Syk is downstream of intercellular adhesion molecule-1 and mediates human rhinovirus activation of p38 MAPK in airway epithelial cells

The airway epithelium is the primary target of inhaled pathogens such as human rhinovirus (HRV). Airway epithelial cells express ICAM-1, the major receptor for HRV. HRV binding to ICAM-1 mediates not only viral entry and replication but also a signaling cascade that leads to enhanced inflammatory mediator production. The specific signaling molecules and pathways activated by HRV-ICAM-1 interactions are not well characterized, although studies in human airway epithelia implicate a role for the p38 MAPK in HRV-induced cytokine production. In the current study, we report that Syk, an important immunoregulatory protein tyrosine kinase, is highly expressed by primary and cultured human airway epithelial cells and is activated in response to infection with HRV16. Biochemical studies revealed that ICAM-1 engagement by HRV and cross-linking Abs enhanced the coassociation of Syk with ICAM-1 and ezrin, a cytoskeletal linker protein. In polarized airway epithelial cells, Syk is diffusely distributed in the cytosol under basal conditions but, following engagement of ICAM-1 by cross-linking Abs, is recruited to the plasma membrane. The enhanced Syk-ICAM-1 association following HRV exposure is accompanied by Syk phosphorylation. ICAM-1 engagement by HRV and cross-linking Abs also induced phosphorylation of p38 in a Syk-dependent manner, and conversely, knockdown of Syk by short interfering (si)RNA substantially diminished p38 activation and IL-8 gene expression. Taken together, these observations identify Syk as an important mediator of the airway epithelial cell inflammatory response by modulating p38 phosphorylation and IL-8 gene expression following ICAM-1 engagement by HRV.     

Isolation and functional analysis of cotton universal stress protein promoter in response to phytohormones and abiotic stresses

The 949 bp promoter fragment upstream from the translation initiation site of the GUSP gene encoding a universal stress protein was isolated from the genomic DNA of Gossypium arboreum. Some putative cis-acting elements involved in stress responses including E-box, ABRE, DPBF-box, and MYB-core elements were found in the promoter region. In an Agrobacterium-mediated transient expression assay, strong activation of the GUSP full promoter region occurred in tobacco leaves following dehydration, abscisic acid, salt, heavy metal, gibberellic acid and dark treatments. Deletion analysis of the promoter revealed that the dehydration, abscisic acid and salt responses were affected by the deletion between −208 and −949 bp and showed 2–4-fold induction. However, in response to dark, gibberellic acid and heavy metals the induction was only 2-fold. These findings further our understanding of the regulation of GUSP expression. This is an important study as no report of this universal stress protein promoter is available in literature.     

Synthesis and antiinflammatory activity of some new 1,3,5-trisubstituted pyrazolines bearing benzene sulfonamide

Nineteen new 2-pyrazoline bearing benzenesulfonamide derivatives were synthesized by condensing chalcones with 4-hydrazinonbenzenesulfonamide hydrochloride. Their chemical structures were proved by means of IR, ¹H NMR, ¹³C NMR, mass spectroscopic and elemental analyses data. These compounds were tested at dose of 20 mg/kg for their anti-inflammatory activity in carrageenan-induced rat paw edema model and volume of paw edema was measured at 0, 3 and 5 h. Two compounds 3k and 3l were found to be more active than celecoxib throughout the study (at 3 and 5 h). While two other compounds 3m and 3n showed more potent activity than celecoxib at 5 h. They are devoid of ulcerogenic potential when administered orally at a dose of 60 mg/kg. Compounds (3k–m) showed COX-1 and COX-2 inhibitory activity at 0.05 μM.     

The bovine lactation genome: insights into the evolution of mammalian milk

Background

The newly assembled Bos taurus genome sequence enables the linkage of bovine milk and lactation data with other mammalian genomes.

Results

Using publicly available milk proteome data and mammary expressed sequence tags, 197 milk protein genes and over 6,000 mammary genes were identified in the bovine genome. Intersection of these genes with 238 milk production quantitative trait loci curated from the literature decreased the search space for milk trait effectors by more than an order of magnitude. Genome location analysis revealed a tendency for milk protein genes to be clustered with other mammary genes. Using the genomes of a monotreme (platypus), a marsupial (opossum), and five placental mammals (bovine, human, dog, mice, rat), gene loss and duplication, phylogeny, sequence conservation, and evolution were examined. Compared with other genes in the bovine genome, milk and mammary genes are: more likely to be present in all mammals; more likely to be duplicated in therians; more highly conserved across Mammalia; and evolving more slowly along the bovine lineage. The most divergent proteins in milk were associated with nutritional and immunological components of milk, whereas highly conserved proteins were associated with secretory processes.

Conclusions

Although both copy number and sequence variation contribute to the diversity of milk protein composition across species, our results suggest that this diversity is primarily due to other mechanisms. Our findings support the essentiality of milk to the survival of mammalian neonates and the establishment of milk secretory mechanisms more than 160 million years ago.     

Synthesis,biological evaluation,and docking studies of novel heterocyclic diaryl compounds as selective COX-2 inhibitors

Three novel series of diaryl heterocyclic derivatives bearing the 2-oxo-5H-furan, 2-oxo-3H-1,3-oxazole, and 1H-pyrazole moieties as the central heterocyclic ring were synthesized and their in vitro inhibitory activities on COX-1 and COX-2 isoforms were evaluated using a purified enzyme assay. The 2-oxo-5H-furan derivative 6b was identified as potent COX inhibitor with selectivity toward COX-1 (COX-1 IC₅₀ = 0.061 μM and COX-2 IC₅₀ = 0.325 μM; selectivity index (SI) = 0.19). Among the 1H-pyrazole derivatives, 11b was found to be a potent COX-2 inhibitor, about 38 times more potent than Rofecoxib (COX-2 IC₅₀ = 0.011 μM and 0.398 μM, respectively), but showed no selectivity for COX-2 isoform. Compound 11c demonstrated strong and selective COX-2 inhibitory activity (COX-1 IC₅₀ = 1 μM, COX-2 IC₅₀ = 0.011 μM; SI = ～92). Molecular docking studies of compounds 6b and 11b–d into the binding sites of COX-1 and COX-2 allowed to shed light on the binding mode of these novel COX inhibitors.     

Gossypium arboreum GHSP26 enhances drought tolerance in Gossypium hirsutum

Heat‐shock proteins (HSP) are molecular chaperones for protein molecules. These proteins play an important role in protein–protein interactions such as, folding and assisting in the establishment of proper protein conformation and prevention of unwanted protein aggregation. A small HSP gene GHSP26 present in Gossypium arboreum responds to dehydration. In the present study, an attempt was made to overcome the problem of drought stress in cotton. A cDNA of GHSP26 was isolated from G. arboreum, cloned in plant expression vector, pCAMBIA‐1301 driven by the cauliflower mosaic virus 35S promoter and introduced into Gossypium hirsutum. The integration and expression studies of putative transgenic plants were performed through GUS assay; PCR from genomic DNA, and quantitative real‐time PCR analysis. Transgenic cotton plants showed an enhanced drought tolerance, suggesting that GHSP26 may play a role in plant responsiveness to drought. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Multiple traits of agronomic importance in transgenic indica rice plants: analysis of transgene integration patterns,expression levels and stability

We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) -endotoxin genes cry1Ac and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R₀ and R₁ plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.     

Effect of dietary n-3 and n-6 fatty acids on tobramycin induced nephrotoxicity

Post fish oil(n-3 fatty acids) treatment (5mg/kg/day for 12 days) was effective in bringing the reversal of tobramycin (160mg/kg/day,ip for 12 days) induced nephrotoxicity in albino rats as was evident by normal urea, creatinine, cholesterol and inorganic phosphate levels in the serum of the treatment group compared with group receiving tobramycin only. The return of normal levels of alkaline and acid phosphatase in kidney homogenates of post fish oil treatment group also indicated the beneficial effect of dietary n-3 fatty acids(fish oil) more than n-6 fatty acids(olive oil).The results suggest that oral supplements of dietary n-3 fatty acids (fish oil) for nearly two weeks after tobramycin exposure is more beneficial than n-6 fatty acids (olive oil) as it results in reversal of nephrotoxicity induced by tobramycin.     

Prediction of possible sites for posttranslational modifications in human gamma crystallins: effect of glycation on the structure of human gamma-B-crystallin as analyzed by molecular modeling

Crystallins are recognized as one of the long-lived proteins of lens tissue that might serve as the target for several posttranslational modifications leading to cataract development. We have studied several such sites present in the human gamma-crystallins based either on PROSITE pattern search results or earlier experimental evidences. Their probabilities were examined on the basis of the database analysis of the gamma-crystallin sequences and on their specific locations in the constructed homology models. An N-glycosylation site in human gammaD-crystallin and several phosphorylation sites in all four human gamma-crystallins were predicted by the PROSITE search. Some of these sites were found to be strongly conserved in the gamma-crystallin sequences from different sources. An extensive analysis of these sites was performed to predict their probabilities as potential sites for protein modifications. Glycation studies were performed separately by attaching sugars to the human gammaB-crystallin model, and the effect of binding was analyzed. The studies showed that the major effect of alphaD-glucose (alphaD-G) and alphaD-glucose-6-phosphate (alphaD-G6P) binding was the disruption of charges not only at the surface but also within the molecule. Only a minor alteration in the distances of sulfhydryl groups of cysteines and on their positions in the three-dimensional models were observed, leading us to assume that glycation alone is not responsible for intra- and intermolecular disulfide bond formation.