The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars

The effect of increasing NaCl concentrations was studied on two different cultivars (cv. Orhangazi and cv. Cumhuriyet) of Sesamum indicum. Seedlings were grown for 40 days in half strength Hoagland solution and after 40 days treated with different NaCl concentrations (0, 50 and 100 mM) for 21 days. Differences in growth parameters, lipid peroxidation, antioxidative enzyme activities and proline accumulation were tested in order to put forward the relative tolerance or sensitivity of the cultivars. Results indicated that both parameters differ according to the cultivar's ability in coping oxidative stress caused by salinity. Constitutive levels of antioxidative enzyme activities were almost the same between the cultivars; however, cv. Cumhuriyet was able to induce antioxidative enzyme activities more efficiently when subjected to salt stress. Growth parameters, lipid peroxidation and proline accumulation results are also in good correlation with supporting this cultivar's being relatively tolerant.     

Grass cover on forest clear-cut areas ameliorates some soil chemical properties

Clear-cut areas formed after forest decline due to acid deposition, pest attacks, or wind-breaks in temperate mountainous regions are often populated by grass (mainly Calamagrostis villosa). This study focused on the changes of soil chemical characteristics under the grass cover replacing the forest, focusing mainly on aluminium (Al) speciation. Clear-cut area due to strong acid deposition in the Jizera Mountains (Northern Bohemia) was studied. The soils under grass cover exhibit higher pH values and lower exchangeable Al content compared to adjacent surviving forest. Mobile Al species under the grass have larger proportion of non-toxic organic complexes. The content of exchangeable base cations is slightly higher under the grass. The positive effect of grass on soil chemistry was enhanced by liming. The temporary grass cover can therefore improve soil chemical quality for following reforestation. However, the differences are generally limited to surface organic horizons. Similar results were found also on a bark-beetle clear-cut area in the Bohemian Forest (Southern Bohemia) with smaller acid deposition; nevertheless, most differences were not significant there.     

An analysis of spontaneous behavior following acute MDMA treatment in male and female rats

3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is a widely abused drug that impairs behavioral, emotional and cognitive functions in humans and animals. The aim of this study was to evaluate MDMA effects on the spontaneous behavioral repertoire of rats with a focus on the gender differences. MDMA was given subcutaneously in a single dose of 2.5, 5 and 10 mg/kg and the spontaneous behavior of male and female rats was studied using the open field test. Behavioral patterns (locomotion, rearing, floor-sniffing, air-sniffing, grooming, immobility and stereotypy) were registered in two sessions - 30 and 60 min following MDMA administration; each session lasting 5 min. We found that MDMA totally disrupted the structure and timing of spontaneous behavioral patterns in both genders; no evident differences were measured between either of the sessions. MDMA irrespective of the dosage produced hyperlocomotion, excessive floor-sniffing and almost absolute suppression of grooming and immobility. A biphasic effect of MDMA was found in rearing. Gender differences were present namely in rearing and sniffing stereotypy. This study also confirms that behavioral experiments should focus on more behavioral elements than only on e.g. locomotion and that the observer-based approach still gives the most reliable results.     

Novel modulators of amyloid-beta precursor protein processing

Proteolytic processing of the amyloid precursor protein (APP) is modulated by the action of enzymes alpha-, beta- and gamma-secretases, with the latter two mediating the amyloidogenic production of amyloid-beta (Abeta). Cellular modulators of APP processing are well known from studies of genetic mutations (such as those found in APP and presenilins) or polymorphisms (such as the apolipoprotein E4 epsilon-allele) that predisposes an individual to early or late-onset Alzheimer's disease. In recent years, several classes of molecule with modulating functions in APP processing and Abeta secretion have emerged. These include the neuronal Munc-18 interacting proteins (Mints)/X11s, members of the reticulon family (RTN-3 and RTN-4/Nogo-B), the Nogo-66 receptor (NgR), the peptidyl-prolyl isomerase Pin1 and the Rho family GTPases and their effectors. Mints and NgR bind to APP directly, while RTN3 and Nogo-B interact with the beta-secretase BACE1. Phosphorylated APP is a Pin1 substrate, which binds to its phosphor-Thr668-Pro motif. These interactions by and large resulted in a reduction of Abeta generation both in vitro and in vivo. Inhibition of Rho and Rho-kinase (ROCK) activity may underlie the ability of non-steroidal anti-inflammatory drugs and statins to reduce Abeta production, a feat which could also be achieved by Rac1 inhibition. Detailed understanding of the underlying mechanisms of action of these novel modulators of APP processing, as well as insights into the molecular neurological basis of how Abeta impairs leaning and memory, will open up multiple avenues for the therapeutic intervention of Alzheimer's disease.     

Sperm DNA adducts impair fertilization during ICSI but not during IVF

Many studies emphasize the influence of the status of spermatozoal nucleus on fertilization, mainly with regard to DNA fragmentation. This study was undertaken to analyze the influence of DNA adducts content in spermatozoa on fertilization during assisted reproduction. Ovarian hyperstimulation, oocyte retrieval and laboratory work-up in 61 IVF (in vitro fertilization) and 118 ICSI (intracytoplasmic sperm injection) first cycles were performed according to the same protocol. Semen analysis was made according to WHO Manual (1999). DNA adducts assay in spermatozoa was performed by 32Ppostlabeling method. In total 331 fertilizable oocytes were obtained during IVF and 659 during ICSI. Both groups differed significantly by sperm count, motility and morphology but not by the concentration of DNA adducts in spermatozoa (0.0306 +/- 0.0217 in IVF versus 0.0373 +/- 0.0321 in ICSI). The fertilization rate during IVF was significantly influenced by sperm count (p=0.0002) and motility (p=0.0037) but not by DNA adducts concentration (p=0.30528), whereas during ICSI was positively influenced by sperm motility (p=0.04669) and negatively by DNA adducts concentration (p=0.00796). DNA adducts concentration in spermatozoa significantly negatively influences fertilization rate during ICSI, but not during IVF.     

SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity

The accumulation of cytosolic lipid droplets in muscle and liver cells has been linked to the development of insulin resistance and type 2 diabetes. Such droplets are formed as small structures that increase in size through fusion, a process that is dependent on intact microtubules and the motor protein dynein. Approximately 15% of all droplets are involved in fusion processes at a given time. Here, we show that lipid droplets are associated with proteins involved in fusion processes in the cell: NSF (N-ethylmaleimide-sensitive-factor), alpha-SNAP (soluble NSF attachment protein) and the SNAREs (SNAP receptors), SNAP23 (synaptosomal-associated protein of 23 kDa), syntaxin-5 and VAMP4 (vesicle-associated membrane protein 4). Knockdown of the genes for SNAP23, syntaxin-5 or VAMP4, or microinjection of a dominant-negative mutant of alpha-SNAP, decreases the rate of fusion and the size of the lipid droplets. Thus, the SNARE system seems to have an important role in lipid droplet fusion. We also show that oleic acid treatment decreases the insulin sensitivity of heart muscle cells, and this sensitivity is completely restored by transfection with SNAP23. Thus, SNAP23 might be a link between insulin sensitivity and the inflow of fatty acids to the cell.     

Emerging subject for chiral separation science: cluster boron compounds

The structural chirality is an inherent feature of fully synthetic boron cluster compounds that sometimes exhibit unique biochemical effects. HPLC studies with zwitter-ionic cluster boron compounds and electrophoretic studies with boron cluster anions reveal that the chiral separability of these species is remarkably dissimilar to that of organic species, if uncharged cyclodextrins are used as chiral selectors. Furthermore, marked differences were found between the analytical characteristics of the chiral separations of the boron cluster species and those of the organic species with uncharged cyclodextrins. The present-day experimental database indicates that the rules valid for the chiral separations of the organic species cannot be applied to the chiral separations of the boron cluster species without experimental verification. The current extent of research work devoted to the investigation of chirality and chiral separations of boron cluster species is negligibly small in comparison with that devoted to the investigation of chirality and chiral separations of organic species. This makes difficult a reliable explanation of both the particularities observed in chiral separations of boron cluster species with cyclodextrins as chiral selectors and the strange effects related to these separations at the moment.     

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).     

Morphology and molecular phylogeny of Pseudotrichonympha hertwigi and Pseudotrichonympha paulistana (Trichonymphea, parabasalia) from neotropical rhinotermitids

Pseudotrichonympha is a large hypermastigote parabasalian found in the hindgut of several species of rhinotermitid termites. The genus was discovered more than 100 years ago, and although over a dozen species have since been described, this represents only a small fraction of its likely diversity: the termite genera from which Pseudotrichonympha is known are all species rich, and in most cases their hindgut symbionts have not been examined. Even formally described species are mostly lacking in detailed microscopic data and/or sequence data. Using small subunit ribosomal RNA gene sequences and light and scanning electron microscopy we describe here the morphology and molecular phylogenetic position of two Pseudotrichonympha species: the type species for the genus, Pseudotrichonympha hertwigi from Coptotermes testaceus (described previously in line drawing only), and Pseudotrichonympha paulistana from Heterotermes tenuis (described previously based on light microscopy only).