<Emphasis Type="Italic">CBS</Emphasis> mutations and <Emphasis Type="Italic">MTFHR</Emphasis> SNPs causative of hyperhomocysteinemia in Pakistani children

Three index patients with hyperhomocysteinemia and ocular anomalies were screened for cystathionine beta synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR) polymorphisms. Genotyping of hyperhomocysteinemia associated MTHFR polymorphisms C677T (rs1801133) and A1298C (rs1801131) was done by PCR-restriction fragment length polymorphism. Sanger sequencing was performed for CBS exonic sequences along with consensus splice sites. In the case of MTHFR polymorphisms, all the patients were heterozygous CT for the single nucleotide polymorphism (SNP) C677T and were therefore carriers of the risk allele (T), while the patients were homozygous CC for the risk genotype of the SNP A1298C. CBS sequencing resulted in the identification of two novel mutations, a missense change (c.467T>C; p.Leu156Pro) in exon 7 and an in-frame deletion (c.808_810del; p.Glu270del) in exon 10. In addition, a recurrent missense mutation (c.770C>T; p.Thr257Met) in exon 10 of the gene was also identified. The mutations were present homozygously in the patients and were inherited from the carrier parents. This is the first report from Pakistan where novel as well as recurrent CBS mutations causing hyperhomocysteinemia and lens dislocation in three patients from different families are being reported with the predicted effect of the risk allele of the MTHFR SNP in causing hyperhomocysteinemia.     

Decreased exhaled nitric oxide as a marker of postinsult immune paralysis.

Nitric oxide (NO) regulates neutrophil migration and alveolar macrophage functions such as cytokine synthesis and bacterial killing, both of which are impaired in immune paralysis associated with critical illness. The aim of this study was to determine whether NO is involved in immune paralysis and whether exhaled NO measurement could help to monitor pulmonary defenses. NO production (protein expression, enzyme activity, end products, and exhaled NO measurements) was assessed in rats after cecal ligation and puncture to induce a mild peritonitis (leading to approximately 20% mortality rate). An early and sustained decrease in exhaled NO was found after peritonitis (from 1 to 72 h) compared with healthy rats [median (25th-75th percentile), 1.5 parts per billion (ppb) (1.2-1.7) vs. 4.0 ppb (3.6-4.3), P < 0.05], despite increased NO synthase-2 and unchanged NO synthase-3 protein expression in lung tissue. NO synthase-2 activity was decreased in lung tissue. Nitrites and nitrates in supernatants of isolated alveolar macrophages decreased after peritonitis compared with healthy rats, and an inhibitory experiment suggested arginase overactivity in alveolar macrophages bypassing the NO substrate. Administration of the NO synthase-2 inhibitor aminoguanidine to healthy animals reproduced the decreased neutrophil migration toward alveolar spaces that was observed after peritonitis, but L-arginine administration after peritonitis failed to correct the defect of neutrophil emigration despite increasing exhaled NO compared with D-arginine administration [4.8 (3.9-5.7) vs. 1.6 (1.3-1.7) ppb, respectively, P < 0.05]. In conclusion, the decrease in exhaled NO observed after mild peritonitis could serve as a marker for lung immunodepression.     

Promising Role of Moderate Soil Drying and Subsequent Recovery Through Moderate Wetting at Grain-Filling Stage for Rice Yield Enhancement

The study was conducted at the grain-filling stage to elucidate the physiological and molecular mechanisms of the root to enhance yield under alternate wetting and drying (AWD) compared with conventional irrigation. Measurements of root dry weight (RDW), seed setting rate, total kernel weight, and grain yield were determined along with 2D electrophoresis to detect altered protein expression in response to moderate soil drying (MD) and the subsequent recovery phase as moderate wetting (MW) under AWD compared with continuous wetting under CI. We found significant enhancement in RDW as well as 14.30 % increase in inferior spikelets^, seed setting and 10.32 g m⁻² increase in final yield. Among the total 55 differentially expressed proteins, 26 proteins were differentially expressed under both MD treatment and MW treatment, whereas 14 proteins under MD and 15 proteins under MW showed distinct expression. Differentially expressed proteins were involved in redox homeostasis, signaling, defense, energy, photoassimilate remobilization and included 14-3-3 proteins, cysteine-rich receptor-like protein kinase, monodehydroascorbate reductase, ascorbate peroxidase, glutathione S-transferases, translationally controlled tumor protein, remorin C-terminal domain containing protein, protein disulfide isomerase, DnaK family protein, cysteine synthase, aminotransferase, phosphoglycerate mutase, pyruvate phosphate dikinase, ATP synthase, and abscisic acid stress ripening (ASR1). The differential expression ratio of the signaling, redox, and defense group proteins was almost the same under MD and MW. ABA signaling, amino acid synthesis, and N remobilization were upregulated under MD, and the enzymes involved in carbohydrate, energy, and transportation metabolism were upregulated under MW. In conclusion, at the rice grain-filling stage, AWD is a potential technique to trigger signaling and the enzymatic protein network for systematic senescence initiation, root enlargement for maximum nutrient uptake, and maximize photoassimilate remobilization for yield enhancement.

     

Maize Brittle Stalk2-Like3, encoding a COBRA protein,functions in cell wall formation and carbohydrate partitioning

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with ¹⁴C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.

Mutations in Bk2L3 result in dwarfed plants with decreased anisotropic cell growth, cellulose deposition, phloem pressure, sucrose export, and carbohydrate hyperaccumulation in mature maize leaves.     

Haloperidol treatment at pre-exposure phase reduces the disturbance of latent inhibition in rats with neonatal ventral hippocampus lesions

Animals with neonatal ventral hippocampal lesions develop during or after adolescence abnormal behaviors related to schizophrenia such as anxiety and latent inhibition disruption. The aim of this study was to test whether haloperidol injection prior to pre-exposure session in the latent inhibition test would facilitate latent inhibition.     

Proteomic analysis of positive influence of alternate wetting and moderate soil drying on the process of rice grain filling

Rice (Oryza sativa L.) is one of the most important crops in the world to feed ever increasing world population. An increase in output of rice crop per unit is imminent. Alternate wetting and moderate soil drying (AWD) irrigation technology has been recommended as a good practice method to improve grain filling of rice crop at late growing stages. Physiological, molecular and agronomic parameters were adopted to elucidate the role of rice stem and sheath under AWD treatment as compared to the conventional irrigation during the grain filling stage. AWD treatment significantly increased stem and sheath dry weight, carbohydrate reserves and their remobilization to the grain, especially inferior spikelet grains. The results showed that the transport and conversion rate of the stem and sheath photoassimilates increased by 9.87 and 8.37%, respectively. Furthermore, protein expression profiles of the stem and sheath at 10, 20 and 30 days after flowering were analyzed. We examined 220 differentially expressed proteins, and successfully identified 166 proteins, including 71 proteins in the stem and 95 proteins in the sheath involved in thirteen important functional groups. Our results suggest that the AWD treatment at the rice grain filling stage is highly conducive to trigger the mobilization of the N assimilates from leave and root to the stem and sheath, and then promotes to remobilize the reserves to the grain through coordinately expressed proteins involved in photosynthesis, systematic senescence, oxidative stress defense, signal transduction and other metabolisms. This study reveals the metabolic mechanism of the stem and sheath in response to AWD at grain filling stage, and provides theoretical evidence for better quality control and scientific improvement of rice in practice.     

Validated spectrofluorimetric method for determination of two phosphodiesterase inhibitors tadalafil and vardenafil in pharmaceutical preparations and spiked human plasma

A valid, sensitive and rapid spectrofluorimetric method has been developed and validated for determination of both tadalafil (TAD) and vardenafil (VAR) either in their pure form, in their tablet dosage forms or spiked in human plasma. This method is based on measurement of the native fluorescence of both drugs in acetonitrile at λ_em 330 and 470 nm after excitation at 280 and 275 nm for tadalafil and vardenafil, respectively. Linear relationships were obtained over the concentration range 4–40 and 10–250 ng/mL with a minimum detection of 1 and 3 ng/mL for tadalafil and vardenafil, respectively. Various experimental parameters affecting the fluorescence intensity were carefully studied and optimized. The developed method was applied successfully for the determination of tadalafil and vardenafil in bulk drugs and tablet dosage forms. Moreover, the high sensitivity of the proposed method permitted their determination in spiked human plasma. The developed method was validated in terms of specificity, linearity, lower limit of quantification (LOQ), lower limit of detection (LOD), precision and accuracy. The mean recoveries of the analytes in pharmaceutical preparations were in agreement with those obtained from the comparison methods, as revealed by statistical analysis of the obtained results using Student's t‐test and the variance ratio F‐test. Copyright © 2015 John Wiley & Sons, Ltd.     

Mutation of the highly conserved tryptophan in the serpin breach region alters the inhibitory mechanism of plasminogen activator inhibitor-1

We have demonstrated that interactions within the conserved serpin breach region play a direct role in the critical step of the serpin reaction in which the acyl-enzyme intermediate must first be exposed to hydrolyzing water and aqueous deacylation. Substitution of the breach tryptophan in PAI-1 (Trp175), a residue found in virtually all known serpins, with phenylalanine altered the kinetics of the reaction mechanism and impeded the ability of PAI-1 to spontaneously become latent without compromising the inherent rate of cleaved loop insertion or partitioning between the final inhibited serpin-proteinase complex and hydrolyzed serpin. Kinetic dissection of the PAI-1 inhibitory mechanism using multiple target proteinases made possible the identification of a single rate-limiting intermediate step coupled to the molecular interactions within the breach region. This step involves the initial insertion of the proximal reactive center loop hinge residue(s) into beta-sheet A and facilitates translocation of the distal P'-side of the cleaved reactive center loop from the substrate cleft of the proteinase. Substitution of the tryptophan residue raised the kinetic barrier restricting the initial loop insertion event, significantly retarding the rate-limiting step in tPA reactions in which strong exosite interactions must be overcome for the reaction to proceed.