Functional interaction of the adenovirus IVa2 protein with adenovirus type 5 packaging sequences

Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.     

Mast cell stabilizator and antioxidant effects of epidermal growth factor (EGF) on gastric mucosal injury induced by ethanol in rats

The role of epidermal growth factor (EGF), a polypeptide containing 53 amino acids, on protection and repair of ethanol-induced gastric mucosal injury was investigated in rats. In addition, the effects of EGF on the gastric damage were evaluated histopathologically. We used 48 Spraque-Dawley rats which were divided into [corrected] three groups as control rats, ethanol treated rats and ethanol+EGF treated rats. The ethanol group was given a gastric gavage containing 1 ml of 80% ethanol (v/v) prepared in distilled water. EGF (100 microg/kg) was given by intragastric gavage 30 min before the administration of ethanol. We studied histopathological evaluation and the histochemical heterogeneity of mast cells and its degree of degranulation. Besides, gastric tissue malondialdehyde (MDA), protein sulfhydryl groups (SH), and protein carbonyl levels were measured. EGF treatment stabilized mast cells degranulation and had lower polymorphonuclear leukocytes (PMNL) infiltration, ulcer index, histamine, and MDA; protein carbonyl levels were also lower, compared to the non-treated animals. EGF exerts a protective effect on gastric mucosa to ethanol-induced gastric injury probably through antioxidant and mast cell stabilizing mechanism.     

Periostin Limits Tumor Response to VEGFA Inhibition

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Use of BODIPY‐Cholesterol (TF‐Chol) for Visualizing Lysosomal Cholesterol Accumulation

Dipyrromethene difluoride‐cholesterol (TopFluor‐Cholesterol, TF‐Chol) is a widely used cholesterol analogue due to its excellent fluorescence properties and considerable similarity with natural cholesterol in terms of membrane partitioning. However, the suitability of TF‐Chol for detecting lysosomal cholesterol deposition has recently been questioned. Here, we highlight the fact that the method of lipid delivery and the analysis of time‐point both affect the membrane distribution and labeling pattern of TF‐Chol, similarly as with radiolabeled cholesterol. Lysosomal sterol accumulation characteristic to a lysosomal storage disease is most readily detected when the probe is introduced via the physiological route, i.e. as a sterol fatty acid ester in low‐density lipoprotein particles. When administered to cells from solvent, lysosomal sterol sequestration becomes evident after an overnight equilibration between membranes.      

Synthesis and characterization of cyclodextrin-based polymers as a support for immobilization of Candida rugosa lipase

The objective of this study was to prepare cross-linked β-cyclodextrin polymers for immobilization of Candida rugosa lipase. The structures of synthesized macrocyclic compounds were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. Properties of the immobilized systems were assessed and their performance on hydrolytic reaction were evaluated and compared with the free enzyme. The influence of activation agents (glutaraldehyde (GA) and hexamethylene diisocyanate (HMDI)) and thermal and pH stabilities of the biocatalyst was evaluated. After the optimization of immobilization process, the physical and chemical characterization of immobilized lipase was performed. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme. It can be observed that the free lipase loses its initial activity within around 80 min at 60 °C, while the immobilized lipases retain their initial activities of about 56% by HMDI and 82% by GA after 120 min of heat treatment at 60 °C.Results showed that the specific activity of the immobilized lipase with glutaraldehyde was 62.75 U/mg protein, which is 28.13 times higher than that of the immobilized lipase with HMDI.     

Signals,Regulatory Networks,and Materials That Build and Break Bacterial Biofilms

Summary: Biofilms are communities of microorganisms that live attached to surfaces. Biofilm formation has received much attention in the last decade, as it has become clear that virtually all types of bacteria can form biofilms and that this may be the preferred mode of bacterial existence in nature. Our current understanding of biofilm formation is based on numerous studies of myriad bacterial species. Here, we review a portion of this large body of work including the environmental signals and signaling pathways that regulate biofilm formation, the components of the biofilm matrix, and the mechanisms and regulation of biofilm dispersal.     

Changes in peroxidase activities and soluble proteins in strawberry varieties under salt-stress

Effects of salt stress on the activity of peroxidase (PRX) isozyme and leaf proteins were studied in the three strawberry varieties, Camarosa, Tioga and Chandler. Plants were watered with modified 1/3 Hoagland nutrient solution containing 0 (control), 8.5, 17.0 and 34.0 mM NaCl for 30 days fallowing 20 days acclimation. PRX activity was assayed in leaf extracts and PRX profiles of control and salt treated plants were performed by native polyacrylamide gel electrophoresis (PAGE). During the salt stress Tioga and Camarosa tolerated the cellular damage with less electrolyte leakage while Chandler exhibited a severe cellular damage with the highest (87.5 % in 34.0 mM NaCl treatment) electrolyte leakage. Total soluble protein content was decreased by salinity in Tioga (from 11.35 to 5.86 mg g⁻¹ DW) and Chandler (from 9.35 to 3.90 mg g⁻¹ DW) while it was almost unchanged in Camarosa. Salt stress increased total and specific PRX activity as compared with the control in all the varieties. In native PAGE, one basic isoperoxidase band (Rf=0.27) was observed commonly with different band intensity in all the treatments. Therefore, it might be associated with lignification and recovery of cell membrane damage in strawberry plants under salt stress.     

In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells

Previous studies in several laboratories have demonstrated inadvertent chromosomal abnormalities in long-term cultured human embryonic stem cells (HESC). Here, using a two-step selection process we report a functional adaptation of a HESC line, HS181, towards a decreased dependence of extra cellular matrix (ECM) for in vitro survival, that is for growth directly onto a plastic surface. Successful adaptation was paralleled with a karyotype change in 100% of the cells to 47,XX,del(7)(q11.2),+i(12)(p10). The resulting adapted population showed increased survival and growth on plastic and also maintained expression of HESC markers, but showed a decreased pluripotency, as demonstrated by results from embryoid body (EB) formation in vitro. The finding of reduced pluripotency may not be totally unexpected since the variant cells were selected for self-renewal and proliferation, not differentiation during the adaptation to growth on plastic. In the light of recent models of a germ cell origin of HESC it is of particular interest that similar to many of the reported spontaneous HESC mutants, one of the identified specific chromosome abnormalities, i(12p), has also been strongly implicated for human germ cell cancer. However, the mutated HESC variant carrying this mutation failed to grow as a xeno-graft in a mouse model in vivo. This is surprising and needs a further mechanistic analysis for its explanation. Increased knowledge of genetic integrity of HESC may have significance on the understanding of mechanisms for tumor progression and thus strategy for treatments, particularly for tumors occurring in early life.     

The effects of desferrioxamine and quercetin on hepatic ischemia-reperfusion induced renal disturbance

BACKGROUND: The aim of this study was to analyze the effects of 45min of hepatic ischemia and 1h of reperfusion on renal oxidative stress parameters, on renal tissue damage, and the role of Desferrioxamin (Dfx) and Q on these parameters. METHODS: Thirty Wistar albino rats were randomized to five groups. Group I was the control group. Group II received no treatment. Groups III and IV received intramuscular injections of desferrioxamine (100mg/kg) and quercetin (50mg/kg), respectively. Group V was administered Dfx and quercetin in combination. After treatment for 3 days, groups II, III, IV, and V were exposed to total hepatic ischemia for 45min. Plasma alanine aminotransferase levels, renal malondialdehyde and reduced glutathione (GSH) activities were measured after reperfusion for 1h. Histopathological and ultrastructural analysis of renal tissues was carried out. RESULTS: Plasma creatinine and BUN levels were markedly increased in the IR group and pretreated groups. Kidney MDA increased in the IR group, Q and Dfx+Q significantly decreased kidney MDA Kidney GSH levels markedly decreased in the IR group, Dfx significantly increased kidney GSH. No evidence of overt injury was observed in any renal tissue under light and electron microscopy. CONCLUSIONS: Our data demonstrated that 45min of hepatic ischemia and 1h of reperfusion may alter renal functions and may cause oxidative stress on renal tissue. Q and Dfx seem to have a beneficial effect via the GSH system and modulation of MDA levels.     

Mutations of the Gene Encoding Otogelin Are a Cause of Autosomal-Recessive Nonsyndromic Moderate Hearing Impairment

Already 40 genes have been identified for autosomal-recessive nonsyndromic hearing impairment (arNSHI); however, many more genes are still to be identified. In a Dutch family segregating arNSHI, homozygosity mapping revealed a 2.4 Mb homozygous region on chromosome 11 in p15.1-15.2, which partially overlapped with the previously described DFNB18 locus. However, no putative pathogenic variants were found in USH1C, the gene mutated in DFNB18 hearing impairment. The homozygous region contained 12 additional annotated genes including OTOG, the gene encoding otogelin, a component of the tectorial membrane. It is thought that otogelin contributes to the stability and strength of this membrane through interaction or stabilization of its constituent fibers. The murine orthologous gene was already known to cause hearing loss when defective. Analysis of OTOG in the Dutch family revealed a homozygous 1 bp deletion, c.5508delC, which leads to a shift in the reading frame and a premature stop codon, p.Ala1838ProfsX31. Further screening of 60 unrelated probands from Spanish arNSHI families detected compound heterozygous OTOG mutations in one family, c.6347C>T (p.Pro2116Leu) and c. 6559C>T (p.Arg2187X). The missense mutation p.Pro2116Leu affects a highly conserved residue in the fourth von Willebrand factor type D domain of otogelin. The subjects with OTOG mutations have a moderate hearing impairment, which can be associated with vestibular dysfunction. The flat to shallow “U” or slightly downsloping shaped audiograms closely resembled audiograms of individuals with recessive mutations in the gene encoding α-tectorin, another component of the tectorial membrane. This distinctive phenotype may represent a clue to orientate the molecular diagnosis.