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.     

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.     

Shoot organogenesis in elite clones of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis>

An efficient shoot organogenesis system has been developed from mature plants of selected elite clones of Eucalyptus tereticornis Sm. Cultures were established using nodal explants taken from freshly coppice shoots cultured on Murashige and Skoog medium containing 58 mM sucrose, 0.7% (w/v) agar (MS medium) and supplemented with 2.5 μM benzyladenine (BA) and 0.5 μM α-naphthaleneacetic acid (NAA). Shoot organogenesis was achieved from leaf segments taken from elongated microshoots on MS medium supplemented with 5.0 μM BA and 1.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of cefotaxime to the medium promoted shoot differentiation, whereas carbenicillin and cephalexin inhibited shoot differentiation. Maximum shoot bud organogenesis (44.6%) occurred in explants cultured on MS medium supplemented with 5.0 μM BA, 1.0 μM 2,4-D and 500 mg/l cefotaxime. Leaf maturity influenced shoot regeneration, with maximum shoot organogeneisis (40.5%) occurring when the source of explants was the fifth leaf (14–16 days old) from the top of microshoot. Shoot organogenic potential also varied amongst the different clones of E. tereticornis. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses indicated clonal uniformity of the newly formed shoots/plants, and these were also found to be true-to-type.     

Biometry of frozen-thawed sperm from eight breeds of Indian buffaloes (Bubalus bubalis)

Sperm morphometry, in combination with other objective traits, can be useful for developing a fertility index. The objective of the present study was to measure various biometric end points of frozen-thawed sperm from eight breeds of Indian buffaloes (Murrah, Surti, Tarai, Mehsana, Jaffrabadi, Bhadawari, Pandharpuri and Nili-Ravi). The sperm head of Pandharpuri buffaloes had the greatest length (10.21 microm), width (6.05 microm), area (52.31 microm(2)) and perimeter (31.86 microm). The ratio of sperm width to length was also greatest (0.61) in Pandharpuri as well as in two other breeds, viz. Nili-Ravi and Jaffrabadi. Murrah had the smallest sperm head width (4.75 microm), area (41.65 microm(2)) and perimeter (29.17 microm), but its sperm tail was longest (57.02 microm), along with that of Jaffrabadi buffaloes (56.96 microm). Based on mean values of sperm tail length, mid piece length and its width the eight buffalo breeds were categorized into three, four and five groups, respectively. Multivariate analysis and clustering put six breeds (Surti, Tarai, Mehsana, Jaffrabadi, Bhadawari and Nili-Ravi) in one cluster, whereas Murrah and Pandharpuri appeared as separate entities.     

Simvastatin potentiates TNF-alpha-induced apoptosis through the down-regulation of NF-kappaB-dependent antiapoptotic gene products: role of IkappaBalpha kinase and TGF-beta-activated kinase-1

Numerous recent reports suggest that statins (hydroxy-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. Therefore, in this article, we investigated the effects of simvastatin on TNF-alpha-induced cell signaling. We found that simvastatin potentiated the apoptosis induced by TNF-alpha as indicated by intracellular esterase activity, caspase activation, TUNEL, and annexin V staining. This effect of simvastatin correlated with down-regulation of various gene products that mediate cell proliferation (cyclin D1 and cyclooxygenase-2), cell survival (Bcl-2, Bcl-x(L), cellular FLIP, inhibitor of apoptosis protein 1, inhibitor of apoptosis protein 2, and survivin), invasion (matrix mellatoproteinase-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor); all known to be regulated by the NF-kappaB. We found that simvastatin inhibited TNF-alpha-induced NF-kappaB activation, and l-mevalonate reversed the suppressive effect, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. Simvastatin suppressed not only the inducible but also the constitutive NF-kappaB activation. Simvastatin inhibited TNF-alpha-induced IkappaBalpha kinase activation, which led to inhibition of IkappaBalpha phosphorylation and degradation, suppression of p65 phosphorylation, and translocation to the nucleus. NF-kappaB-dependent reporter gene expression induced by TNF-alpha, TNFR1, TNFR-associated death domain protein, TNFR-associated factor 2, TGF-beta-activated kinase 1, receptor-interacting protein, NF-kappaB-inducing kinase, and IkappaB kinase beta was abolished by simvastatin. Overall, our results provide novel insight into the role of simvastatin in potentially preventing and treating cancer through modulation of IkappaB kinase and NF-kappaB-regulated gene products.     

Mononuclear cobalt(II) carboxylate complexes: Synthesis, molecular structure and selective oxygenation study

Some cobalt carboxylate (both mononuclear as well as binuclear) complexes have been prepared by using hindered hydrotris(3,5-diisopropyl-1-pyrazolyl)borate (Tp^iPr2) as supporting ligand. The reaction of [Tp^iPr2Co(NO₃)] (2) with sodium benzoate resulted in the formation of acetonitrile coordinated complex [Tp^iPr2Co(OBz)(CH₃CN)] (3) whereas the reaction of 2 with sodium fluorobenzoate gave coordinately unsaturated five coordinate complex of the type [Tp^iPr2Co(F-OBz)] (4). The oxidation of compound 4 in the presence of 3,5-diisopropylpyrazole resulted in the formation of a unique compound (5) where only one methine carbon of isopropyl group on pyrazole ring of hydrotris(3,5-diisopropyl-1-pyrazolyl)borate oxidized and coordinated with cobalt center. In compound 5, the binding behavior of fluorobenzoate also changes from bidentate to monodentate and the nonbonded oxygen atom formed intramolecular hydrogen bond with the hydrogen atom of the NH fragment of the coordinated . X-ray crystallography and IR studies confirmed the existence of hydrogen bonding in complex 5. The pyrazolato bridged binuclear cobalt(II) complex (6) was prepared by the reaction of hydrated cobalt(II) nitrate, 3,5-diisopropylpyrazole and sodium nitrobenzoate where, each cobalt is four coordinate. The X-ray structure of 6 showed that the NH fragment of terminally coordinated formed intramolecular hydrogen bonding with nonbonded oxygen atom of monodentately coordinated nitrobenzoate.