Renal Effects and Underlying Molecular Mechanisms of Long-Term Salt Content Diets in Spontaneously Hypertensive Rats

Several evidences have shown that salt excess is an important determinant of cardiovascular and renal derangement in hypertension. The present study aimed to investigate the renal effects of chronic high or low salt intake in the context of hypertension and to elucidate the molecular mechanisms underlying such effects. To this end, newly weaned male SHR were fed with diets only differing in NaCl content: normal salt (NS: 0.3%), low salt (LS: 0.03%), and high salt diet (HS: 3%) until 7 months of age. Analysis of renal function, morphology, and evaluation of the expression of the main molecular components involved in the renal handling of albumin, including podocyte slit-diaphragm proteins and proximal tubule endocytic receptors were performed. The relationship between diets and the balance of the renal angiotensin-converting enzyme (ACE) and ACE2 enzymes was also examined. HS produced glomerular hypertrophy and decreased ACE2 and nephrin expressions, loss of morphological integrity of the podocyte processes, and increased proteinuria, characterized by loss of albumin and high molecular weight proteins. Conversely, severe hypertension was attenuated and renal dysfunction was prevented by LS since proteinuria was much lower than in the NS SHRs. This was associated with a decrease in kidney ACE/ACE2 protein and activity ratio and increased cubilin renal expression. Taken together, these results suggest that LS attenuates hypertension progression in SHRs and preserves renal function. The mechanisms partially explaining these findings include modulation of the intrarenal ACE/ACE2 balance and the increased cubilin expression. Importantly, HS worsens hypertensive kidney injury and decreases the expression nephrin, a key component of the slit diaphragm.     

Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells

The ability to edit the genome is essential for many state-of-the-art experimental paradigms. Since DNA breaks stimulate repair, they can be exploited to target site-specific integration. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system from Streptococcus pyogenes has been harnessed into an efficient and programmable nuclease for eukaryotic cells. We thus combined DNA cleavage by cas9, the generation of homologous recombination donors by polymerase chain reaction (PCR) and transient depletion of the non-homologous end joining factor lig4. Using cultured Drosophila melanogaster S2-cells and the phosphoglycerate kinase gene as a model, we reached targeted integration frequencies of up to 50% in drug-selected cell populations. Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial. We confirmed established rules for S. pyogenes cas9 sgRNA design and demonstrate that the complementarity region allows length variation and 5′-extensions. This enables generation of U6-promoter fusion templates by overlap-extension PCR with a standardized protocol. We present a series of PCR template vectors for C-terminal protein tagging and clonal Drosophila S2 cell lines with stable expression of a myc-tagged cas9 protein. The system can be used for epitope tagging or reporter gene knock-ins in an experimental setup that can in principle be fully automated.     

Reproductive performance of Eurasian Kestrel Falco tinnunculus in an agricultural landscape with a mosaic of land uses

There are serious concerns about the environmental and ecological degradation caused by modern agriculture and its impact on animal populations. There is therefore a need to assess the reproductive performance of free‐living animals in agricultural landscapes. We undertook a 4‐year study on the reproductive biology of Eurasian Kestrel Falco tinnunculus in the Parma district of Italy. Kestrels breeding in areas of intensive agriculture delayed egg‐laying and their offspring were in poorer condition compared with those breeding in areas of temporary or permanent grasslands. However, egg volume and fledging success were similar among sites. The availability of temporary and permanent grasslands may limit the negative impacts of intensive agriculture on the breeding activity of Kestrels.     

S-glutathionylation: from redox regulation of protein functions to human diseases

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an integral role in the modulation of several physiological functions but can also be potentially destructive if produced in excessive amounts. Protein cysteinyl thiols appear especially sensitive to ROS/RNS attack. Experimental evidence started to accumulate recently, documenting that S-glutathionylation occurs in a number of physiologically relevant situations, where it can produce discrete modulatory effects on protein function. The increasing evidence of functional changes resulting from this modification, and the growing number of proteins shown to be S-glutathionylated both in vitro and in vivo support this contention, and confirm this as an attractive area of research. S-glutathionylated proteins are now actively investigated with reference to problems of biological interest and as possible biomarkers of human diseases associated with oxidative/nitrosative stress.     

2-aminoethoxydiphenyl borate alters selectivity of Orai3 channels by increasing their pore size

Stim1 in the endoplasmic reticulum and the three Orai (also termed CRACM) channels in the plasma-membrane are main components of native Ca(2+) release-activated Ca(2+) channels. A pharmacological hallmark of these channels is their distinct sensitivity to 2-aminoethoxydiphenyl borate (2-APB). Here we report that Orai3 currents can be robustly stimulated by 75 microm 2-APB independent of Stim1, whereas 2-APB at similar concentrations inhibited store-operated Orai1 currents. 2-APB did not only promote currents through Orai3 channels but also dramatically altered ion selectivity of Orai3 channels. This allowed for permeation of monovalent cations both in the inward as well as outward direction, which is in sharp contrast to the high Ca(2+) selectivity of store-operated Orai3 currents. An Orai3-R66W mutant, which lacked in analogy to the severe combined immune deficiency mutant Orai1-R91W store-operated activation, was also found to be resistant to 2-APB stimulation. The change in selectivity by 2-APB was associated with an increase in Orai3 minimum pore size from about 3.8A to more than 5.34 A. In line with a potential interaction of 2-APB with the Orai3 pore, among three pore mutants tested, the Orai3 E165Q mutant particularly resembled in its permeation properties those of 2-APB stimulated Orai3 and additionally exhibited a reduced response to 2-APB. In aggregate, stimulation of Orai3 currents by 2-APB occurred along with an alteration of the permeation pathway that represents a unique mechanism for regulating ion channel selectivity by chemical compounds.     

Structure-function studies of the G-domain from human gem, a novel small G-protein

Gem, a member of the Rad,Gem/Kir subfamily of small G-proteins, has unique sequence features. We report here the crystallographic structure determination of the Gem G-domain in complex with nucleotide to 2.4 A resolution. Although the basic Ras protein fold is maintained, the Gem switch regions emphatically differ from the Ras paradigm. Our ensuing biochemical characterization indicates that Gem G-domain markedly prefers GDP over GTP. Two known functions of Gem are distinctly affected by spatially separated clusters of mutations.     

Cell-line specific chromatin acetylation at the Sox10-Pax3 enhancer site modulates the RET proto-oncogene expression

IscA homologs are known to be involved in iron-sulfur cluster formation in various organisms. Recombinant proteins of two IscA homologs from the cyanobacterium Synechocystis PCC 6803, designated SLR1417 and SLR1565, were purified. The absorption spectrum of purified SLR1565 was typical for [2Fe-2S] cluster-containing proteins, whereas that of SLR1417 predominantly showed the presence of the iron ion alone. In the cyanobacterial cell extracts, only SLR1565 was found to form a complex with a novel prokaryotic HEAT-repeats-containing protein, SLR1098. Thus, the two cyanobacterial IscA protein homologs exist in distinct molecular states, suggesting different cellular roles for these proteins.