Beneficial effects of carbon monoxide-releasing molecules on post-ischemic myocardial recovery

There is increasing evidence corroborating a protective role of carbon monoxide releasing molecules (CORMs) in injured tissues. Carbon monoxide (CO) carriers have been recently developed as a pharmacological tool to simulate the effect of heme oxygenase-1-derived CO. The effects of CORM-3, a water-soluble CO releaser, on the incidence of reperfusion-induced ventricular fibrillation (VF) and tachycardia (VT) were studied in isolated rat hearts. Hearts were treated with different doses of CORM-3 before the induction of 30 min global ischemia followed by 120 min reperfusion. We found that at concentrations of 25 μM and 50 μM of CORM-3 promoted a significant reduction in the incidence of VF and VT. Thus, the incidence of VF was reduced by 67% (p < 0.05) and 92% (p < 0.05) with 25 μM and 50 μM of CORM-3, respectively. The protective effect of CORM-3 on the incidence of VT followed the same pattern. The antiarrhythmic protection was associated with a marked attenuation in infarct size, significant decreases in cellular Na⁺ and Ca²⁺ gains and K⁺ loss. Consequently, the recovery of post-ischemic function was significantly improved. In conclusion, CORM-3 exerts beneficial effects against ischemia/reperfusion-induced injury through its abilities to release CO which mediates a cardioprotective action by regulating tissue Na⁺, K⁺, and Ca²⁺ levels.     

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) analyses. First, the salt gradient (using K⁺ as displacing agent) was evaluated from 25 to 500 mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC–MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.     

Specific Depletion of Ly6Chi Inflammatory Monocytes Prevents Immunopathology in Experimental Cerebral Malaria

Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage. In other parasitic infection models, inflammatory monocytes have been shown to regulate Th1 responses but their role in ECM remains poorly defined, whereas neutrophils are reported to contribute to ECM immune pathology. Making use of the recent development of specific monoclonal antibodies (mAb), we depleted in vivo Ly6C^hi inflammatory monocytes (by anti-CCR2), Ly6G⁺ neutrophils (by anti-Ly6G) or both cell types (by anti-Gr1) during infection with Ovalbumin-transgenic PbA parasites (PbTg). Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development. In addition, depletion of Ly6C^hi inflammatory monocytes but not neutrophils led to decreased IFNγ levels and IFNγ⁺CD8⁺ T effector cells in the brain. Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses. In conclusion, the specific depletion of Ly6C^hi inflammatory monocytes attenuated brain inflammation and immune cell recruitment to the CNS, which prevented ECM following Plasmodium infection, pointing out a substantial role of Ly6C⁺ monocytes in ECM inflammatory processes.     

FGFRL1 is a neglected putative actor of the FGF signalling pathway present in all major metazoan phyla

Background  

Fibroblast Growth Factors (FGF) and their receptors are well known for having major implications in cell signalling controlling embryonic development. Recently, a gene coding for a protein closely related to FGFRs (Fibroblast Growth Factor Receptors) called FGFR5 or FGFR-like 1 (FGFRL1), has been described in vertebrates. An orthologous gene was also found in the cephalochordate amphioxus, but no orthologous genes were found by the authors in other non-vertebrate species, even if a FGFRL1 gene was identified in the sea urchin genome, as well as a closely related gene, named nou-darake, in the planarian Dugesia japonica. These intriguing data of a deuterostome-specific gene that might be implicated in FGF signalling prompted us to search for putative FGFRL1 orthologues in the completely sequenced genomes of metazoans.     

Dihydropyridine neuropeptide Y Y(1) receptor antagonists

Dihydropyridine 5a was found to be an inhibitor of neuropeptide Y(1) binding in a high throughput (125)I-PYY screening assay. Structure-activity studies around certain portions of the dihydropyridine chemotype identified BMS-193885 (6e) as a potent and selective Y(1) receptor antagonist. In a forskolin-stimulated c-AMP production assay using CHO cells expressing the human Y(1) receptor, 6e demonstrated full functional antagonism (K(b)=4.5 nM). Compound 6e inhibited NPY-induced feeding in satiated rats when dosed at 3.0 and 10.0 mg/kg (ip), and also decreased spontaneous overnight food consumption in rats at doses of 10 and 20 mg/kg (ip).     

Polyubiquitinated PCNA Recruits the ZRANB3 Translocase to Maintain Genomic Integrity after Replication Stress

Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in?vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA?structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.     

Exercise changes volatiles in exhaled breath assessed by an electronic nose

Exercise-caused metabolic changes can be followed by monitoring exhaled volatiles; however it has not been previously reported if a spectrum of exhaled gases is modified after physical challenge. We have hypothesized that changes in volatile molecules assessed by an electronic nose may be the reason for the alkalization of the exhaled breath condensate (EBC) fluid following physical exercise.Ten healthy young subjects performed a 6-minute running test. Exhaled breath samples pre-exercise and post-exercise (0 min, 15 min, 30 min and 60 min) were collected for volatile pattern ("smellprint") determination and pH measurements (at 5.33 kPa CO2), respectively. Exhaled breath smellprints were analyzed using principal component analysis and were related to EBC pH.Smellprints (p=0.04) and EBC pH (p=0.01) were altered during exercise challenge. Compared to pre-exercise values, smellprints and pH differed at 15 min, 30 min and 60 min following exercise (p<0.05), while no difference was found at 0 min post-exercise. In addition, a significant correlation was found between volatile pattern of exhaled breath and EBC pH (p=0.01, r=-0.34).Physical exercise changes the pattern of exhaled volatiles together with an increase in pH of breath. Changes in volatiles may be responsible for increase in EBC pH.     

Fibronectin matrix assembly suppresses dispersal of glioblastoma cells

Glioblastoma (GBM), the most aggressive and most common form of primary brain tumor, has a median survival of 12-15 months. Surgical excision, radiation and chemotherapy are rarely curative since tumor cells broadly disperse within the brain. Preventing dispersal could be of therapeutic benefit. Previous studies have reported that increased cell-cell cohesion can markedly reduce invasion by discouraging cell detachment from the tumor mass. We have previously reported that α5β1 integrin-fibronectin interaction is a powerful mediator of indirect cell-cell cohesion and that the process of fibronectin matrix assembly (FNMA) is crucial to establishing strong bonds between cells in 3D tumor-like spheroids. Here, we explore a potential role for FNMA in preventing dispersal of GBM cells from a tumor-like mass. Using a series of GBM-derived cell lines we developed an in vitro assay to measure the dispersal velocity of aggregates on a solid substrate. Despite their similar pathologic grade, aggregates from these lines spread at markedly different rates. Spreading velocity is inversely proportional to capacity for FNMA and restoring FNMA in GBM cells markedly reduces spreading velocity by keeping cells more connected. Blocking FNMA using the 70 KDa fibronectin fragment in FNMA-restored cells rescues spreading velocity, establishing a functional role for FNMA in mediating dispersal. Collectively, the data support a functional causation between restoration of FNMA and decreased dispersal velocity. This is a first demonstration that FNMA can play a suppressive role in GBM dispersal.     

Introgression of an intermediate <Emphasis Type="Italic">VRNH1</Emphasis> allele in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) leads to reduced vernalization requirement without affecting freezing tolerance

The process of vernalization is mainly controlled by two genes in winter barley (Hordeum vulgare L.), VRNH1 and VRNH2. A recessive allele at VRNH1 and a dominant allele at VRNH2 must be present to induce a vernalization requirement. In addition, this process is usually associated with greater low-temperature tolerance. Spanish barleys originated in areas with mild winters and display a reduced vernalization requirement compared with standard winter cultivars. The objective of this study was to investigate the genetic origin of this reduced vernalization requirement and its effect on frost tolerance. We introgressed the regions of a typical Spanish barley line that carry VRNH1 and VRNH2 into a winter cultivar, Plaisant, using marker-assisted backcrossing. We present the results of a set of 12 lines introgressed with all four possible combinations of VRNH1 and VRNH2, which were evaluated for vernalization requirement and frost tolerance. The reduced vernalization requirement of the Spanish parent was confirmed, and was found to be due completely to the effect of the VRNH1 region. The backcross lines showed no decline in frost tolerance compared with that of the recurrent parent unless they carried an extra segment of chromosome 5H. This extra segment, a carryover of the backcross process, apparently contained the well-known frost tolerance quantitative trait locus Fr-H2. We demonstrate that it is possible to manipulate the vernalization requirement with only minor effects on frost tolerance. This finding opens the path to creating new types of barley cultivars that are better suited to specific environments, especially in a climate-change scenario.