Effect of maternal micronutrients (folic acid, vitamin B12) and omega 3 fatty acids on liver fatty acid desaturases and transport proteins in Wistar rats

A disturbed fatty acid metabolism increases the risk of adult non-communicable diseases. This study examines the effect of maternal micronutrients on the fatty acid composition, desaturase activity, mRNA levels of fatty acid desaturases and transport proteins in the liver. Pregnant female rats were divided into 6 groups at 2 levels of folic acid both in the presence and absence of vitamin B(12). The vitamin B(12) deficient groups were supplemented with omega 3 fatty acid. An imbalance of maternal micronutrients reduces liver docosahexaenoic acid, increases Δ5 desaturase activity but decreases mRNA levels, decreases Δ6 desaturase activity but not mRNA levels as compared to control. mRNA level of Δ5 desaturase reverts back to the levels of the control group as a result of omega 3 fatty acid supplementation. Our data for the first time indicates that maternal micronutrients differentially alter the activity and expression of fatty acid desaturases in the liver.     

Effective targeted gene delivery to dendritic cells via synergetic interaction of mannosylated lipid with DOPE and BCAT

The efficient delivery of plasmids encoding antigenic determinants into dendritic cells (DCs) that control immune response is a promising strategy for rapid development of new vaccines. In this study, we prepared a series of targeted cationic lipoplex based on two synthetic lipid components, mannose-poly(ethylene glycol, MW3000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (Mannose-PEG3000-DSPE) and O-(2R-1,2-di-O-(1'Z-octadecenyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), that were formulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for evaluation as nonviral vectors for transgene expression in DCs. First, we optimized the N/P ratio for maximum transfection and then screened the effects of mannose targeting for further enhancement of transfection levels. Our results indicate that efficient delivery of gWIZ GFP plasmid into DCs was observed for mannose compositions of ～10%, whereas low transfection efficiencies were observed with nontargeted formulations. Mannose-targeted lipofectamine complexes also showed high GFP expression levels in DCs relative to nontargeted lipofectamine controls. The best transfection performance was observed using 10 mol % Mannose-PEG3000-DSPE, 60 mol % BCAT, and 30 mol % DOPE, indicating that the most efficient delivery into DCs occurs via synergistic interaction between mannose targeting and acid-labile, fusogenic BCAT/DOPE formulations. Our data suggest that mannose-PEG3000-DSPE/BCAT/DOPE formulations may be effective gene delivery vehicles for the development of DC-based vaccines.     

Molecular characterization and expression analysis of two crucial MAPKs- jnk1 and erk1 as cellular signal transducers in Labeo rohita in response to PAMPs stimulation and pathogenic invasion

Mitogen-activated protein kinases (MAPKs) are crucial Ser/Thr protein kinases that play important roles in innate immunity by converting extracellular stimuli into a wide range of cellular responses, including the production of cytokines. In this study, two MAPK genes, jnk1 and erk1, were cloned and characterized in rohu (Labeo rohita), a commercially important freshwater fish species in the Indian subcontinent. In healthy rohu, both jnk1 and erk1 gene expressions were highest in the spleen as compared to gill, liver, blood and kidney tissues. In vitro stimulation of the L. rohita gill (LRG) cell line with γ-D-glutamyl-meso-diaminopimelic acid, muramyl dipeptide and polyinosinic: polycytidylic acid (poly I:C) resulted in significantly enhanced expressions of jnk1 and erk1 genes. In the in vivo experiments, jnk1 and erk1 gene expressions were also enhanced in lipopolysaccharides and poly I:C-treatment. Infection of rohu fingerlings with Aeromonas hydrophila and Bacillus subtilis revealed significantly enhanced expressions of the jnk1 and erk1 genes in all of the tested organs/tissues. Together these results imply the important role of jnk1 and erk1 genes in fish during pathogenic invasion and diseases.     

Design and synthesis of novel heterobiaryl amides as metabotropic glutamate receptor subtype 5 antagonists

A series of heterobiaryl amides was designed and synthesized as novel mGluR5 antagonists. The synthesis using palladium catalyzed Suzuki-Miyaura cross-coupling reactions provided an array of compounds with a range of in vitro activities. In particular, compound 9e, 4(3,5-difluorophenyl)-N-(6-methylpyridin-1-yl)picolinamide, exhibited nanomolar affinity at the mGluR5 and will serve as a template for future drug design.     

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization

BACKGROUND: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.     

Hepatoprotective action of abhrak bhasma, an ayurvedic drug in albino rats against hepatitis induced by CCl4

Abhrak bhasma is a commonly used ayurvedic drug against many diseases including hepatitis. It is tested in albino rats using a model of hepatitis induced by a single dose of CCl4 (3 ml/kg body wt). Different doses of abhrak bhasma (10, 20, 30 and 40 mg/kg body wt) were tested to decide the dose related hepatoprotective efficacy. The centrolobular necrosis induced by single dose of CCl4 was reduced significantly by abhrak bhasma (10 mg) and liver histology was also protected by 20 mg dose. Liver acid lipase activity was lowered, while alkaline and lipoprotein lipase activities were elevated due to treatment of single dose of CCl4. Abhrak bhasma counteracted the action of CCl4 on liver lipolytic enzymes. CCl4 did not alter the kidney histologically. Activities of three lipases of rat kidney (acid, alkaline and lipoprotein lipases) were reduced by CCl4 treatment and were reversed by administration of abhrak bhasma. Acid lipase activity of rat adipose tissue was reduced by CCl4 treatment. On the contrary alkaline, lipoprotein and hormone sensitive lipases were enhanced after 24 hr of administration of CCl4. Acid lipase activity was raised by administration of different doses of abhrak bhasma concurrent with CCl4. Abhrak bhasma treatment along with CCl4 enhanced alkaline lipase activity at 10 and 20 mg dose and later it was reduced at 30 and 40 mg doses and came to normal levels. Lipoprotein and hormone sensitive lipases were reduced by the counteraction of increasing doses of abhrak bhasma.     

Characterization of high temperature-tolerant rhizobia isolated from Prosopis juliflora grown in alkaline soil

A method was developed for the fast screening and selection of high-temperature tolerant rhizobial strains from root nodules of Prosopis juliflora growing in alkaline soils. The high-temperature tolerant rhizobia were selected from 2,500 Rhizobium isolates with similar growth patterns on yeast mannitol agar plates after 72 h incubation at 30 and 45 degrees C, followed by a second screening at 47.5 degrees C. Seventeen high-temperature tolerant rhizobial strains having distinguishable protein band patterns were finally selected for further screening by subjecting them to temperature stress up to 60 degrees C in yeast mannitol broth for 6 h. The high-temperature tolerant strains were NBRI12, NBRI329, NBRI330, NBRI332, and NBRI133. Using this procedure, a large number of rhizobia from root nodules of P. juliflora were screened for high-temperature tolerance. The assimilation of several carbon sources, tolerance to high pH and salt stress, and ability to nodulate P. juliflora growing in a glasshouse and nursery of the strains were studied. All five isolates had higher plant dry weight in the range of 29.9 to 88.6% in comparison with uninoculated nursery-grown plants. It was demonstrated that it is possible to screen in nature for superior rhizobia exemplified by the isolation of temperature-tolerant strains, which established effective symbiosis with nursery-grown P. juliflora. These findings indicate a correlation between strain performance under in vitro stress in pure culture and strain behavior under symbiotic conditions. Pure culture evaluation may be a useful tool in search for Rhizobium strains better suited for soil environments where high temperature, pH, and salt stress constitutes a limitation for symbiotic biological nitrogen fixation.     

Spatial and temporal activity of the dentin sialophosphoprotein gene promoter: differential regulation in odontoblasts and ameloblasts

Dentin sialoprotein and dentin phosphoprotein are non-collagenous proteins that are cleavage products of dentin sialophosphoprotein (DSPP). Although these two protein products are believed to have a crucial role in the process of tooth mineralization, their precise biological functions and the molecular mechanisms of gene regulation are not clearly understood. To understand such functions, we have developed a transgenic mouse model expressing a reporter gene (lacZ) under the control of approximately 6 kb upstream sequences of Dspp. The transgenic fusion protein was designed to reside within the cells to facilitate the precise identification of cell type and developmental stages at which the Dspp-lacZ gene is expressed. The results presented in this report demonstrate: (a) the 6 kb upstream sequences of Dspp have the necessary regulatory elements to direct the tissue specific expression of the transgene similar to endogenous Dspp, (b) both odontoblasts and ameloblasts exhibit transgene expression in a differentiation dependent manner, and (c) a differential regulation of the transgene in odontoblasts and ameloblasts occurs during tooth development and mineralization.     

Immunolocalization and functional role of Sclerotium rolfsii lectin in development of fungus by interaction with its endogenous receptor

Many fungi are known to secrete lectins, but their functional roles are not clearly understood. Sclerotium rolfsii, a soilborne plant pathogenic fungus capable of forming fruiting bodies called sclerotial bodies, secrete a cell wall-associated Thomsen-Friedenreich antigen-specific lectin. To understand the functional role of this lectin, we examined its occurrence and expression during development of the fungus. Furthermore, putative endogenous receptors of the lectin were examined to substantiate the functional role of the lectin. Immunolocalization studies using FITC-labeled lectin antibodies revealed discrete distribution of lectin sites at the branching points of the developing mycelia and uniformly occurring lectin sites on the mature sclerotial bodies. During development of the fungus the lectin is expressed in small amounts on the vegetative mycelia and reaching very high levels in mature sclerotial bodies with a sudden spurt in secretion at the maturation stage. Capping of the lectin sites on the sclerotial bodies by lectin antibodies or haptens inhibit strongly the germination of these bodies, indicating functional significance of the lectin. At the maturation stage the lectin interacts with the cell wall-associated putative endogenous receptor leading to the aggregation of mycelium to form sclerotial bodies. The lectin-receptor complex probably acts as signaling molecule in the germination process of sclerotial bodies. Using biotinylated lectin, the receptors were identified by determining the specific lectin binding to lipid components, extracted from sclerotial bodies, and separated on thin-layer chromatograms. Preliminary characterization studies indicated that the receptors are glycosphingolipids and resemble inositolphosphoceramides. These findings together demonstrate the importance of lectin-receptor interactions to explain hitherto speculated functional role of the lectins and also the glycosphingolipids of fungi.