VEGF gene polymorphism interactions with dietary trace elements intake in determining the risk of metabolic syndrome

There is a complex association among genetic, metabolic, and environmental factors in determining the risk of metabolic syndrome (MetS). The aim of this study was to investigate the role of the association between the dietary intake of iron, copper, zinc, manganese, selenium, and iodine (assessed by 24 recall) with vascular endothelial growth factor variants (rs6921438, rs4416670, rs6993770, and rs10738760), on the risk of MetS. Two-hundred and forty-eight individuals with MetS and 100 individuals without MetS were recruited. The dietary intake and the daily average of energy and nutrient intake were obtained by a questionnaire and quantified using Diet Plan 6 software. DNA was extracted from EDTA anticoagulated whole blood. The SNPs were assessed using using a Sequenom iPLEX Gold assay. Data analysis was undertaken using the Student t test, χ2 test and logistic regression using SPSS 11.5 software. There was a significant association between low dietary iron intake and rs6993770 (β = .10, P < .05), and a low dietary zinc and a high manganese intake with rs6921438 in relation to the presence of MetS (β = −.17, P < .05, β = −.30, P < .05, respectively). Our data showed the association of rs6993770 with iron intake and rs6921438 with zinc and manganese intake, indicating further investigation in a larger population to evaluate their values.     

Roles for osteocalcin in proliferation and differentiation of spermatogonial cells cocultured with somatic cells

Spermatogonial cells (SCs) are key cells for spermatogenesis. These cells are affected by paracrine signals originated from nearby somatic cells, among them Leydig cells have receptors for osteocalcin, a hormone known for exerting positive roles in the promotion of spermatogenesis. The aim of this study was to evaluate roles for osteocalcin on SCs proliferative and differentiation features after coculture with Leydig cells. SCs and Leydig cells were isolated from neonate NMRI offspring mice and adult NMRI mice, respectively. SCs population were then enriched in a differential attachment technique and assessed for morphological features and identity. Then, SCs were cocultured with Leydig cells and incubated with osteocalcin for 4 weeks. Evaluation of proliferation and differentiation-related factors were surveyed using immunocytochemistry (ICC), Western blot, and quantitative real-time polymerase chain reaction (PCR). Finally, the rate of testosterone release to the culture media was measured at the end of 4th week. Morphological and flow cytometry results showed that the SCs were the population of cells able to form colonies and to express ID4, α6-, and β1-integrin markers, respectively. Leydig cells were also able to express Gprc6α as a specific marker for the cells. Incubation of SCs/Leydig coculture with osteocalcin has resulted in an increase in the rate of expressions for differentiation-related markers. Levels of testosterone in the culture media of SCs/Leydig was positively influenced by osteocalcin. It could be concluded that osteocalcin acts as a positive inducer of SCs in coculture with Leydig cells probably through stimulation of testosterone release from Leydig cells and associated signaling.     

Role of thrombin in the pathogenesis of atherosclerosis

Atherosclerosis is an arterial disease associated with inflammation. Thrombin is a procoagulant and proinflammatory serine protease that contributes to the pathology of atherosclerosis by enhancing the expression of cell adhesion molecules, inducing the secretion of proinflammatory cytokines, activating inflammatory responses in atherosclerotic plaques, stimulating proliferation of aortic smooth muscle cells, and exacerbating vascular lesions at sites of injury. Hence, thrombin appears to be an important target for treatment of atherosclerosis and thrombin pharmacological inhibitors have significant therapeutic potency for suppressing inflammatory responses in cardiovascular diseases. This review summarizes the proinflammatory signaling functions of thrombin as well as the therapeutic potency of thrombin inhibitors in the pathogenesis of atherosclerosis and hence their potential therapeutic value in this condition.     

Combined anticancer effects of simvastatin and arsenic trioxide on prostate cancer cell lines via downregulation of the VEGF and OPN isoforms genes

Arsenic trioxide (ATO) and statins have been demonstrated to have anti‐neoplastic properties; however, the data regarding their combination therapy is limited. Thus, we aimed to study the effects of ATO, Simvastatin and their combination in proliferation, apoptosis and pathological angiogenesis in prostate cancer cell lines. The human prostate cell lines were treated with different concentrations of Simvastatin and ATO alone and combined to find effective doses and IC50 values. In addition, the percentage of apoptotic cells was evaluated by annexin/PI staining, and mRNA expression levels of the apoptotic gene, including OPN isoforms and VEGF, were investigated using real‐time PCR. Our data displayed that Simvastatin (12 and 8 μM in PC3 and LNCaP cell lines respectively), ATO (8 and 5 μM in PC3 and LNCaP cell lines respectively), and also their combination (12 μM Simvastatin and 8 μM ATO in PC3, 8 μM Simvastatin and 5 μM ATO in LNCaP cell lines respectively) significantly increased the percentage of apoptotic cells. Also, we showed that the combination therapy by Simvastatin and ATO increased cell apoptosis and inhibited cell proliferation, providing anti‐proliferative and anti‐angiogenic properties, possibly via downregulation of the expression of VEGF and OPN genes. These results provide new perceptions regarding the anticancer roles of ATO and statins’ combination therapy in prostate cancer.     

Chemoprotective effects of carnosine against genotoxicity induced by cyclophosphamide in mice bone marrow cells

The protective effects of carnosine as a natural dipeptide were investigated in mouse bone marrow cells against genotoxicity induced by cyclophosphamide. Mice were injected with solutions of carnosine at three different doses (10, 50 and 100?mg kg(-1) bw) for five consecutive days. On the fifth day of treatment, mice were injected cyclophosphamide and killed after 24?h. The frequency of micronuclei in polychromatic erythrocytes and the ratio of polychromatic erythrocyte/polychromatic erythrocyte?+?normochromatic erythrocyte [PCE/(PCE?+?NCE)] were evaluated by May-Grunwald/Giemsa staining. Histopathology of bone marrow was examined in mice treated with cyclophosphamide and carnosine. Carnosine significantly reduced micronucleated polychromatic erythrocytes (MnPCEs) induced by cyclophosphamide at all three doses. Carnosine at dose of 100?mg kg(-1) bw reduced MnPCEs 3.76-fold and completely normalized the PCE/(PCE?+?NCE) ratio. Administration of carnosine inhibited bone marrow toxicity induced by cyclophosphamide. It appeared that carnosine with protective activity reduced the oxidative stress and genotoxicity induced by cyclophosphamide in bone marrow cells of mice. Copyright ? 2012 John Wiley & Sons, Ltd.     

Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ-carboxylation of a human vitamin K-dependent protein by the insect enzyme

The Drosophila γ-glutamyl carboxylase (dγC) has substrate recognition properties similar to that of the vertebrate γ-carboxylase (γC), and its carboxylated product yield, in vitro, was shown to be more than that obtained with the human enzyme. However, whether the Drosophila enzyme is able to γ-carboxylate the human vitamin K-dependent (VKD) proteins, such as the human coagulation factor IX (hFIX), as synthesized in cultured Drosophila cells was not known. To examine this possibility, the Drosophila Schnider (S2) cell line was transfected with a metallothionein promoter-regulated hFIX-expressing plasmid. After induction with copper ion, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assey (ELISA) and coagulation test on the culture supernatant of the transfected S2 cells during 72 h of postinduction. In comparison with Chinese hamster ovary cell line, S2 cells showed higher (≈ 12-fold) expression level of the hFIX. The γ-carboxylation of the Drosophila-derived hFIX was confirmed by evaluation of the expressed protein, after being precipitated with barium citrate. The biological activity of the S2 cell-derived hFIX indicated the capability of S2 cells to fulfill the required γ-carboxylation of the expressed hFIX. Coexpression of the human γ-glutamyl carboxylases (hγC) was also shown to improve both expression and γ-carboxylation of the hFIX. This is the first in vivo data to describe the ability of the dγC to recognize the human-based propeptide as substrate, which is an essential step for production of biologically active γ-carboxylated VKD proteins.     

Glutamate oxidative injury to RGC-5 cells in culture is necrostatin sensitive and blunted by a hydrogen sulfide (H2S)-releasing derivative of aspirin (ACS14)

Oxidative stress to RGC-5 cells in culture was delivered by exposure to a combination of glutamate (Glu) and buthionine-S,R-sulfoximine (BSO). The effect of the insult on cell survival was quantified by the resazurin-reduction and a dead/live assays. Moreover, breakdown of DNA, the localisation of phosphatidylserine and reactive radical species (ROS) and its quantification were determined. In addition, various proteins and mRNAs were studied using Western blot, real time PCR and immunocytochemistry. ACS14, its sulfurated moiety ACS1 and aspirin were tested for their ability to blunt the negative effects of Glu/BSO on RGC-5 cells. In addition assays were carried out to see whether any of these substances influenced glutathione (GSH). Glu/BSO dose-dependently kills RGC-5 cells by a mechanism that involves an elevation of ROS accompanied by a breakdown of DNA, expression of phosphatidylserine and the activation of p38 MAPK. The process is unaffected by the pan caspase inhibitor z-VAD-fmk, does not involve the activation of apoptosis inducing factor (AIF) but is sensitive to active necrostatin-1. In cell viability studies (resazurin-reduction assay), ACS1 and ACS14 equally counteracted the negative effects of 5mM Glu/BSO to RGC-5 cells but aspirin was only effective with a milder oxidative stress (1 mM Glu/BSO). In all other assays ACS14 was very much more effective than aspirin at counteracting the influence of 5mM Glu/BSO. Moreover, ACS14 and ACS1 directly stimulated GSH while aspirin was ineffective. In addition the neuroprotecive effect of ACS14 was specifically blunted by the non-specific potassium channel blocker glibenclamide. Also the up-regulation of Bcl-2, HO-1 and XIAP induced by 5mM Glu/BSO were all attenuated to a greater extent by ACS14 (20 μM) than aspirin (20 μM). These data show that ACS14 is a very effective neuroprotectant when compared with aspirin. ACS14 maintains its aspirin characteristics and has the ability to release H(2)S. The combined multiple actions of aspirin and H(2)S in the form of ACS14 is worthy to consider for possible use in the treatment of glaucoma.     

Environmental Risk Assessment of Dams in Construction Phase Using a Multi-Criteria Decision-Making (MCDM) Method

This study was conducted to identify environmental and human health risks caused by Balarood Dam, in construction phrase. The first step, all risk-generating factors were identified using a Delphi Questionnaire. Afterwards, the identified criteria were prioritized once using the Analytical Hierarchy Process (AHP) method and then by the Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS). Due to the complex and uncertain nature of decision-making in times of risk, it was necessary to use more than one weighting method to ensure accuracy of weights. The results from AHP and TOPSIS revealed a mismatch in priorities; therefore, an integration method was presented blending Mean-Rank, Borda, and Copeland methods. According to the TOPSIS results, factors including cut and fill, explosion, and transportation, were first to third highest-priority risk-generating factors, respectively. Considering the results from the AHP method, factors cut and fill, drilling, and explosion were identified as first to third top-priority risk-generating factors, respectively. The results obtained from the integration method suggested that cut and fill, explosion, and drilling are the most important environmental risks at construction phase. As a general conclusion, different weighting methods can lead to different results by which the fate of a decision may be changed and it is essential to control final scores by applying more than one weighting method.     

Design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin-1 against influenza viruses

Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1(HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte(CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin(HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.