CRYAB and HSPB2 deficiency alters cardiac metabolism and paradoxically confers protection against myocardial ischemia in aging mice

The abundantly expressed small molecular weight proteins, CRYAB and HSPB2, have been implicated in cardioprotection ex vivo. However, the biological roles of CRYAB/HSPB2 coexpression for either ischemic preconditioning and/or protection in situ remain poorly defined. Wild-type (WT) and age-matched ( approximately 5-9 mo) CRYAB/HSPB2 double knockout (DKO) mice were subjected either to 30 min of coronary occlusion and 24 h of reperfusion in situ or preconditioned with a 4-min coronary occlusion/4-min reperfusion x 6, before similar ischemic challenge (ischemic preconditioning). Additionally, WT and DKO mice were subjected to 30 min of global ischemia in isolated hearts ex vivo. All experimental groups were assessed for area at risk and infarct size. Mitochondrial respiration was analyzed in isolated permeabilized cardiac skinned fibers. As a result, DKO mice modestly altered heat shock protein expression. Surprisingly, infarct size in situ was reduced by 35% in hearts of DKO compared with WT mice (38.8 +/- 17.9 vs. 59.8 +/- 10.6% area at risk, P < 0.05). In DKO mice, ischemic preconditioning was additive to its infarct-sparing phenotype. Similarly, infarct size after ischemia and reperfusion ex vivo was decreased and the production of superoxide and creatine kinase release was decreased in DKO compared with WT mice (P < 0.05). In permeabilized fibers, ADP-stimulated respiration rates were modestly reduced and calcium-dependent ATP synthesis was abrogated in DKO compared with WT mice. In conclusion, contrary to expectation, our findings demonstrate that CRYAB and HSPB2 deficiency induces profound adaptations that are related to 1) a reduction in calcium-dependent metabolism/respiration, including ATP production, and 2) decreased superoxide production during reperfusion. We discuss the implications of these disparate results in the context of phenotypic responses reported for CRYAB/HSPB2-deficient mice to different ischemic challenges.     

Identification and in silico analysis of functional SNPs of the BRCA1 gene

Single-nucleotide polymorphisms (SNPs) play a major role in the understanding of the genetic basis of many complex human diseases. Also, the genetics of human phenotype variation could be understood by knowing the functions of these SNPs. It is still a major challenge to identify the functional SNPs in a disease-related gene. In this work, we have analyzed the genetic variation that can alter the expression and the function of the BRCA1 gene using computational methods. Of the total 477 SNPs, 65 were found to be nonsynonymous (ns) SNPs. Among the 14 SNPs in the untranslated region, 4 were found in the 5' and 10 were found in the 3' untranslated region (UTR). It was found that 16.9% of the nsSNPs were damaging, by both the SIFT and the PolyPhen servers. The UTR Resource tool suggested that 2 of 4 SNPs in the 5' UTR and 3 of 10 SNPs in the 3' UTR might change the protein expression levels. We identified major mutations from proline to serine at positions 1776 and 1812 of the native protein of the BRCA1 gene. From a comparison of the stabilizing residues of the native and mutant proteins, we propose that an nsSNP (rs1800751) could be an important candidate for the breast cancer caused by the BRCA1 gene.     

In silico detection of binding mode of J-superfamily conotoxin pl14a with Kv1.6 channel

A novel conotoxin pl14a containing 25 amino acid residues with an amidated C-terminus from vermivorous cone snail, Conus planorbis belongs to J-conotoxin superfamily and this is the first conotoxin, which inhibits both nicotinic acetylcholine receptor subtypes and Kv1.6 channel. We have attempted through bioinformatics approaches to elucidate the extent of specificity of pl14a towards Kv1 channel subtypes (Kv1.1-Kv1.6). Our work provides rationale for the relatively high specificity and binding mode of pl14a to Kv1.6 channel. The pl14a peptide contains two types of structural elements, namely the putative dyad (Lys18 and Tyr19) and basic residue ring constituted of arginine residues. We have carried out in silico docking studies so as to assess the contribution of one or combination of both structural elements of pl14a in blocking of Kv1.6 channel. For this purpose, we have built by homology modelling, the theoretical 3D structure of Kv1.6 channel based on the available crystal structure of mammalian shaker Kv1.2 channel. Docking studies suggest that positively charged residues ring may be involved in the blocking mechanism of Kv1.6 channel. The models suggest that the peptide interacts with negatively charged extracellular loops and pore-mouth of the potassium channel and blocks the channel by covering the pore as a lid, akin to previously proposed blocking mechanism of kappaM-conotoxin RIIIK from Conus radiatus to Tsha1 potassium channel. The newly detected pharmacophore for pl14a interacting with Kv1.6 channel provides a pointer to experimental work to validate the observations made here. Based on differences in the number and distribution of the positively-charged residues in other conopeptides from the J-superfamily, we hypothesize different selectivity profiles against subtypes of the potassium channels for these conopeptides.     

Natural variability of minimotifs in 1092 people indicates that minimotifs are targets of evolution

Since the function of a short contiguous peptide minimotif can be introduced or eliminated by a single point mutation, these functional elements may be a source of human variation and a target of selection. We analyzed the variability of ∼300 000 minimotifs in 1092 human genomes from the 1000 Genomes Project. Most minimotifs have been purified by selection, with a 94% invariance, which supports important functional roles for minimotifs. Minimotifs are generally under negative selection, possessing high genomic evolutionary rate profiling (GERP) and sitewise likelihood-ratio (SLR) scores. Some are subject to neutral drift or positive selection, similar to coding regions. Most SNPs in minimotif were common variants, but with minor allele frequencies generally <10%. This was supported by low substation rates and few newly derived minimotifs. Several minimotif alleles showed different intercontinental and regional geographic distributions, strongly suggesting a role for minimotifs in adaptive evolution. We also note that 4% of PTM minimotif sites in histone tails were common variants, which has the potential to differentially affect DNA packaging among individuals. In conclusion, minimotifs are a source of functional genetic variation in the human population; thus, they are likely to be an important target of selection and evolution.     

Enhancement of the anti‐inflammatory activity of temporin‐1Tl‐derived antimicrobial peptides by tryptophan,arginine and lysine substitutions

Temporin‐1Tl (TL) is a 13‐residue frog antimicrobial peptide (AMP) exhibiting potent antimicrobial and anti‐inflammatory activity. To develop novel AMP with improved anti‐inflammatory activity and antimicrobial selectivity, we designed and synthesized a series of TL analogs by substituting Trp, Arg and Lys at selected positions. Except for Escherichia coli and Staphylococcus epidermidis, all TL analogs exhibited retained or increased antimicrobial activity against seven bacterial strains including three methicillin‐resistant Staphylococcus aureus strains compared with TL. TL‐1 and TL‐4 showed a little increase in antimicrobial selectivity, while TL‐2 and TL‐3 displayed slightly decreased antimicrobial selectivity because of their about twofold increased hemolytic activity. All TL analogs demonstrated greatly increased anti‐inflammatory activity, evident by their higher inhibition of the production tumor necrosis factor‐α (TNF‐α) and nitric oxide and the mRNA expression of inducible nitric oxide synthase and TNF‐α in lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophage cells, compared with TL. Taken together, the peptide anti‐inflammatory activity is as follows: TL‐2 ≈ TL‐3 ≈ TL‐4 > TL‐1 > TL. In addition, LPS binding ability of the peptides corresponded with their anti‐inflammatory activity. These results apparently suggest that the anti‐inflammatory activity of TL analogs is associated with the direct binding ability between these peptides and LPS. Collectively, our designed TL analogs possess improved anti‐inflammatory activity and retain antimicrobial activity without a significant increase in hemolysis. Therefore, it is evident that our TL analogs constitute promising candidates for the development of peptide therapeutics for gram‐negative bacterial infection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Endogenous and exogenous ethylene induces needle abscission and cellulase activity in post-harvest balsam fir (Abies balsamea L.)

Post-harvest needle loss is a major problem for balsam fir and other Christmas tree species. Recent evidence has implicated ethylene as a signal responsible for post-harvest needle abscission, but enzymological changes remain unknown. The objective of this study was to identify and quantify cellulase activity associated with endogenous and exogenous ethylene-induced abscission. An experiment was designed with three treatments (control, endogenous ethylene, or exogenous ethylene) with five replicates. Key response variables include needle retention duration, xylem pressure potential, ethylene evolution rate, and cellulase activity. Two complimentary methods were used to assess cellulase activity: a cellulose plate digestion and zymography. The results confirm ethylene as a signal for post-harvest abscission and identify ethylene-induced cellulase. Ethylene evolution was typically between 15 and 16 μL g⁻¹ h⁻¹, but there was no difference among the three treatments. However, exogenous ethylene significantly decreased needle retention by 60% and resulted in a sixfold decrease in xylem pressure potential. In addition, cellulase activity increased by 8- and 12-fold in endogenous and exogenous ethylene-induced abscission, respectively, compared to the control. Identification of ethylene-induced cellulase activity has increased our understanding of the post-harvest needle abscission process and confirms ethylene’s role as a signal molecule.     

Synthesis of polyfunctionalized piperidone oxime ethers and their cytotoxicity on HeLa cells

A series of twenty 2,6-diarylpiperidin-4-one O-methyloximes were synthesized with fluoro/chloro/bromo/methyl/methoxy/ethoxy/isopropyl substituents on various positions of the phenyl at C-2 and C-6 in association with/without methyl substituent on the secondary amino group and methyl/ethyl/isopropyl substituents on the active methylene centers. Regardless of their substitution all compounds predominantly exist in the chair conformation except 3m, which adopts a twist-boat conformation. All the synthesized compounds were evaluated for their in vitro antiproliferative activity against human cervical carcinoma (HeLa) cell line. The cytotoxicity of the test compounds was determined by measuring the number of live cells after 24 h of treatment by MTT assay method. This preliminary SAR suggests some lead molecules 3c-f, 3j-k, 4d-g, and 4i with a scope of further structural optimization of the piperidone pharmacophore toward the development of anticancer drug synthesis.     

Renal clear-cell carcinoma: an ultrastructural study on the junctional complexes

Junctional complexes such as tight junctions, adherens junctions, and desmosomes play crucial roles in the structure and function of epithelial cells. These junctions are involved in increasing cell-cell contact and as well serve as signaling centers regulating multiple functions in epithelial cells. Carcinoma cell lines cultured in the laboratory generally lack junctional complexes. However, studies directed towards understanding the distribution of junctional complexes in human cancer tissues are lacking. In this study, we analyzed by electron microscopy the distribution of junctional complexes in patients diagnosed with renal clear-cell carcinoma. We found that both tight junctions and adherens junctions were drastically reduced in patients with cancer compared to normal tissues. Desmosomes were not detected in normal proximal tubules while distinctly present in cancer tissues. These results suggest that analysis of junctional complexes in human tumors should provide valuable information that might have prognostic and diagnostic value.     

Mineral contents of Aloe vera leaf gel and their role on streptozotocin-induced diabetic rats

The role of some inorganic elements like vanadium, zinc, sodium, potassium, calcium, copper, manganese, and traces of chromium in the improvement of impaired glucose tolerance and their indirect role in the management of diabetes mellitus are being increasingly recognized. In traditional methods, medicinal plants are being used, which contain both organic and inorganic constituents. In the present study, an attempt has been made to analyze the inorganic elements present in Aloe vera leat gel and their role on diabetes-related biochemical alterations in experimental rats. Special emphasis was given to the inorganic parts by carefully preparing ash of the leaf gel. The results clearly indicate the presence of several hypoglycemic-activity-possessing elements in the gel. The ash treatment also resulted in hypoglycemic action. In conclusion, the presence of various inorganic trace elements in the gel might account for the hypoglycemic nature of the plant.