Modifying L-type calcium current kinetics: consequences for cardiac excitation and arrhythmia dynamics

The L-type Ca current (I_Ca,L), essential for normal cardiac function, also regulates dynamic action potential (AP) properties that promote ventricular fibrillation. Blocking I_Ca,L can prevent ventricular fibrillation, but only at levels suppressing contractility. We speculated that, instead of blocking I_Ca,L, modifying its shape by altering kinetic features could produce equivalent anti-fibrillatory effects without depressing contractility. To test this concept experimentally, we overexpressed a mutant Ca-insensitive calmodulin (CaM₁₂₃₄) in rabbit ventricular myocytes to inhibit Ca-dependent I_Ca,L inactivation, combined with the ATP-sensitive K current agonist pinacidil or I_Ca,L blocker verapamil to maintain AP duration (APD) near control levels. Cell shortening was enhanced in pinacidil-treated myocytes, but depressed in verapamil-treated myocytes. Both combinations flattened APD restitution slope and prevented APD alternans, similar to I_Ca,L blockade. To predict the arrhythmogenic consequences, we simulated the cellular effects using a new AP model, which reproduced flattening of APD restitution slope and prevention of APD/Ca_i transient alternans but maintained a normal Ca_i transient. In simulated two-dimensional cardiac tissue, these changes prevented the arrhythmogenic spatially discordant APD/Ca_i transient alternans and spiral wave breakup. These findings provide a proof-of-concept test that I_Ca,L can be targeted to increase dynamic wave stability without depressing contractility, which may have promise as an antifibrillatory strategy.     

Alteration of superoxide- and nitric oxide-mediated antimicrobial function of macrophages by in vivo cocaine exposure

Cocaine is a popular drug of abuse and despite impressive advances in the understanding of its physiological, pharmacological, and toxic effects, its mechanism of immunosuppression at the cellular level is not well understood. In this paper we report the role of effector molecules like superoxide and nitric oxide in the antibacterial function of macrophages exposed to acute and chronic doses of cocaine in vivo. Bacterial killing by acute cocaine-exposed macrophages (ACE-Mphis) increased significantly, with a concomitant rise in respiratory burst and generation of superoxide and nitric oxide, compared with control macrophages. In contrast, chronic cocaine-exposed macrophages (CCE-Mphis) exhibited limited antimicrobial activity, which correlated closely with diminished respiratory burst and reduced production of superoxide and nitric oxide. Further, a killing assay was carried out in the presence of N(G)-methyl-L-arginine acetate, an inhibitor of iNOS, to evaluate the role of nitric oxide in the killing process. The results obtained indicate that while about 30% killing of input bacteria by control and ACE-Mphis was attributable to NO-mediated killing, only about 6% killing from NO was found with CCE-Mphis. The findings indicate that acute exposure to cocaine possibly caused upregulation of enzymes responsible for the generation of ROI (reactive oxygen intermediates) and RNI (reactive nitrogen intermediates), leading to enhanced antimicrobial function. On the other hand, chronic exposure to cocaine impaired the oxygen-dependent microbicidal capacity of macrophages, possibly through impaired expression of enzymes responsible for ROI and RNI formation. Proinflammatory cytokines may play a key role in cocaine-mediated immunosuppression, since exposure of macrophages to cocaine impairs the ability of the cells to produce these cytokines.     

Cr(VI) Imposed Toxicity in Maize Seedlings Assessed in Terms of Disruption in Carbohydrate Metabolism

The present study examined the toxic effects of Cr(VI; 100, 250 and 500 μM) in maize seedlings by investigating the changes in carbohydrate metabolism after 48, 96, and 144 h of exposure. Cr-stress results in severe alterations in the contents of carbohydrates and reducing sugars and the activities of carbohydrate metabolizing enzymes, amylases, phosphatases and phosphorylases, and invertases in maize seedlings. Under Cr stress, the contents of carbohydrates and reducing sugars declined in roots, whereas an increase was noticed in leaves. The catalytic activity of carbohydrate metabolizing enzymes, except invertases, in roots declined in the presence of Cr(VI) in a concentration- and exposure time-dependent manner. In contrast, the activities of these enzymes were enhanced in leaves under Cr(VI) stress. The activity of invertases increased with increasing amount of Cr(VI) but declined with an increase in the time interval. In conclusion, our results show that carbohydrate metabolism is severely affected under Cr(VI) toxicity. The study suggests that Cr-induced perturbations in the carbohydrate metabolism are one of the factors resulting in growth inhibition under Cr(VI) stress.     

Pancreatitis severity in mice with impaired CFTR function but pancreatic sufficiency is mediated via ductal and inflammatory cells-Not acinar cells

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) are an established risk factor for cystic fibrosis (CF) and chronic pancreatitis. Whereas patients with CF usually develop complete exocrine pancreatic insufficiency, pancreatitis patients with CFTR mutations have mostly preserved exocrine pancreatic function. We therefore used a strain of transgenic mice with significant residual CFTR function (CFTR^tm1HGU) to induce pancreatitis experimentally by serial caerulein injections. Protease activation and necrosis were investigated in isolated acini, disease severity over 24h, pancreatic function by MRI, isolated duct stimulation and faecal chymotrypsin, and leucocyte function by ex vivo lipopolysaccharide (LPS) stimulation. Pancreatic and lung injury were more severe in CFTR^tm1HGU but intrapancreatic trypsin and serum enzyme activities higher than in wild-type controls only at 8h, a time interval previously attributed to leucocyte infiltration. CCK-induced trypsin activation and necrosis in acini from CFTR^tm1HGU did not differ from controls. Fluid and bicarbonate secretion were greatly impaired, whereas faecal chymotrypsin remained unchanged. LPS stimulation of splenocytes from CFTR^tm1HGU resulted in increased INF-γ and IL-6, but decreased IL-10 secretion. CFTR mutations that preserve residual pancreatic function significantly increase the severity of experimental pancreatitis—mostly via impairing duct cell function and a shift towards a pro-inflammatory phenotype, not by rendering acinar cells more susceptible to pathological stimuli.     

Identification of a 148-kDa surface lectin from Giardia lamblia with specificity for α-methyl-d-mannoside

Abstract A lectin specific for α-methyl-d-mannoside was purified from the membrane extract of Giardia lamblia by a combination of gel filtration chromatography on Sephadex G-75 and Superose 6-HR 10/30. The homogeneity of the lectin was established by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass of the native protein was 148 kDa. The lectin agglutinated rabbit erythrocytes in the presence of Ca²⁺ at 37 °C and pH 7.O. The maximum activity of the lectin was obtained after trypsin treatment. The inhibition study clearly suggests that the binding site of the lectin recognizes α-methyl-d-mannoside as the immunodominant sugar.     

1-Halo analogs of dihydroxyacetone 3-phosphate. The effects of the fluoro analog on cytosolic glycerol-3-phosphate dehydrogenase and triosephosphate isomerase.

Fluoro-o-hydorxyacetone phosphate (fluoroacetol phosphate) has been prepared by oxidation of 1-fluoro-3-chloro-2-propanol to 1-fluoro-3-chloroacetone, phosphorylation with silver dibenzylphosphate, and the intermediate isolation of 1-fluoro-3-hydroxyacetone phosphate dibenzyl ester, followed by catalytic hydrogenation and preparation of the stable monosodium salt. The chloro analog as the pure, stable monosodium salt has been prepared by a similar route from 1,3-dichloroacetone. 1-Fluoro-3-hydroxyacetone-P is substrate for cytosolic NAD+-linked glycerol-3-P dehydrogenese (EC 1.1.1.8) from rabbit skeletal muscle with an apparent Km of 50 mM under conditions in which dihydroxyacetone-P exhibits an apparent Km of 0.15 mM. Under these conditions the fluoro analog is 85% hydrated wheras dihydroxyacetone-P has been shown by others to be 44% hydrated. The turnover numbers are 49,000 molecules of NADH oxidized per minute per molecule of enzyme at 25 degrees with the fluoro analog as substrate, and 60,000 with dihydrocyacetone-P as substrate. The product of the reduction of the fluoro analog has been identified as 1-fluorodeoxyglycerol-3-P. 1-Fluoro-3-hydroxyacetone-P is comparatively weak irreversible inhibitor at 4 degrees of rabbit muscle triosephosphate isomerase (EC 5.3.1.1) with second-order rate constant of 2.6 M minus 1 sec minus 1. Inhibition by pyrazole in vivo of alcohol dehydrogenese catalyzed oxidation of 1-fluorodeoxyglecerol-3-P indicates in mice the reduction of 1-fluoro-3-hydroxyacetone-P to -l-1-fluorodexoxyglycerol-3-P is not significant metabolic route, or that an alternative route exists when the alcohol dehydrogenase dependent pathway is inhibited.     

Pediatric glioblastoma cells inhibit neurogenesis and promote astrogenesis,phenotypic transformation and migration of human neural progenitor cells within cocultures

Neural progenitor cell (NPC) fate is influenced by a variety of biological cues elicited from the surrounding microenvironment and recent studies suggest their possible role in pediatric glioblastoma multiforme (GBM) development. Since a few GBM cells also display NPC characteristics, it is not clear whether NPCs transform to tumor cell phenotype leading to the onset of GBM formation, or NPCs migrate to developing tumor sites in response to paracrine signaling from GBM cells. Elucidating the paracrine interactions between GBM cells and NPCs in vivo is challenging due to the inherent complexity of the CNS. Here, we investigated the interactions between human NPCs (ReNcell) and human pediatric GBM-derived cells (SJ-GBM2) using a Transwell^® coculture setup to assess the effects of GBM cells on ReNcells (cytokine and chemokine release, viability, phenotype, differentiation, migration). Standalone ReNcell or GBM cultures served as controls. Qualitative and quantitative results from ELISA^®, Live/Dead^® and BrdU assays, immunofluorescence labeling, western blot analysis, and scratch test suggests that although ReNcell viability remained unaffected in the presence of pediatric GBM cells, their morphology, phenotype, differentiation patterns, neurite outgrowth, migration patterns (average speed, distance, number of cells) and GSK-3β expression were significantly influenced. The cumulative distance migrated by the cells in each condition was fit to Furth's formula, derived formally from Ornstein-Uhlenbeck process. ReNcell differentiation into neural lineage was compromised and astrogenesis promoted within cocultures. Such coculture platform could be extended to identify the specific molecules contributing to the observed phenomena, to investigate whether NPCs could be transplanted to replace lesions of excised tumor sites, and to elucidate the underlying molecular pathways involved in GBM-NPC interactions within the tumor microenvironment.     

Characterisation of wheat germplasm for slow rust resistance against Puccinia striiformis

Thirty-seven wheat germplasm were screened under artificial epiphytotic conditions against stripe rust of wheat at University Research Farm, Chatha, during Rabi, 2013–2014 and 2014–2015. On the basis of final rust severity (FRS), AURPC (Area Under Rust Progress Curve) and CI (Coefficient of Infection), 8 genotypes ((DWR 16, HD2281, VL616, K65, UP2121, HD2329, HD2307 and Lal Bahadur) exhibited partial resistance against the disease. PCR profiles revealed co-dominant pattern with distinct fragment of 150 bp in 14 germplasm, showing the presence of Yr18 gene and 229 bp band in 16 germplasm exhibited absence of Yr18.     

Genetic Mapping in Escherichia coli K-12 by Radiation-Induced Crossing-Over

Conventional methods for chromosomal mapping in Escherichia coli are (i) interruption of matings to obtain minimum marker entry times, (ii) linkage analysis of recombinants, and (iii) cotransduction. Method (i) has a resolution of about 0.5 min (5 x 10(4) nucleotides) and is not useful for distances less than about 1 min; methods (ii) and (iii) are capable of better resolution but are generally not very reproducible and no general theory is available for translating crossing-over and cotransduction frequencies into physical chromosomal distances. We found that when merozygotes are irradiated (X rays or ultraviolet light) soon after marker transfer, high linkage values (0.8 to 1.0) between nearby marker pairs decrease with radiation dose to 0.5. Our results are quantitatively consistent with the idea that radiations induce crossing-over lesions proportional to dose, and the number of such lesions between two markers is proportional to the physical separation of the markers in the range that can also be measured by interruption of mating (0.5 to 4.0 min). Additivity relations among markers are also satisfied. We used this technique to measure the distances (0.1 to 1.0 min) between several pairs of closely linked markers.