Protein kinase C-lambda knockout in embryonic stem cells and adipocytes impairs insulin-stimulated glucose transport

Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.     

Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.     

Mn‐catalase (Alr0998) protects the photosynthetic,nitrogen‐fixing cyanobacterium Anabaena PCC7120 from oxidative stress

Role of the non‐haem, manganese catalase (Mn‐catalase) in oxidative stress tolerance is unknown in cyanobacteria. The ORF alr0998 from the Anabaena PCC7120, which encodes a putative Mn‐catalase, was constitutively overexpressed in Anabaena PCC7120 to generate a recombinant strain, AnKat⁺. The Alr0998 protein could be immunodetected in AnKat⁺ cells and zymographic analysis showed a distinct thermostable catalase activity in the cytosol of AnKat⁺ cells but not in the wild‐type Anabaena PCC7120. The observed catalase activity was insensitive to inhibition by azide indicating that Alr0998 protein is indeed a Mn‐catalase. In response to oxidative stress, the AnKat⁺ showed reduced levels of intracellular ROS which was also corroborated by decreased production of an oxidative stress‐inducible 2‐Cys‐Prx protein. Treatment of wild‐type Anabaena PCC7120 with H₂O₂ caused (i) RNA degradation in vivo, (ii) severe reduction of photosynthetic pigments and CO₂ fixation, (iii) fragmentation and lysis of filaments and (iv) loss of viability. In contrast, the AnKat⁺ strain was protected from all the aforesaid deleterious effect under oxidative stress. This is the first report on protection of an organism from oxidative stress by overexpression of a Mn‐catalase.     

The roles of the shikimate pathway genes,aroA and aroB,in virulence,growth and UV tolerance of Burkholderia glumae strain 411gr‐6

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B. glumae strains showed pigmentation phenotypes producing at least two (yellow–green and purple) pigment compounds in casein–peptone–glucose agar medium. The B. glumae strains LSUPB114 and LSUPB116 are pigment‐deficient mutant derivatives of the virulent and pigment‐proficient strain 411gr‐6, having mini‐Tn5gus insertions in aroA encoding 3‐phosphoshikimate 1‐carboxyvinyltransferase and aroB encoding 3‐dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B. glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.     

Advances in Arachis genomics for peanut improvement

Peanut genomics is very challenging due to its inherent problem of genetic architecture. Blockage of gene flow from diploid wild relatives to the tetraploid; cultivated peanut, recent polyploidization combined with self pollination, and the narrow genetic base of the primary genepool have resulted in low genetic diversity that has remained a major bottleneck for genetic improvement of peanut. Harnessing the rich source of wild relatives has been negligible due to differences in ploidy level as well as genetic drag and undesirable alleles for low yield. Lack of appropriate genomic resources has severely hampered molecular breeding activities, and this crop remains among the less-studied crops. The last five years, however, have witnessed accelerated development of genomic resources such as development of molecular markers, genetic and physical maps, generation of expressed sequenced tags (ESTs), development of mutant resources, and functional genomics platforms that facilitate the identification of QTLs and discovery of genes associated with tolerance/resistance to abiotic and biotic stresses and agronomic traits. Molecular breeding has been initiated for several traits for development of superior genotypes. The genome or at least gene space sequence is expected to be available in near future and this will further accelerate use of biotechnological approaches for peanut improvement.     

Influence of cytoplasmic male sterility on expression of different mechanisms of resistance in sorghum to Atherigona soccata (Diptera: Muscidae)

Atherigona soccata (Rondani) (Diptera: Muscidae) is one of the most important pests of sorghum, Sorghum bicolor (L.) Moench, in Asia, Africa, and the Mediterranean Europe. Exploitation of cytoplasmic male sterility (CMS) for hybrid production has resulted in considerable narrowing of the genetic base and may increase the vulnerability of this crop to insect pests. Therefore, we studied the expression of different mechanisms of resistance in sorghum to A. soccata in CMS (A) and maintainer (B) lines of 12 genotypes under field and greenhouse conditions. The CMS lines of A. soccata-resistant genotypes were preferred for oviposition (78.5 versus 71.5% plants with eggs) and suffered greater deadheart incidence (47.6 versus 41.6%) than the corresponding maintainer lines, whereas such differences were not apparent in CMS lines belonging to the susceptible genotypes (92.7 versus 92.3% plants with eggs and 75.6 versus 74.6% deadhearts) under multichoice field conditions. Similar differences also were observed under controlled conditions in the greenhouse. The larval period (9.0 versus 8.8 d) and pupal mortality (18.4 versus 13.4%) were greater on maintainer lines than that on the CMS lines in the resistant group. The male and female pupal weights, fecundity, and antibiosis index were greater on the CMS than on the maintainer lines. The maintainer lines showed better recovery resistance than the CMS lines, but no such differences were observed in tiller deadhearts. The differences in susceptibility to A. soccata were greater in the A. soccata resistant CMS and maintainer lines than in the CMS and maintainer lines belonging to susceptible genotypes. Conversion of A. soccata-resistant genotypes into alternate less susceptible cytoplasmic backgrounds may be undertaken for developing sorghum hybrids with stable resistance to A. soccata.     

A computational approach to design energetic ionic liquids

The present work deals with the theoretical estimation of ion-pair binding energies and the energetic properties of four ion pairs formed by combining the 1-butyl-2,4-dinitro-3-methyl imidazolium ion with nitrate (I), perchlorate (II), dinitramide (III), or 3,5-dinitro-1,2,4-triazolate (IV) anions. The counterpoise-corrected ion-pair binding energies were calculated for each ion pair at the B3LYP/6-311+G(d,p) level of theory. Results show that the cation–anion interaction is strongest for ion pair I and weakest for IV, indicating that the nitrate (I) has a greater tendency to exist as a stable ionic salt whereas the 3,5-dinitro-1,2,4-triazolate (IV) may exist as an ionic liquid. Natural bond orbital (NBO) analysis and electrostatic potential (ESP) mapping revealed that charge transfer occurs in all of the ion pairs, but is greatest (0.25e) for ion pair I and smallest (0.03e) for IV, resulting in ion pair I being the least polarized. A nucleus-independent chemical shift (NICS) study revealed that the aromaticity of the 1-butyl-2,4-dinitro-3-methyl imidazolium ion significantly increases in ion pair IV, indicating that this has the greatest charge delocalization among all of the four ion pairs considered. Studies of thermodynamic and detonation properties showed that ion pair II is the most energetic ion pair in terms of its detonation velocity (D = 7.5 km s^?1) and detonation pressure (P = 23.1 GPa). It is also envisaged that ion pair IV would exist as an energetic azolium azolate type ionic liquid that could be conveniently used as a secondary explosive characterized by detonation parameters D and P of 6.9 km s^?1 and 19.3 GPa, respectively. These values are comparable to those of conventional explosives such as TNT.     

Comparative Genomic Analysis of Buffalo (Bubalus bubalis) NOD1 and NOD2 Receptors and Their Functional Role in In-Vitro Cellular Immune Response

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo—a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.     

A Longitudinal Study: Changes in Cortical Thickness and Surface Area during Pubertal Maturation

Sex hormones have been shown to contribute to the organization and function of the brain during puberty and adolescence. Moreover, it has been suggested that distinct hormone changes in girls versus boys may contribute to the emergence of sex differences in internalizing and externalizing behavior during adolescence. In the current longitudinal study, the influence of within-subject changes in puberty (physical and hormonal) on cortical thickness and surface area was examined across a 2-year span, while controlling for age. Greater increases in Tanner Stage predicted less superior frontal thinning and decreases in precuneus surface area in both sexes. Significant Tanner Stage and sex interactions were also seen, with less right superior temporal thinning in girls but not boys, as well as greater decreases in the right bank of the superior temporal sulcus surface area in boys compared to girls. In addition, within-subject changes in testosterone over the 2-year follow-up period were found to relate to decreases in middle superior frontal surface area in boys, but increases in surface area in girls. Lastly, larger increases in estradiol in girls predicted greater middle temporal lobe thinning. These results show that within-subject physical and hormonal markers of puberty relate to region and sex-specific changes in cortical development across adolescence.     

Comparative study of perturbations of peripheral markers in different stressors in rats

Stress has been implicated in the etiopathogenesis of several diseases. In the present study, the effects of acute (AS), chronic (CS), and chronic unpredictable stress (CUS) were studied on the ulcer index, adrenal gland mass, and biochemical and hormonal changes in rats. The stress was provided in the form of immobilization-immobilization for 150 min, once only, and for 10 consecutive days in CS and CUS. In CUS, animals received variable unpredictable stressors. Immediately after stress, animals were decapitated, blood was collected, and plasma was separated for the estimation of plasma glucose, triglyceride, cholesterol, creatine kinase (CK), corticosterone, and insulin. The adrenal gland and stomach were also dissected for mass and ulcer scoring, respectively. AS significantly increased the ulcer index, plasma glucose, CK, corticosterone, and insulin. CS and CUS significantly increased the ulcer index, adrenal gland mass, and corticosterone. In CS, a significant decrease in plasma triglyceride and cholesterol levels was found, but in CUS only cholesterol was decreased significantly. High CK activity and hyperglycemia maintain the energy demands of metabolism, and elevated corticosterone desensitizes the insulin receptor in AS. In CS and CUS, prolonged elevation of corticosterone shifts metabolism to utilization of lipids as a secondary substrate by gluconeogenesis. From our experiment, it is clear that AS causes maximum activation of energy metabolism, which becomes specific after habituation in prolonged CS. These biochemical manipulations in the body by using different types of stressors are good markers that can be of great use to understand, target, and manage stress-induced etiologies.