Phylogenetic reconstruction of endophytic fungal isolates using internal transcribed spacer 2 (ITS2) region

Endophytic fungi are inhabitants of plants, living most part of their lifecycle asymptomatically which mainly confer protection and ecological advantages to the host plant. In this present study, 48 endophytic fungi were isolated from the leaves of three medicinal plants and characterized based on ITS2 sequence – secondary structure analysis. ITS2 secondary structures were elucidated with minimum free energy method (MFOLD version 3.1) and consensus structure of each genus was generated by 4SALE. ProfDistS was used to generate ITS2 sequence structure based phylogenetic tree respectively. Our elucidated isolates were belonging to Ascomycetes family, representing 5 orders and 6 genera. Colletotrichum/Glomerella spp., Diaporthae/Phomopsis spp., and Alternaria spp., were predominantly observed while Cochliobolus sp., Cladosporium sp., and Emericella sp., were represented by singletons. The constructed phylogenetic tree has well resolved monophyletic groups with >50% bootstrap value support. Secondary structures based fungal systematics improves not only the stability; it also increases the precision of phylogenetic inference. Above ITS2 based phylogenetic analysis was performed for our 48 isolates along with sequences of known ex-types taken from GenBank which confirms the efficiency of the proposed method. Further, we propose it as superlative marker for reconstructing phylogenetic relationships at different taxonomic levels due to their lesser length.     

Deficiency of Cardiomyocyte-specific MicroRNA-378 Contributes to the Development of Cardiac Fibrosis Involving a Transforming Growth Factor β (TGFβ1)-dependent Paracrine Mechanism

Understanding the regulation of cardiac fibrosis is critical for controlling adverse cardiac remodeling during heart failure. Previously we identified miR-378 as a cardiomyocyte-abundant miRNA down-regulated in several experimental models of cardiac hypertrophy and in patients with heart failure. To understand the consequence of miR-378 down-regulation during cardiac remodeling, our current study employed a locked nucleic acid-modified antimiR to target miR-378 in vivo. Results showed development of cardiomyocyte hypertrophy and fibrosis in mouse hearts. Mechanistically, miR-378 depletion was found to induce TGFβ1 expression in mouse hearts and in cultured cardiomyocytes. Among various secreted cytokines in the conditioned-media of miR-378-depleted cardiomyocytes, only TGFβ1 levels were found to be increased. The increase was prevented by miR-378 expression. Treatment of cardiac fibroblasts with the conditioned media of miR-378-depleted myocytes activated pSMAD2/3 and induced fibrotic gene expression. This effect was counteracted by including a TGFβ1-neutralizing antibody in the conditioned-medium. In cardiomyocytes, adenoviruses expressing dominant negative N-Ras or c-Jun prevented antimiR-mediated induction of TGFβ1 mRNA, documenting the importance of Ras and AP-1 signaling in this response. Our study demonstrates that reduction of miR-378 during pathological conditions contributes to cardiac remodeling by promoting paracrine release of profibrotic cytokine, TGFβ1 from cardiomyocytes. Our data imply that the presence in cardiomyocyte of miR-378 plays a critical role in the protection of neighboring fibroblasts from activation by pro-fibrotic stimuli.     

Identification of quantitative trait loci controlling linolenic acid concentration in PI483463 (Glycine soja)

Key message

The QTLs controlling alpha-linolenic acid concentration from wild soybean were mapped on nine soybean chromosomes with various phenotypic variations. New QTLs for alpha-linolenic acid were detected in wild soybean.

Abstract

Alpha-linolenic acid (ALA) is a polyunsaturated fatty acid desired in human and animal diets. Some wild soybean (Glycine soja) genotypes are high in ALA. The objective of this study was to identify quantitative trait loci (QTLs) controlling ALA concentration in a wild soybean accession, PI483463. In total, 188 recombinant inbred lines of F_5:6, F_5:7, and F_5:8 generations derived from a cross of wild soybean PI483463 (~15 % ALA) and cultivar Hutcheson (~9 % ALA) were planted in four environments. Harvested seeds were used to measure fatty acid concentration. Single nucleotide polymorphism markers of the universal soybean linkage panel (USLP 1.0) and simple sequence repeat markers were used for molecular genotyping. Nine putative QTLs were identified that controlled ALA concentration by model-based composite interval mapping and mapped to different soybean chromosomes. The QTLs detected in four environments explained 2.4–7.9 % of the total phenotypic variation (PV). Five QTLs, qALA5_3, qALA6_1, qALA14_1, qALA15_1, and qALA17_1, located on chromosomes 5, 6, 14, 15, and 17 were identified by model-based composite interval mapping and composite interval mapping in two individual environments. Among them, qALA6_1 showed the highest contribution to the PV with 10.0–10.2 % in two environments. The total detected QTLs for additive and epistatic effects explained 52.4 % of the PV for ALA concentration. These findings will provide useful information for understanding genetic structure and marker-assisted breeding programs to increase ALA concentration in seeds derived from wild soybean PI483463.     

A mutational analysis of the cytosolic domain of the tomato Cf‐9 disease‐resistance protein shows that membrane‐proximal residues are important for Avr9‐dependent necrosis

The tomato Cf‐9 gene encodes a membrane‐anchored glycoprotein that imparts race‐specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N‐terminal half of the extracellular leucine‐rich repeat (eLRR) domain of the Cf‐9 protein determines its specificity for Avr9, the C‐terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf‐9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9‐dependent necrosis triggered by Cf‐9. Our results indicate that the membrane‐proximal region of the cytosolic domain of Cf‐9 plays an important role in Cf‐9‐mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf‐9 function. An alanine mutation of T835 had no effect on Cf‐9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf‐9 function. We therefore postulate that phosphorylation/de‐phosphorylation of T835 could act as a molecular switch to determine whether Cf‐9 is in a primed or inactive state. Yeast two‐hybrid analysis was used to show that the cytosolic domain of Cf‐9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline‐induced kink in the membrane‐proximal region of the cytosolic domain of Cf‐9 may be important for interaction with VAP27, which may, in turn, be important for Cf‐9 function.     

Structures of the dI2dIII1 complex of proton-translocating transhydrogenase with bound, inactive analogues of NADH and NADPH reveal active site geometries

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The enzyme comprises three components; dI binds NAD(H), dIII binds NADP(H), and dII spans the membrane. The 1,4,5,6-tetrahydro analogue of NADH (designated H2NADH) bound to isolated dI from Rhodospirillum rubrum transhydrogenase with similar affinity to the physiological nucleotide. Binding of either NADH or H2NADH led to closure of the dI mobile loop. The 1,4,5,6-tetrahydro analogue of NADPH (H2NADPH) bound very tightly to isolated R. rubrum dIII, but the rate constant for dissociation was greater than that for NADPH. The replacement of NADP+ on dIII either with H2NADPH or with NADPH caused a similar set of chemical shift alterations, signifying an equivalent conformational change. Despite similar binding properties to the natural nucleotides, neither H2NADH nor H2NADPH could serve as a hydride donor in transhydrogenation reactions. Mixtures of dI and dIII form dI2dIII1 complexes. The nucleotide charge distribution of complexes loaded either with H2NADH and NADP+ or with NAD+ and H2NADPH should more closely mimic the ground states for forward and reverse hydride transfer, respectively, than previously studied dead-end species. Crystal structures of such complexes at 2.6 and 2.3 A resolution are described. A transition state for hydride transfer between dihydronicotinamide and nicotinamide derivatives determined in ab initio quantum mechanical calculations resembles the organization of nucleotides in the transhydrogenase active site in the crystal structure. Molecular dynamics simulations of the enzyme indicate that the (dihydro)nicotinamide rings remain close to a ground state for hydride transfer throughout a 1.4 ns trajectory.     

Modulation of A and B cell functions by tolbutamide and arginine in the pancreas of thiamine-deficient rats

The effects of administration of glucose orally and tolbutamide or arginine intravenously on insulin and glucagon secretion and blood glucose level were studied in normal and thiamine-deficient rats. In thiamine deficiency, insulin secretion and glucose tolerance were impaired during glucose ingestion. Tolbutamide decreased the blood glucose level in both control and thiamine-deficient rats but its stimulatory effect on insulin secretion was minimal in thiamine-deficient rats unlike the control animals. Arginine did not alter substantially the blood glucose or insulin in thiamine-deficient rats, whereas it increased the insulin level in control rats. The fasting plasma glucagon level was high in thiamine deficiency. Tolbutamide increased the plasma glucagon in control rats, but did so only marginally in thiamine-deficient rats. Arginine also increased the glucagon secretion throughout the period of study in control rats. In thiamine-deficient rats the glucagon secretion was pronounced only after 20 min of arginine administration. These results suggest that an unimpaired glucose metabolism is a prerequisite to induce proper insulin secretion. Only proper insulin secretion can check the glucagon secretion rather than the increased glucose level. Hypoglycemia can induce glucagon secretion independent of the insulin level.     

Somatic excision of the Mu1 transposable element of maize.

The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.     

The Molecular Basis for Modulation of Human Vγ9Vδ2 T Cell Responses by CD277/Butyrophilin-3 (BTN3A)-specific Antibodies

Human Vγ9Vδ2 T cells are well known for their rapid and potent response to infection and tumorigenesis when in the presence of endogenous or exogenous phosphoisoprenoids. However, the molecular mechanisms behind the activation of this γδ T cell population remains unclear. Evidence pointing to a role for the CD277/butyrophilin-3 (BTN3A) molecules in this response led us to investigate the structures of these molecules and their modifications upon binding to an agonist antibody (20.1) that mimics phosphoisoprenoid-mediated Vγ9Vδ2 activation and an antagonist antibody (103.2) that inhibits this reactivity. We find that the three BTN3A isoforms: BTN3A1, BTN3A2, and BTN3A3, have high structural homology to the B7 superfamily of proteins and exist as V-shaped homodimers in solution, associating through the membrane proximal C-type Ig domain. The 20.1 and 103.2 antibodies bind to separate epitopes on the BTN3A Ig-V domain with high affinity but likely with different valencies based on their binding orientation. These structures directly complement functional studies of this system that demonstrate that BTN3A1 is necessary for Vγ9Vδ2 activation and begin to unravel the extracellular events that occur during stimulation through the Vγ9Vδ2 T cell receptor.     

Glutamate metabolism in the brain of young rats exposed to organic and inorganic lead

The synthesis of glutamate and its conversion to glutamine and GABA were studied using labelled glucose in cerebral cortex, cerebellum and brainstem of rats intoxicated acutely with tetraethyl lead and chronically with lead acetate. To assess the interconversion and the synaptosomal accumulation of these amino acids, the labelling of glutamate, glutamine and GABA were measured in whole tissue and synaptosomes after giving labelled glutamate. The radioactive carbon dioxide production from labelled glutamate by brain slices was measured to evaluate the oxidation of glutamate. The tissue levels of glutamate, glutamine and GABA and the activity of glutamate decarboxylase were also measured in both conditions.In inorganic lead toxicity, even though the glutamate pool size was reduced, the glutamate-glutamine cycling between synaptosomes and astrocytes was increased. The oxidation of glutamate and the glutamate-GABA cycling were reduced. These findings suggest that brain tries to maintain the endogenous glutamate levels by decreasing the oxidation of glutamate and increasing the uptake systems and the cycling through glutamine in inorganic lead toxicity. In organic lead toxicity, the glutamate pool as well as glutamate turnover was reduced markedly resulting in complete distortion of glutamate metabolism.     

Electrokinetic behaviour of subpopulations of T lymphocytes in allografted mice.

In AKR(H-2k) mice transplanted with DBA/2(H-2d) skin grafts, the mean electrophoretic mobilities (EPM) of total lymph node cells (LNC) and T cells were significantly reduced. Subpopulations of T lymphocytes, viz. CD4- (CD8- (CD4+) T cells were obtained by depletion treatment of T cells with monoclonal antibodies specific for these surface antigens and complement. Determination of EPM of these two subpopulations revealed that the electrokinetic change following immunostimulation equally afflicted these two subpopulations. These data thus confirmed that CD4+ as well as CD8+ T cells were activated in MHC unmatched allograft rejection.