News in fusion

Syncytial muscles arise by the fusion of mononucleated myoblasts. Main cellular events during the fusion of mammalian and Drosophila myoblasts are the recognition and adhesion of myoblasts, F‐actin polymerization, formation of the fusion pore, blending of the cytoplasm and the integration of the fusing myoblast into the growing myotube. During the last twenty years many key players of myoblast fusion have been identified in the model organism Drosophila melanogaster. However, none of these proteins showed fusogenic characteristics. During the last five years, two new proteins have been identified in mice that control membrane remodeling and that possess fusogenic properties. These proteins might in the future help to increase our knowledge about the fundamental mechanism of myoblast fusion.     

Transformation of the dermal collagen framework under laser heating

The aim of this study was to compare between the changes undergone by the dermal collagen framework when heated by IR laser radiation and by traditional means and to reveal the specific features of the dermal matrix modification under moderate IR laser irradiation. Rabbit skin specimens were heated to 50°C, 55°C, 60°C and 65°C in a calorimeter furnace and with a 1.68‐μm fiber Raman laser. The proportion of the degraded collagen macromolecules was determined by differential scanning calorimetry. Changes in the architectonics of the collagen framework were revealed by using standard, phase‐contrast, polarization optical and scanning electron microscopy techniques. The collagen denaturation and dermal matrix amorphization temperature in the case of laser heating proved to be lower by 10°C than that for heating in the calorimeter furnace. The IR laser treatment of the skin was found to cause a specific low‐temperature (45°C‐50°C) transformation of its collagen framework, with some collagen macromolecules remaining intact. The transformation reduces to the splitting of collagen bundles and distortion of the course of collagen fibers. The denaturation of collagen macromolecules in the case of traditional heating takes its course in a threshold manner, so that their pre‐denaturation morphological changes are insignificant.     

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

Background

Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6^th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells.

Results

Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 μg/100 g) were found in cells treated with 1.0 mM CdCl₂ and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl₂ gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 μg/100 g) were detected in the cell cultures collected at day 6.

Conclusions

As a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.     

Localization of pan-cadherin immunoreactivity in adult rat tissues

Cadherins, being responsible for selective cell recognition and normal tissue integrity in adults, regulate morphogenesis in a variety of organs during development. In this study, anti-rat pan-cadherin antibody, specific to all subgroups of the cadherin family, was used to map the distribution of the pan-cadherin immunoreactivity in adult rat organs. Pan-cadherin immunoreactivity positive tissues were: secretory cells of the adenohypophysis, autonomic nerve, corneal epithelium, oesophageal nerve plexus, stomach and pyloric glandular cells, epithelium of the ileum and its nerve plexus, alveolar cells of the lung, proximal convoluted tubules of the kidney, islet cells of Langerhans, and the acinar cells of the exocrine pancreas. For the first time, positive pan-cadherin immunoreactivity was demonstrated in the epithelial cells of the corpus ciliaris and in the nerve plexus of corpus cavernosum of the penis. In conclusion, our results suggest that cells in many tissues and organs of the adult rat synthesize cadherins.     

ATXN2 and Its Neighbouring Gene SH2B3 Are Associated with Increased ALS Risk in the Turkish Population

Expansions of the polyglutamine (polyQ) domain (≥34) in Ataxin-2 (ATXN2) are the primary cause of spinocerebellar ataxia type 2 (SCA2). Recent studies reported that intermediate-length (27–33) expansions increase the risk of Amyotrophic Lateral Sclerosis (ALS) in 1–4% of cases in diverse populations. This study investigates the Turkish population with respect to ALS risk, genotyping 158 sporadic, 78 familial patients and 420 neurologically healthy controls. We re-assessed the effect of ATXN2 expansions and extended the analysis for the first time to cover the ATXN2 locus with 18 Single Nucleotide Polymorphisms (SNPs) and their haplotypes. In accordance with other studies, our results confirmed that 31–32 polyQ repeats in the ATXN2 gene are associated with risk of developing ALS in 1.7% of the Turkish ALS cohort (p = 0.0172). Additionally, a significant association of a 136 kb haplotype block across the ATXN2 and SH2B3 genes was found in 19.4% of a subset of our ALS cohort and in 10.1% of the controls (p = 0.0057, OR: 2.23). ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observations suggest that genotyping of SNPs at this locus may be useful for the study of ALS risk in a high percentage of individuals and that ATXN2 and SH2B3 variants may interact in modulating the disease pathway.     

Threonine-120 phosphorylation regulated by phosphoinositide-3-kinase/Akt and mammalian target of rapamycin pathway signaling limits the antitumor activity of mammalian sterile 20-like kinase 1

Mst1/Stk4, a hippo-like serine-threonine kinase, is implicated in many cancers, including prostate cancer. However, the mechanisms regulating Mst1 remain obscure. Here, we characterized the effects of phospho-Thr-120 on Mst1 in prostate cancer cells. We demonstrated that phospho-Thr-120 did not alter the nuclear localization or cleavage of Mst1 in a LNCaP or castration-resistant C4-2 prostate tumor cell model, as revealed by a mutagenesis approach. Phospho-Thr-120 appeared to be specific to cancer cells and predominantly localized in the nucleus. In contrast, phospho-Thr-183, a critical regulator of Mst1 cell death, was exclusively found in the cytoplasm. As assessed by immunohistochemistry, a similar distribution of phospho-Mst1-Thr-120/Thr-183 was also observed in a prostate cancer specimen. In addition, the blockade of PI3K signaling by a small molecule inhibitor, LY294002, increased cytoplasmic phospho-Mst1-Thr-183 without having a significant effect on nuclear phospho-Mst1-Thr-120. However, the attenuation of mammalian target of rapamycin (mTOR) activity by a selective pharmacologic inhibitor, Ku0063794 or CCI-779, caused the up-regulation of nuclear phospho-Mst1-Thr-120 without affecting cytoplasmic phospho-Mst1-Thr-183. This suggests that PI3K and mTOR pathway signaling differentially regulate phospho-Mst1-Thr-120/Thr-183. Moreover, mutagenesis and RNAi data revealed that phospho-Thr-120 resulted in C4-2 cell resistance to mTOR inhibition and reduced the Mst1 suppression of cell growth and androgen receptor-driven gene expression. Collectively, these findings indicate that phospho-Thr-120 leads to the loss of Mst1 functions, supporting cancer cell growth and survival.     

Low doses of selenium specifically stimulate the repair of oxidative DNA damage in LNCaP prostate cancer cells

Epidemiological studies have demonstrated an inverse relationship between selenium (Se) intake and cancer incidence and/or mortality. However, the molecular mechanisms underlying the cancer chemopreventive activity of Se compounds remain largely unknown. The objective of this study was to investigate the effect of low doses of Se on the stimulation of DNA repair systems in response to four different qualities of DNA damage. P53-proficient LNCaP human prostate adenocarcinoma cells were grown either untreated or in the presence of low concentrations of two Se compounds (30° nM sodium selenite, or 10 μM selenomethionine) and exposed to UVA, H2O2, methylmethane sulfonate (MMS) or UVC. Cell viability as well as DNA damage induction and repair were evaluated by the alkaline Comet assay. Overall, Se was shown to be a very potent protector against cell toxicity and genotoxicity induced by oxidative stress (UVA or H2O2) but not from the agents that induce other types of deleterious lesions (MMS or UVC). Furthermore, Se-treated cells exhibited increased oxidative DNA repair activity, indicating a novel mechanism of Se action. Therefore, the benefits of Se could be explained by a combination of antioxidant activity, the reduction in DNA damage and the enhancement of oxidative DNA repair capacity.     

The role of lipids for the functional integrity of porin: an FTIR study using lipid and protein reporter groups

We have investigated the temperature-dependent interaction of the porins OmpF from Paracoccus denitrificans and OmpG from Escherichia coli with lipid molecules after reconstitution in lecithin. Effects of incubation at increased temperatures on activity were tested by functional experiments for OmpG and compared with previously published results of OmpF in order to understand the activity loss of OmpF with monomerization. Protein-lipid interaction was monitored by different reporter groups both from lipid molecules and from protein. OmpF loses its activity by approximately 90% at 50 degrees C while OmpG does not show a temperature-dependent change in activity between room temperature and 50 degrees C. The interaction between OmpF and lipid molecules is severely altered in a two-step mechanism at 55 and approximately 75 degrees C for OmpF. The first step is attributed to changes in the degree of interaction between the aromatic girdle of OmpF and the interfacial region of the lipid bilayer, leading to monomerization of this trimeric porin. The second step at 75 degrees C is attributed to the changes in lipid-porin monomer interaction. Around 90 degrees C, reconstituted porin aggregates. For OmpG, changes in lipid-protein interaction were observed starting from approximately 80 degrees C because of temperature-induced breakdown of its folding. This study provides deeper understanding of porin-lipid bilayer interaction as a function of temperature and can explain the functional breakdown by monomerization while porin secondary structure is still preserved.     

Response of the cherry rootstock to water stress induced <Emphasis Type="Italic">in vitro</Emphasis>

The in vitro response of sweet cherry (Prunus cerasus × P. canescens) rootstock Gisela 5 to increasing water deficit in the culture medium was studied. Water stress induced by the incorporation of 1, 2 and 4 % polyethylene glycol (PEG-8000) into the Murashige and Skoog medium was applied for 6 weeks. PEG-induced water stress reduced shoot dry mass, length, water content and relative chlorophyll content. Water stress also induced leaf necrosis without causing loss of viability in the explants. The increase in malondialdehyde content indicated oxidative stress. The activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POX) and glutathione reductase (GR) were also significantly elevated. The concentrations of K, Ca, Fe and Mn of shoots were decreased.     

Methylenetetrahydrofolate reductase gene polymorphisms in patients with nonalcoholic steatohepatitis (NASH)

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal hepatic steatosis in the absence of a history of alcohol use. Nonalcoholic steatohepatitis (NASH) is the progressive form of NAFLD. Hyperhomocysteinemia causes steatosis, and the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms result in hyperhomocysteinemia. To examine whether the C677T and A1298C polymorphisms of the MTHFR gene were associated with NASH, we analysed the allele and genotype distribution of the MTHFR C677T and A1298C polymorphisms in 57 well-diagnosed NASH patients, 324 healthy controls in a case-control study of Turkish subjects of Caucasian origin. The diagnosis of the NASH patients was based on liver biopsy. The method used in the analysis of genotypes was PCR-RFLP. The MTHFR A1298C polymorphism was significantly associated with NASH (chi(2) = 8.439; p = 0.015) in the total NASH patients compared with healthy controls. The MTHFR 1298C allele (odds ratio (OR) = 2.480; 95%CI = 1.286-4.782; chi(2) = 7.703; df = 1; p = 0.006) was significantly associated with NASH in the total NASH patients. The MTHFR C677C/A1298C compound genotype (OR = 2.218; 95%CI = 1.003-4.906; chi(2) = 3.998; df = 1; p = 0.046) in men patients was also significantly associated with NASH. Likewise the MTHFR C1298C genotype was significantly associated with NASH in women patients with NASH (OR = 2.979; 95%CI = 1.027-8.641; chi(2) = 4.343; df = 1; p = 0.037). In conclusion, the MTHFR 1298C allele in all NASH patients, C1298C genotype, C677C/C1298C compound genotype in women NASH patients and C677C/A1298C compound genotype in men NASH patients were genetic risk factors for NASH.