects on beta cell viability. The most potent toxicity exhibited IL-1β and its combinations with other cytokines. The toxic effects of IL-1β towards cell viability, caspase activation and iNOS activity were dependent on nitric oxide and abolished by an iNOS blocker. IL-1β was the strongest inducer of the NFκB activation. An iNOS blocker inhibited IL-1β-mediated NFκB activation in the first, initial phase of cytokine action, but did not affect significantly NFκB activation after prolonged incubation. Interestingly iNOS protein expression was induced predominantly by IL-1β and decreased in the presence of an iNOS blocker in the case of a short time exposure. The changes in the expression of ER stress markers were also almost exclusively dependent on the IL-1β presence and counteracted by iNOS blockade. Thus cytokine-induced beta cell death is primarily IL-1β mediated with a NO-independent enhancement by TNFα and IFNγ. The deleterious effects on cell viability and function are crucially dependent on IL-1β-induced nitric oxide formation.     

Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution

Background  

New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-validation of 454 sequencing and conventional pyrosequencing, both of which offer exact quantification of methylation levels with a single CpG dinucleotide resolution, was performed.     

Protein expression changes in skeletal muscle in response to growth promoter abuse in beef cattle

The fraudulent treatment of cattle with growth promoting agents (GPAs) is a matter of great concern for the European Union (EU) authorities and consumers. It has been estimated that 10% of animals are being illegally treated in the EU. In contrast, only a much lower percentage of animals (<0.5%) are actually found as being noncompliant by conventional analytical methods. Thus, it has been proposed that methods should be developed that can detect the use of the substances via the biological effects of these substances on target organs, such as the alteration of protein expression profiles. Here we present a study aimed at evaluating if a correlation exists between the treatment with GPAs and alterations in the two-dimensional electrophoresis (2DE) protein pattern obtained from the biceps brachii skeletal muscle from mixed-bred cattle. After image analysis and statistical evaluation, protein spots that differentiate between treated and control groups were selected for analysis by mass spectrometry. A set of proteins could be defined that accurately detect the use of glucocorticoids and β(2)-agonists as growth promoters through the changes caused in muscle differentiation. As a further validation, we repeated the analysis using an independent set of samples from a strain of pure-bred cattle and verified these proteins by Western blot analysis.     

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.The evolving maturation of the field of proteomics has, in the same way as in genomics, highlighted the need of better sampling procedures and sample preparation methodologies to minimize the effect of post mortem alterations. The aspect of sample quality is not new in any way and is relevant in most biomedical fields but has only lately started to receive adequate attention. The main factors influencing sample quality is storage temperature of the body until tissue removal (foremost a problem in clinical settings and extraction of less accessible tissue samples from model organisms) and post mortem interval (PMI)¹ (1–3). Post mortem degradation in during PMI is a well known compromising problem when studying endogenous peptides (2, 3) and has also been proven to affect the results of polypeptide (here defined as proteins larger than 10 kDa) studies (3–8). PMI degradation has mainly been studied on human or mouse brain tissue, using two-dimensional electrophoresis (2-DE), SDS-PAGE, and immunoblotting (1, 3–12). There are also a few proteomic studies on muscle tissue degradation in livestock (13–16).We and others have previously explored the effect of focused microwave irradiation with regard to sample quality, demonstrating that this method is more reliable than snap freezing in liquid nitrogen, especially with regard to post-translational modification (PTM) stability (2, 3, 17–20). An alternative method based on cryostat dissection with subsequent heat treatment through boiling has also been reported to improve endogenous peptide sample quality (21). Besides focused microwave irradiation, which is specifically used for rodent brain tissue sampling, we have also demonstrated the efficiency of rapid heat stabilization through conductivity with regard to sample degradation (3, 22). Although somewhat constrained by its dependence on how quickly the tissue is harvested from the body, the latter procedure has the added advantage that it can be used on any type of tissue and species, fresh as well as frozen. This study will compare effects of sampling procedures on the liver and pancreas degradome following rapid heat stabilization, the more traditional snap freezing, or the combination of snap freezing with subsequent heat stabilization.To summarize, this study investigated the effects of post mortem degradation in pancreas and liver. Both tissues are well studied because of their multiple functions in the body and their involvement in different diseases such as diabetes or hepatocarcinoma. Pancreas is especially interesting in this context as it displays endocrine secretion of peptides, and exocrine secretion of digestive enzymes, the later making it a protease rich tissue. We used both two-dimensional difference in gel electrophoresis (2D-DIGE) and label free liquid chromatography mass spectrometry (LC-MS) based differential peptide display (2, 18), the later to better investigate changes in small molecular fragment that are not easily detectable by gel-based methods. 2D-DIGE is an unrivaled methodology to characterize alterations in isoform patterns, which is an important aspect considering that post-translational modifications (PTMs) such as phosphorylations are especially sensitive to post mortem influence within a few minutes PMI (3). The peptidomics approach has been used in several studies to point out early post mortem changes and protein degradation that tissue undergo following sampling and is therefore a well-suited method (3, 18, 22).     

Male fertility, chromosome abnormalities, and nuclear organization

Numerous studies have implicated the role of gross genomic rearrangements in male infertility, e.g., constitutional aneuploidy, translocations, inversions, Y chromosome deletions, elevated sperm disomy, and DNA damage. The primary purpose of this paper is to review male fertility studies associated with such abnormalities. In addition, we speculate whether altered nuclear organization, another chromosomal/whole genome-associated phenomenon, is also concomitant with male factor infertility. Nuclear organization has been studied in a range of systems and implicated in several diseases. For many applications the measurement of the relative position of chromosome territories is sufficient to determine patterns of nuclear organization. Initial evidence has suggested that, unlike in the more usual 'size-related' or 'gene density-related' models, mammalian (including human) sperm heads display a highly organized pattern including a chromocenter with the centromeres located to the center of the nucleus and the telomeres near the periphery. More recent evidence, however, suggests there may be size- and gene density-related components to nuclear organization in sperm. It seems reasonable to hypothesize therefore that alterations in this pattern may be associated with male factor infertility. A small handful of studies have addressed this issue; however, to date it remains an exciting avenue for future research with possible implications for diagnosis and therapy.     

Thyrsiferol Inhibits Mitochondrial Respiration and HIF-1 Activation

The cytotoxic marine red algal metabolite thyrsiferol (1) was found to inhibit hypoxia-induced hypoxia-inducible factor-1 (HIF-1) activation in T47D human breast tumor cells (66% inhibition at 3 μM). Compound 1 also suppressed hypoxic induction of HIF-1 target genes (VEGF, GLUT-1) at the mRNA level, and displayed tumor cell line-selective time-dependent inhibition of cell viability/proliferation. Mechanistic studies revealed that 1 selectively suppressed mitochondrial respiration at Complex I (IC(50) 3 μM). Thyrsiferol represents a prototypical, structurally unique electron transport chain inhibitor. The apparent rotenone-like activity may contribute to the observed cytotoxicity of 1 and play an important role in Laurencia chemical defense.