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.     

A Recombinant Probiotic, <Emphasis Type="Italic">Lactobacillus casei,</Emphasis> Expressing the <Emphasis Type="Italic">Clostridium perfringens</Emphasis> α-toxoid,as an Orally Vaccine Candidate Against Gas Gangrene and Necrotic Enteritis

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa _247-370 ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p < 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.     

A new solution for maximal clique problem based sticker model

In this paper, we use stickers to construct a solution space of DNA for the maximal clique problem (MCP). Simultaneously, we also apply the DNA operation in the sticker-based model to develop a DNA algorithm. The results of the proposed algorithm show that the MCP is resolved with biological operations in the sticker-based model for the solution space of the sticker. Moreover, this work presents clear evidence of the ability of DNA computing to solve the NP-complete problem. The potential of DNA computing for the MCP is promising given the operational time complexity of O(nxk).     

Discovery of N-substituted 7-azaindoles as PIM1 kinase inhibitors – Part I

Novel N-substituted azaindoles have been discovered as PIM1 inhibitors. X-ray structures have played a significant role in orienting the chemistry effort in the initial phase of hit confirmation. Disclosure of an unconventional binding mode for 1 and 2, as demonstrated by X-ray crystallography, is presented and was an important factor in selecting and advancing a lead series.     

Use of SRAP markers to assess genetic diversity and population structure of wild, cultivated, and ornamental pomegranates (Punica granatum L.) in different regions of Iran

Sequence-related amplified polymorphism (SRAP) was used to assess the genetic diversity of 63 cultivated, wild, and ornamental pomegranate genotypes from five different geographical regions of Iran. A total of 250 fragments were amplified using 13 primer combinations; among these, 133 bands (53?%) were polymorphic. The average PIC value was 0.28 over all PCs. The genetic distance among genotypes ranged from 0.10 to 0.37 with an average of 0.24. Cluster analysis using the neighbor-joining (NJ) method suggested there are close relationships between ornamental and some wild genotypes. Although AMOVA results revealed significant differences in the genetic diversity among the regions (P?=?0.0048), the genetic variation was mainly caused by variation of intra regions. The results indicated low genetic differentiation (Fst?=?0.025) and high gene flow (Nm?=?2.28) among regions. These results confirmed that SRAP markers could be powerful tools and an effective marker system for determining the genetic diversity and population genetic structure of the pomegranate.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Aminolevulinic acid and nitric oxide regulate oxidative defense and secondary metabolisms in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under drought stress

To minimize the damaging effects of stresses, plant growth regulators (PGRs) are widely used to sustain the plant life under stress-prone environments. So, a study was carried out to evaluate the response of two canola (Brassica napus L.) cultivars, Dunkeld and Cyclone, to foliar-applied two potential PGRs, nitric oxide (NO) and 5-aminolevulinic acid, under water deficit conditions. In this study, the levels of NO and ALA used were 0.02 and 0.895 mM, respectively. Plants of both canola cultivars were subjected to control (100% field capacity) and water deficit (60% field capacity). Drought stress significantly decreased growth, chlorophyll pigments, relative water contents (RWC), and soluble proteins, while it increased relative membrane permeability (RMP), proline, glycinebetaine (GB), malondialdehyde (MDA), total phenolics, and activities of catalase (CAT) and peroxidase (POD) enzymes in both cultivars. Foliar application of PGRs improved growth, chlorophyll a, GB, total phenolics, CAT activity, and total soluble proteins, while it decreased RMP, MDA, and POD activity in both canola cultivars. Other physio-biochemical attributes such as chlorophyll b, RWC, hydrogen peroxide (H₂O₂) and proline contents as well as superoxide dismutase (SOD) activity remained unaffected due to application of PGRs. So, the results of the present study suggest that exogenous application of NO and ALA could be useful to enhance the drought tolerance of canola plants by up-regulating the oxidative defense system, osmoprotectant accumulation, and minimizing the lipid peroxidation.     

Effects of Cadmium,Copper, Lead,and Zinc Contamination on Metal Accumulation by Safflower and Wheat

Accumulation of heavy metals (HMs) in cultivated soils is a continuing environmental problem in many parts of the world. An increase in HM concentration can enhance uptake of toxic metals by crops and enter the human food chain. In this study, the uptake behavior of wheat and safflower was evaluated in a calcareous soil by using 12 undisturbed columns in which half were artificially contaminated. Heavy metals in the form of CdCl₂ (15 mg Cd kg^{? 1}), CuSO₄ (585 mg Cu kg^{? 1}), Pb(NO₃)₂ (117 mg Pb kg^{? 1}), and ZnCl₂ (1094 mg Zn kg^{? 1}) were sprayed on the soil surface and completely mixed in the top 10 cm. The background total concentrations of Cd, Cu, Pb and Zn were 1.6, 29.5, 17.5 and 61.2 mg kg^{? 1}, respectively. After metal application, half of the columns (3 contaminated and 3 uncontaminated) were sown with wheat (Triticum aestivum) and the other half with safflower (Carthamus tinctorious) and grown for 74 days until maturity. After harvesting, soil columns were cut into 10-cm sections and analyzed for HNO₃- and DTPA-extractable metal concentrations. Metal concentrations were also measured in different plant tissues. The results showed that artificial contamination of topsoil decreased the transpiration rate of wheat by 12% and that of safflower by 6%. In contaminated columns, Cd, Cu, Pb, and Zn accumulation in wheat shoot was greater by 8.0-, 1.9-, 3.0-, and 2.1-fold than the control, respectively. Accordingly, these numbers were 46.0-, 1.3-, 1.7-, and 1.6-fold in safflower shoot. Soil contamination with HMs resulted in a 55% decrease in shoot dry matter yield of wheat while it had no significant effect on shoot dry matter of safflower. The normalized water consumption for safflower was therefore not affected by metal contamination (≈ 13 mm H₂O g^{? 1} of dry weight for all safflower and uncontaminated wheat treatments), while contaminated wheat was much less water efficient at about 27 mm H₂O g^{? 1} dry weight. It was concluded that although artificial contamination had a negative effect on wheat growth, it did not affect safflower's normal growth and water efficiency.     

Interactions of the NPXY microdomains of the low density lipoprotein receptor‐related protein 1

The low density lipoprotein receptor‐related protein 1 (LRP1) mediates internalization of a large number of proteins and protein–lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1‐CT) can be phosphorylated by activated protein‐tyrosine kinases at two NPXY motifs in LRP1‐CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull‐down experiments from brain lysate revealed numerous proteins binding to LRP1‐CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non‐phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC‐MS/MS, and confirmed by Western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY₄₅₀₇ (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine‐phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY₄₄₇₃ (membrane proximal) bound many fewer proteins and only to the phosphorylated form.     

Bioaugmentation and coexistence of two functionally similar bacterial strains in aerobic granules

The survival of the inoculated microbial culture is critical for successful bioaugmentation but impossible to predict precisely. As an alternative strategy, bioaugmentation of a group of microorganisms may improve reliability of bioaugmentation. This study evaluated simultaneous bioaugmentation of two functionally similar bacterial strains in aerobic granules. The two strains, Pandoraea sp. PG-01 and Rhodococcus erythropolis PG-03, showed high phenol degradation and growth rates in phenol medium, but they were characterized as having a poor aggregation activity and weak bioflocculant-producing and biofilm-forming abilities. In the spatially homogeneous batch conditions, strain PG-01 with higher growth rates outcompeted strain PG-03. However, the two strains could stably coexist in the spatially heterogeneous conditions. Then the two strains were mixed and bioaugmented into activated sludge in two sequencing batch reactors, which were operated with the different settling times of 5 and 30 min, respectively. Aerobic granules were developed only in the reactor with a settling time of 5 min. Fluorescence in situ hybridization and denaturing gradient gel electrophoresis showed that the two strains could coexist in aerobic granules but not in activated sludge. These findings suggested that the compact structure of aerobic granules provided spatial isolation for coexistence of competitively superior and inferior strains with similar functions.