The prevalence of factor V Leiden,prothrombin G20210A and methylenetetrahydrofolate reductase polymorphism C677T among G6PD deficient individuals from Western Iran

It has been suggested that the allele frequency of thrombophilic mutations is affected by glucose-6-phosphate dehydrogenase (G6PD) deficiency. The prevalence of thrombophilic mutations were studied in sixty G6PD deficient individuals including 57 males and three females with the mean age of 15 ± 3.08 and 110 age and sex matched healthy individuals consisted of 95 males and 15 females with the mean age of 16.19 ± 2.17 from the Kermanshah Province of Iran. Using a combination of PCR-RFLP technique, single strand conformation polymorphism (SSCP) analysis and DNA sequencing polymorphic G6PD mutations were identified. The factor V Leiden, prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T were detected by PCR-RFLP method using MnlI, HindIII and HinfI restriction enzymes, respectively. Three mutations, G6PD Mediterranean, G6PD Chatham and G6PD Cosenza were identified in 60 G6PD deficient individuals with highest prevalence of G6PD Mediterranean (91.6%). In G6PD deficient individuals the prevalence of factor V Leiden tended to be higher (5%) compared to healthy individuals (2.7%). The prevalence of prothrombin G20210A mutation in G6PD deficient individuals was 1.7%. However, in normal subjects the prevalence of this mutation was 2.7%. The frequency of T allele in G6PD deficient individuals were insignificantly higher (29.16%) than those in healthy individuals (26.8%). Our finding indicates that the prevalence of factor V Leiden, prothrombin G20210A and MTHFR C677T in G6PD deficient individuals is not statistically different compared to normal subjects and G6PD deficiency is not associated with these thrombophilic mutations in Western Iran.     

Neuronal apoptosis inhibitory protein,NAIP, is an inhibitor of procaspase-9

Ability of the full length NAIP and its BIR3 domain in inhibition of the proteases of the intrinsic apoptosis pathway was investigated. Activity of endogenous executioner caspases was drastically reduced by both recombinant NAIP-BIR3 (NBIR3) and the full length protein. Western blotting experiments showed that the full length NAIP and its BIR3 domain inhibited the cleavage of procaspase-3 by apoptosome activated caspase-9. Moreover, full length NAIP inhibited autocatalytic processing of procaspase-9 in the apoptosome complex indicating that unlike other inhibitor of apoptosis proteins (IAPs) human NAIP is an inhibitor of procaspase-9. Furthermore, inhibition of single-chain caspase-9 (human caspase-9, D315, D330/A point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9) by the BIR3 domain indicated that the this domain is the caspase-9 interacting moiety. Consistently, pull-down experiments of single-chain capsase-9 in apoptosome complex by the NBIR3 but not the X-linked inhibitor of apoptosis protein (XIAP)-BIR3 domain confirmed that the protein can associate with procaspase-9 prior to its autoproteolysis upon apoptosome formation. Interaction studies revealed the association of C338W variant of the NBIR3, but not the wild type protein with both SMAC-peptide and the SMAC protein. These data indicate that mutation of C338 to Trp is sufficient to accommodate the interaction of NAIP-BIR3 with SMAC-peptide and protein. Taken together, these results demonstrate that NAIP is evolved to prevent apoptosis right at the initiation stage of apoptosome formation and this inhibition cannot be antagonized by SMAC-type proteins.     

Chronic morphine treatment induces oxidant and apoptotic damage in the mice liver

Recently many researchers have proposed a protective role for morphine against tumor growth and metastasis, especially through induction of apoptosis in tumoral cells. These findings may lead to underestimation of cytotoxic effects of opioid drugs which are usually expected only at high doses. The present study was conducted to clarify whether repeated morphine administration, which is commonly used for relief from chronic pain, would interfere with liver antioxidant defence and hepatocytes vitality. Morphine was injected repeatedly at doses that have been reported to relieve cancer pain and reduce tumor spread in mice (5 and 10 mg/kg/day for nine consecutive days). The changes in hepatic glutathione concentration, its synthesis pathway and enzymatic antioxidant defense revealed the pro-oxidant effects of chronic morphine treatment on the liver. None of these changes were observed in those mice that were co-treated with naltrexone (opioid antagonist) and same doses of morphine. However induction of liver conjugating enzymes following morphine treatment was not receptor mediated. Moreover, chronic morphine treatment induced hepatocytes apoptosis. Interestingly, the apoptotic changes were antagonized by co-administration of either naltrexone or thiol antioxidant. In conclusion, although hepatotoxic effects of morphine at high doses have been reported previously, our findings propose that repeated morphine administration even at lower doses would induce oxidative stress in the liver, which may contribute to induction of apoptosis in hepatocytes. Since many of the observed adverse effects were mediated by opioid receptors, our results suggest that other opioid analgesics should also be used more cautiously.     

Effects of precipitation and temperature on crop production variability in northeast Iran

Climate variability adversely impacts crop production and imposes a major constraint on farming planning, mostly under rainfed conditions, across the world. Considering the recent advances in climate science, many studies are trying to provide a reliable basis for climate, and subsequently agricultural production, forecasts. The El Niño-Southern Oscillation phenomenon (ENSO) is one of the principle sources of interannual climatic variability. In Iran, primarily in the northeast, rainfed cereal yield shows a high annual variability. This study investigated the role played by precipitation, temperature and three climate indices [Arctic Oscillation (AO), North Atlantic Oscillation (NAO) and NINO 3.4] in historically observed rainfed crop yields (1983–2005) of both barley and wheat in the northeast of Iran. The results revealed differences in the association between crop yield and climatic factors at different locations. The south of the study area is a very hot location, and the maximum temperature proved to be the limiting and determining factor for crop yields; temperature variability resulted in crop yield variability. For the north of the study area, NINO 3.4 exhibited a clear association trend with crop yields. In central locations, NAO provided a solid basis for the relationship between crop yields and climate factors.     

Phosphate solubilization potential and modeling of stress tolerance of rhizobacteria from rice paddy soil in northern Iran

The purposes of this study were to evaluate the phosphate solubilization activity of bacteria isolated from the rhizosphere of rice paddy soil in northern Iran, and to study the effect of temperature, NaCl and pH on the growth of these isolates by modeling. Three of the most effective strains from a total of 300 isolates were identified and a phylogenetic analysis was carried out by 16S rDNA sequencing. The isolates were identified as Pantoea ananatis (M36), Rahnella aquatilis (M100) and Enterobacter sp. (M183). These isolates showed multiple plant growth-promoting attributes such as phosphate solubilization activity and indole-3-acetic acid (IAA) production. The M36, M100 and M183 isolates were able to solubilize 172, 263 and 254 µg ml^?1 of Ca₃(PO₄)₂ after 5 days of growth at 28 °C and pH 7.5, and to produce 8.0, 2.0 and 3.0 μg ml^?1 of IAA when supplemented with l-tryptophan (1 mg ml^?1) for 72 h, at 28 °C and pH 7.0, respectively. The solubilization of insoluble phosphate was associated with a drop in the pH of the culture medium and there was an inverse relationship between pH and solubilized P (r = ?0.98, P < 0.0952). There were no significant differences among isolates in terms of acidity tolerance based on their confidence limits as assessed by segmented model analysis and all isolates were able to grow at pH 4.3–11 (with optimum at 7.0–7.5). Based on a sigmoidal trend of a three-parameter logistic model, the salt concentration required for 50 % inhibition was 8.15, 6.30 and 8.23 % NaCl for M36, M100 and M183 isolates, respectively. Moreover, the minimum and maximum growth temperatures estimated by the segmented model were 5.0 and 42.75 °C for M36, 12.76 and 40.32 °C for M100, and 10.63 and 43.66 °C for M183. The three selected isolates could be deployed as inoculants to promote plant growth in an agricultural environment.     

Identifying Ca2+-Binding Sites in Proteins by Liquid Chromatography-Mass Spectrometry Using Ca2+-Directed Dissociations

Here we describe a new method to identify calcium-binding sites in proteins using high-resolution liquid chromatography-mass spectrometry in concert with calcium-directed collision-induced dissociations. Our method does not require any modifications to the liquid chromatography-mass spectrometry apparatus, uses standard digestion protocols, and can be applied to existing high-resolution MS data files. In contrast to NMR, our method is applicable to very small amounts of complex protein mixtures (femtomole level). Calcium-bound peptides can be identified using three criteria: (1) the calculated exact mass of the calcium containing peptide; (2) specific dissociations of the calcium-containing peptide from threonine and serine residues; and (3) the very similar retention times of the calcium-containing peptide and the free peptide.Calcium-dependent protein interactions mostly organized in protein networks are responsible for the regulation of cell cycle progression, cell growth, differentiation, secretion, and cytoskeletal organization (1–3). As many of these proteins are linked to various pathological conditions, they are clinically important. The speed at which calcium can have an interplay between various cellular components is impressive and comes notably detectable in neurological processes and in muscle contraction. Calcium binding sites in proteins can be determined by NMR spectroscopy (4, 5). For example, by such NMR measurements, the Ca²⁺-binding sites of the tellurite-resistance protein TerD from Klebsiella pneumoniae were found to be formed in part by a highly conserved motif of 13 residues specified by the sequence GDN(R/L)TG(E/A)GDGDDE (4).Although NMR is the gold standard to study calcium binding in proteins, this approach has several drawbacks. For instance, protein size is limited (< 30 kDa) and proteins should be pure and isotopically labeled. In addition, although the information content is high, NMR is relatively insensitive compared with other techniques such as MS and fluorescence spectroscopy, and relatively large quantities of material (typically 0.5 ml at 0.5–1.0 mm in biological samples) are needed, although efforts are devoted to improve sensitivity in NMR, such as stripline NMR (6).In bottom-up proteomics, proteolytic peptides, generated by enzymatic digestion of complex protein mixtures, are sequenced by MS-based methods (MS/MS (7, 8)) using collision-induced dissociations. Because of the even higher complexity of these peptide mixtures, liquid chromatography (LC)¹ is used to separate the peptides prior to sequencing. In such an LC-MS/MS procedure, many peptides can be identified belonging to the same protein. It has been stated (9) that by this procedure more peptides are analyzed than strictly necessary for identification purposes, but it can equally well be argued that such large coverages enable more reliable protein identifications; moreover, these larger coverages allow the detection of post-translational modifications, including specific calcium complexation as described here.Considering the need of identifying calcium-bound proteins in complex biological samples at low concentrations, we set out to develop a novel method for detecting Ca²⁺-binding sites in proteins based on LC-MS.     

Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1 enzyme in colorectal cancer

Background

Aberrant DNA methylation as the most important reason making epigenetic silencing of genes is a main mechanism of gene inactivation in patients with colorectal cancer. In this study, we decided to identify promoter methylation status of ten genes encoding WNT negative regulators, and measure the expression of DNMT1 enzyme in colorectal cancer samples.

Results

Aberrant methylation of APC gene was statistically significant associated with age over 50 (p = 0.017), DDK3 with male (p < 0.0001), SFRP4, WIF1, and WNT5a with increasing tumor stage (p = 0.004, p = 0.029, and p = 0.004), SFRP4 and WIF1 with tumor differentiation (p = 0.009 and p = 0.031) and SFRP2 and SFRP5 with histological type (p = 0.001 and p = 0.025). The increasing number of methylated genes correlated with the expression levels of the DNMT1 mRNA.

Conclusions

The rate of gene promoter methylation of WNT pathway regulators is high in colorectal cancer cells. Hyper-methylation is associated with increased expression of the DNMT1 enzyme.