PHI-1 interacts with the catalytic subunit of myosin light chain phosphatase to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force in avian smooth muscle

In avian smooth muscles, GTPgammaS produces a Rho kinase mediated increase in PHI-1 phosphorylation and force, but whether this correlation is causal is unknown. We examined the effect of phosphorylated PHI-1 (P-PHI-1) on force and myosin light chain (MLC(20)) phosphorylation at a constant [Ca(2+)]. P-PHI-1, but not PHI-1, increased MLC(20) phosphorylation and force, and phosphorylation of PHI-1 increased the interaction of PHI-1 with PP1c. Microcystin induced a dose-dependent reduction in the binding of PHI-1 to PP1c. These results suggest PHI-1 inhibits myosin light chain phosphatase by interacting with the active site of PP1c to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force.     

Regulation of gene expression and biochemical changes in small intestine of newborn diabetic rats by exogenous ghrelin

The aim of this study was to investigate (i) the cholecystokinin, somatostatin and apelin mRNA levels, (ii) the changes in levels and localization of these peptides, (iii) relation between these peptides, (iv) antiapoptotic effects and (v) antioxidant effects of ghrelin. The rats were divided into four groups second day after birth. These groups were respectively treated with physiological saline, ghrelin (100μg/kg/day), streptozotocin (100mg/kg), ghrelin and streptozotocin. After four weeks, small intestine and blood samples were taken from rats. Cholecystokinin mRNA and peptide, somatostatin mRNA, release to duodenal lumen of apelin peptide and apelin mRNA signals decreased in ghrelin-treated diabetic rats compared to the diabetic group. There was no statistically significant difference among the four groups for somatostatin and apelin peptides. Caspase-3 signals were not observed only in diabetic group treated with ghrelin. Caspase-8 signals were increased while PCNA signals were decreased in diabetic group given ghrelin compared to diabetic group. Small intestine CAT, SOD, GP(x) and GST activities and GSH levels were decreased and LPO, PC levels were increased in diabetic rats. Administration of ghrelin to diabetic rats caused an increase in intestinal CAT, SOD, GP(x) and GST activities and GSH levels, while PC levels decreased. As a result, we observed positive changes in diabetic rats treated with ghrelin in both microscopic and biochemical studies. We can suggest that ghrelin may be an important hormone for the treatment of diabetes.     

Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis

The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2:997-1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.     

Synthesis and analgesic activity of some acetamide derivatives

In the present study, some acetamide derivatives were synthesized and their potential analgesic activities were investigated. N-(benzothiazol-2-yl)-2-[(1-substituted-1H-tetrazol-5-yl)thio]acetamide derivatives were obtained by the nucleophilic substitution reaction of 2-chloro-N-(benzothiazole-2-yl)acetamides with appropriate tetrazol-5-thioles. The chemical structures of the compounds were elucidated by IR, 1H-NMR, 13C-NMR and FAB?-MS spectral data and elemental analyses. The prepared compounds were investigated for their potential analgesic properties against thermal, mechanical and chemical nociceptive stimuli using hot-plate, tail-clip and acetic acid-induced writhing tests, respectively. The assessment of motor coordination was carried out using Rota-Rod test. Tested compounds applied at 100 mg/kg doses caused significant decrease in acetic acid-induced writhing responses and increase in hot-plate and tail-clip latencies. None of the compounds exhibited destructive effect on motor coordination of the mice in Rota-Rod performance.     

Synthesis and antioxidant activity evaluations of melatonin-based analogue indole-hydrazide/hydrazone derivatives

Melatonin (MLT) is a hormone synthesized from the pineal gland. It is a direct scavenger of free radicals, which is related to its capability to defend cells from oxidative stress. Recently MLT-related compounds are under investigation to establish which exhibit the maximum activity with the lowest side effects. In this study 5-chloroindole hydrazide/hydrazone derivatives were synthesized from 5-chloroindole-3-carboxaldehyde and phenyl hydrazine derivatives. All the compounds characterized and in vitro antioxidant activity was investigated against MLT and BHT. Most of the compounds showed strong inhibitory effect on the superoxide radical scavenging assay at 1?mM concentration (79 to 95%). Almost all the tested compounds possessed strong scavenging activity against the DPPH radical scavenging activity with IC(50) values (2 to 60 μM). Lastly, compound 1j revealed stronger inhibitory activity against MLT in the LP inhibitory assay at 0.1mM concentration (51%) while the rest of the compounds showed moderate inhibition.     

Takayasu's arteritis is associated with HLA-B*52, but not with HLA-B*51, in Turkey

Introduction

HLA-B*51 and HLA-B*52 are two close human leukocyte antigen (HLA) allele groups with minor amino acid differences. However, they are associated with two different vasculitides (HLA-B*51 in Behçet's disease and HLA-B*52 in Takayasu's arteritis (TAK)) and with major clinical and immunological differences. In this study, we aimed to screen a large cohort of TAK patients from Turkey for the presence of HLA-B*51 and HLA-B*52 as susceptibility and severity factors.

Methods

TAK patients (n = 330) followed at a total of 15 centers were included in the study. The mean age of the patients was 37.8 years, and 86% were women. DNA samples from the patients and healthy controls (HC; n = 210) were isolated, and the presence of HLA-B*51 or HLA-B*52 was screened for by using PCR with sequence-specific primers.

Results

We found a significant association of HLA-B*52 with TAK (20.9% vs HC = 6.7%, P = 0.000, OR = 3.7, 95% CI = 2.02 to 6.77). The distribution of HLA-B*51 did not differ between TAK patients and HCs (22.7% vs 24.8%, OR = 0.9, 95% CI = 0.60 to 1.34). The presence of HLA-B*52 decreased in late-onset patients (> 40 years of age; 12.0%, P = 0.024, OR = 0.43, 95% CI = 0.20 to 0.91). Patients with angiographic type I disease with limited aortic involvement also had a lower presence of HLA-B*52 compared to those with all other disease subtypes (13.1% vs 26%, P = 0.005, OR = 0.43, 95% CI = 0.23 to 0.78).

Conclusions

In this study, the previously reported association of TAK with HLA-B*52 in other populations was confirmed in patients from Turkey. The functional relevance of HLA-B*52 in TAK pathogenesis needs to be explored further.     

Misperceptions in the trajectories of objects undergoing curvilinear motion

Trajectory perception is crucial in scene understanding and action. A variety of trajectory misperceptions have been reported in the literature. In this study, we quantify earlier observations that reported distortions in the perceived shape of bilinear trajectories and in the perceived positions of their deviation. Our results show that bilinear trajectories with deviation angles smaller than 90 deg are perceived smoothed while those with deviation angles larger than 90 degrees are perceived sharpened. The sharpening effect is weaker in magnitude than the smoothing effect. We also found a correlation between the distortion of perceived trajectories and the perceived shift of their deviation point. Finally, using a dual-task paradigm, we found that reducing attentional resources allocated to the moving target causes an increase in the perceived shift of the deviation point of the trajectory. We interpret these results in the context of interactions between motion and position systems.     

Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells

This study investigated the involvement of CNP-3, chick homologue for human C-type natriuretic peptide (CNP), in TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells (MSCs). Chondrogenic differentiation of MSCs in pellet cultures was induced by TGF-β1. Chondrogenic differentiation and glycosaminoglycan synthesis were analyzed on the basis of basic histology, collagen type II expression, and Alcian blue staining. Antibodies against CNP and NPR-B were used to block their function during these processes. Results revealed that expression of CNP-3 and NPR-B in MSCs were regulated by TGF-β1 in monolayer cultures at mRNA level. In pellet cultures of MSCs, TGF-β1 successfully induced chondrogenic differentiation and glycosaminoglycan synthesis. Addition of CNP into the TGF-β1 supplemented chondrogenic differentiation medium further induced the glycosaminoglycan synthesis and hypertrophy of differentiated chondrocytes in these pellets. Pellets induced with TGF-β1 and treated with antibodies against CNP and NPR-B, did show collagen type II expression, however, Alcian blue staining showing glycosaminoglycan synthesis was significantly suppressed. In conclusion, CNP-3/NPR-B signaling may strongly be involved in synthesis of glycosaminoglycans of the chondrogenic matrix and hypertrophy of differentiated chondrocytes during TGF-β1 induced chondrogenic differentiation of MSCs.     

miR-376b controls starvation and mTOR inhibition-related autophagy by targeting ATG4C and BECN1

Macroautophagy (autophagy) is the major intracellular degradation pathway for long-lived proteins and organelles. It helps the cell to survive a spectrum of stressful conditions including starvation, growth factor deprivation and misfolded protein accumulation. Moreover, abnormalities of autophagy play a role in major health problems including cancer and neurodegenerative diseases. Yet, mechanisms controlling autophagic activity are not fully understood. Here, we describe hsa-miR-376b (miR-376b) as a new microRNA (miRNA) regulating autophagy. We showed that miR-376b expression attenuated starvation- and rapamycin-induced autophagy in MCF-7 and Huh-7 cells. We discovered autophagy proteins ATG4C and BECN1 (Beclin 1) as cellular targets of miR-376b. Indeed, upon miRNA overexpression, both mRNA and protein levels of ATG4C and BECN1 were decreased. miR-376b target sequences were present in the 3' UTR of ATG4C and BECN1 mRNAs and introduction of mutations abolished their miR-376b responsiveness. Antagomir-mediated inactivation of the endogenous miR-376b led to an increase in ATG4C and BECN1 levels. Therefore, miR-376b controls autophagy by directly regulating intracellular levels of two key autophagy proteins, ATG4C and BECN1.