Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS^-/-) are protected against ALI. In both wild-type and eNOS^-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS^-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS^-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS^-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS^-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr³⁴. Finally, we found that the reduction in NOS uncoupling in eNOS^-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.     

Characterization and Cloning of ODAP Degrading Gene from a Soil Microbe

The strain BYT-1, capable of utilizing ODAP/DAP as a sole source of nitrogen and carbon was identified as Psuedomonas stutzeri by various microbial and biochemical tests. Transformation experiments showed that the ODAP utilizing property Is encoded by the plasmid. Restriction of plasmid DNA with Pstl, followed by cloning of fragments and screening of ODAP containing medium, led to the isolation of a clone with insert size of ?3.3 kb, which encoded ODAP metabolizing property. The growth and ODAP/DAP utilization by this clone (T_B) was almost similar to that of the wild type strain.     

Molecular cloning and characterization of the mouse UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I gene

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.     

Purification of murine oncornaviral phosphoproteins using alkyl agarose derivatives..

Methods for the purification of both murine mammary tumor (type B) and murine leukemia (type C) oncornaviral phosphoproteins are described, in which chromatography on alkyl-agarose derivatives is used as the primary fractionation step. Gel filtration or ion exchange chromatography on phosphocellulose was the only subsequent step required for the purification of the type B and type C viral proteins, respectively. The two-step protocols also resulted in the co-purification of a low molecular weight core protein from each virus. Recoveries of the viral proteins purified by this method, based on per cent contribution of individual polypeptides to total virion proteins, were 70% or greater. Radioimmunocompetition analysis of the purified murine mammary tumor virus major core protein as well as analysis of the RNA binding properties of purified low molecular weight type C virus proteins suggests that neither antigenic reactivity nor specific RNA binding characteristics are altered by the purification protocols. The availability of these procedures should aid studies on the possible function and immunochemical properties of the native murine oncornaviral phosphoproteins and may also be extended to the purification of other oncornaviral proteins.     

Molecular and morphological characterization of somatic hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. and <Emphasis Type="Italic">S. etuberosum</Emphasis> Lindl.

Interspecific potato somatic hybrids between Solanum tuberosum L. (di)haploid C-13 and 1 endosperm balance number non-tuberous wild species S. etuberosum Lindl. were produced by protoplasts electrofusion. The objective was to transfer virus resistance from this wild species into the cultivated potatoes. Post-fusion products were cultured in VKM medium followed by regeneration of calli in MS₁₃ K medium at 20°C under a 16-h photoperiod, and regenerants were multiplied on MS medium. Twenty-one somatic hybrids were confirmed by RAPD, SSR and cytoplasm (chloroplast/mitochondria) type analysis possessing species-specific diagnostic bands of corresponding parents. Tetraploid nature of these somatic hybrids was determined through flow cytometry analysis. Somatic hybrids showed intermediate phenotypes (plant, leaves and floral morphology) to their parents in glass-house grown plants. All the somatic hybrids were male-fertile. ELISA assay of somatic hybrids after artificial inoculation of Potato virus Y (PVY) infection reveals high PVY resistance.     

Impact of hyperglycemia on the expression of GLUT1 during oral carcinogenesis in rats

Molecular Biology Reports - On the background of the epidemiological link between diabetes and oral cancer, the present study aimed to analyze the potential involvement of selected glucose...     

N‐acetylcysteine counteracts oxidative stress and prevents hCG‐induced apoptosis in rat Leydig cells through down regulation of caspase‐8 and JNK

We have earlier reported that following persistent stimulation with hCG, oxidative stress‐induced apoptosis in rat Leydig cells was mainly achieved through the extrinsic pathway. In the present study, the role of N‐acetylcysteine (NAC) in counteracting the oxidative stress and the mechanisms of inhibition of apoptosis under such conditions were investigated. NAC (1 mM) intervention with repeated hCG stimulation (50 ng/ml, four times, each with 30 min challenge) prevented the decline in Leydig cell viability and the rise in lipid peroxidation and reactive oxygen species. Simultaneously, the activities of the enzymes glutathione‐S‐transferase, catalase, superoxide dismutase and the intracellular glutathione and antioxidant capacity of the treated cells improved significantly. Apoptotic markers Fas, FasL, and caspase‐8, up‐regulated following repeated hCG exposure, were significantly down‐regulated following NAC co‐incubation. While Bcl‐2 expression was fully restored, Bax and caspase‐9 remained unchanged. NAC treatment induced down‐regulation of upstream JNK/pJNK and down‐stream caspase‐3 in the target cells. Taken together, the above findings indicate that NAC counteracted the oxidative stress in Leydig cells induced as a result of repeated hCG stimulation, and inhibited apoptosis by mainly regulating the extrinsic and JNK pathways of metazoan apoptosis. Mol. Reprod. Dev. 77:900–909, 2010. © 2010 Wiley‐Liss, Inc.