Genome wide association mapping and candidate gene analysis for pod shatter resistance in Brassica juncea and its progenitor species

Molecular Biology Reports - We investigated phenotypic variations for pod shattering, pod length and number of seeds per pod in large germplasm collections of Brassica juncea (2n?=?36;...     

Plant proteomics in India and Nepal: current status and challenges ahead

Plant proteomics has made tremendous contributions in understanding the complex processes of plant biology. Here, its current status in India and Nepal is discussed. Gel-based proteomics is predominantly utilized on crops and non-crops to analyze majorly abiotic (49 %) and biotic (18 %) stress, development (11 %) and post-translational modifications (7 %). Rice is the most explored system (36 %) with major focus on abiotic mainly dehydration (36 %) stress. In spite of expensive proteomics setup and scarcity of trained workforce, output in form of publications is encouraging. To boost plant proteomics in India and Nepal, researchers have discussed ground level issues among themselves and with the International Plant Proteomics Organization (INPPO) to act in priority on concerns like food security. Active collaboration may help in translating this knowledge to fruitful applications.     

Quantitation of lysolipids, fatty acids, and phospholipase A2 activity and correlation with membrane polarity

Acrylodan-labeled rat-intestinal fatty acid binding protein, ADIFAB, binds both of lysophosphatidylcholines (LPC) and FA. Binding displaces Acrylodan and its fluorescence peak shifts from 432 to 505 nm. A fluorescence assay that relies on this shift is presented for quantitating LPC, FA, and phospholipase A(2) (PLA(2)) activity in phospholipid bilayers in absolute units of μM/min/mg of enzyme. This is a development over an earlier assay that took into account only FA binding. Activities of bee venom PLA(2) on dipalmitoylphosphatidylcholine (DPPC) and dioleylphosphatidylcholine (DOPC) bilayers were measured. Standard pH-Stat assays validated the present assay. Products increase linearly with time for about one minute in DOPC and five minutes in DPPC corresponding to completion of 5 to 8% hydrolysis in DOPC and 20% in DPPC. Membrane polarity and microviscosity measured using electron spin resonance (ESR) exhibited discontinuities at compositions that mimicked similar percentages of hydrolysis products in the respective bilayers. The observed hydrolysis rate decrease following the initial linear period thus correlates to changes in membrane polarity. The ability of the assay to yield actual product concentrations, reveal structure in the reaction progress curves, and interpretation in light of the ESR data bring insight into the shape of the reaction curve.     

Alterations in membrane fluidity and dynamics in experimental colon cancer and its chemoprevention by diclofenac

Acs2p is one of two acetyl-coenzyme A synthetases in Saccharomyces cerevisiae. We have prepared and characterized a monoclonal antibody specific for Acs2p and find that Acs2p is localized primarily to the nucleus, including the nucleolus, with a minor amount in the cytosol. We find that Acs2p is required for replicative longevity: an acs2? strain has a reduced replicative life span compared to wild-type and acs1? strains. Furthermore, replicatively aged acs2? cells contain elevated levels of extrachromosomal rDNA circles, and silencing at the rDNA locus is impaired in an acs2? strain. These findings indicate that Acs2p-mediated synthesis of acetyl-CoA in the nucleus functions to promote rDNA silencing and replicative longevity in yeast.     

Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10⁴–10⁹ cells per g tissue) than those with H or VFR feet.     

Mixed micelles of monomeric and dimeric cationic surfactants with phospholipids: effect of hydrophobic interactions

Surface tension (gamma) and time resolved fluorescence quenching (TRFQ) measurements have been performed on the binary mixtures of monomeric as well as dimeric alkylammonium bromides with l-alpha-dimyristoylphosphatidycholine (DMPC) and L-alpha-dipalmitoylphosphatidycholine (DPPC). The critical micelle concentration (cmc) has been evaluated from the gamma measurements. The gamma plots show two breaks in the gamma versus [total surfactant] curves in most of the cases. The first break (C1) has been attributed to the mixed vesicle formation process. The break down of the vesicles leads to the mixed micellization between the surfactant and phospholipid monomers at the second break (C2). The amount of surfactant used in the vesicle breakdown process (DeltaC) increases linearly with the increase in the amount of phospholipid and depends significantly on the hydrophobicities of the cationic components. The surface area per molecule (a) evaluated from the gamma plots indicates compact monolayer formation in the case of monomeric surfactants with lower hydrophobicities and reverse is observed for dimeric surfactants. The pyrene life time (tau) of the solubilized pyrene in the hydrophobic environment of mixed micelles, fully supports the conclusion that derived from a.     

TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions: EVIDENCE FOR DISTINCT BINDING MECHANISMS FOR DIFFERENT TspanC8 PROTEINS*

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting.     

Per1/Per2–Igf2 axis–mediated circadian regulation of myogenic differentiation

Circadian rhythms regulate cell proliferation and differentiation, but circadian control of tissue regeneration remains elusive at the molecular level. Here, we show that proper myoblast differentiation and muscle regeneration are regulated by the circadian master regulators Per1 and Per2. Depletion of Per1 or Per2 suppressed myoblast differentiation in vitro and muscle regeneration in vivo, demonstrating their nonredundant functions. Both Per1 and Per2 were required for the activation of Igf2, an autocrine promoter of myoblast differentiation, accompanied by Per-dependent recruitment of RNA polymerase II, dynamic histone modifications at the Igf2 promoter and enhancer, and the promoter–enhancer interaction. This circadian epigenetic priming created a preferred time window for initiating myoblast differentiation. Consistently, muscle regeneration was faster if initiated at night, when Per1, Per2, and Igf2 were highly expressed compared with morning. This study reveals the circadian timing as a significant factor for effective muscle cell differentiation and regeneration.     

Nitric Oxide Modulates the Expression of Proteins and Promotes Epiphyllous Bud Differentiation in Kalanchoe pinnata

Nitric oxide (NO), a recent addition to the signaling molecules in plants, plays an important role in mediating both biotic and abiotic stress responses. The occurrence of reproductive/vegetative structures, known as epiphylly, on the surface of leaves is a stress survival mechanism exhibited by some plants, including Kalanchoe pinnata. In the present study the role of NO during epiphyllous bud differentiation was investigated. NO donors l-arginine, NaNO₂, and SNP promoted epiphyllous bud differentiation in a dose-dependent manner, whereas NO inhibitors reversed the effect, suggesting the involvement of NO in the process. Albeit numerous physiological processes are reported to be modulated by NO, but their correlation with NO-responsive genes and proteins needs to be established. To address this issue, NO-responsive proteins (NORPs) were identified using 2D-PAGE and MS analysis. Major NORPs that were identified belonged to photosynthesis, oxidative phosphorylation, signaling, and stress metabolism, aptly indicating their probable role in this stress-induced growth process. The present study clearly indicates the role of NO in this stress survival mechanism, thus strongly affirming its role in stress responses.     

Towards the development of better crops by genetic transformation using engineered plant chromosomes

Plant Biotechnology involves manipulation of genetic material to develop better crops. Keeping in view the challenges being faced by humanity in terms of shortage of food and other resources, we need to continuously upgrade the genomic technologies and fine tune the existing methods. For efficient genetic transformation, Agrobacterium-mediated as well as direct delivery methods have been used successfully. However, these methods suffer from many disadvantages especially in terms of transfer of large genes, gene complexes and gene silencing. To overcome these problems, recently, some efforts have been made to develop genetic transformation systems based on engineered plant chromosomes called minichromosomes or plant artificial chromosomes. Two approaches namely, “top-down” or “bottom-up” have been used for minichromosomes. The former involves engineering of the existing chromosomes within a cell and the latter de novo assembling of chromosomes from the basic constituents. While some success has been achieved using these chromosomes as vectors for genetic transformation in maize, however, more studies are needed to extend this technology to crop plants. The present review attempts to trace the genesis of minichromosomes and discusses their potential of development into plant artificial chromosome vectors. The use of these vectors in genetic transformation will greatly ameliorate the food problem and help to achieve the UN Millennium development goals.