Age-dependent renal accumulation of 4-hydroxy-2-nonenal (HNE)-modified proteins following parenteral administration of ferric nitrilotriacetate commensurate with its differential toxicity: implications for the involvement of HNE-protein adducts in oxidative stress and carcinogenesis

In this study, we show that the toxicity of ferric nitrilotriacetate (Fe-NTA) can be correlated with the tissue accumulation of 4-hydroxy-2-nonenal (HNE)-modified protein adducts. It is observed that the toxic manifestations of Fe-NTA gradually increase with the increasing age of animals. A dose of Fe-NTA which produces almost 100% mortality in aged rats causes 70% mortality in adults, 30% in pups, 20% in litters, and less than 10% in neonates. The age-dependent increase in its toxicity is also evident from the data of renal microsomal lipid peroxidation and hydrogen peroxide generation. No significant difference in the generation of H2O2 and induction of renal microsomal lipid peroxidation between saline- and Fe-NTA-treated neonates, litters, and pups could be observed. However, in adult rats, a significant increase in both of the parameters was observed which was even greater in aged rats. On the contrary, renal glutathione levels in these animals show an inverse relationship with the oxidant generation. In neonates, litters, and pups the maximum decrease of glutathione was up to 22%, whereas in adult and aged rats, the depletion was more than 60% of their respective saline-treated controls. Parallel to this data, blood urea nitrogen and creatinine, the indicators of renal damage, show a significant increase in Fe-NTA-treated adult and aged rats only, whereas no significant alterations were observed in other groups. Similarly, the magnitude of ODC induction and [3H]thymidine incorporation was much higher in aged and adult rats in comparison to other groups of animals after Fe-NTA treatment. Additionally, the immunohistochemical localization studies show a significant increase in HNE-modified protein adducts in kidney of adult and aged rats, whereas no significant staining was observed in other groups. A similar increase in the level of protein carbonyls has also been observed with the increasing age of rats. These data suggest that the toxicity of Fe-NTA increases with the increasing age of rats and correlates with the accumulation of HNE-modified protein adducts. It may also be speculated that Fe-NTA-mediated renal toxicity leading to carcinogenesis may be related to the tissue accumulation of HNE-modified protein adducts. However, further studies are needed to establish a definite role of HNE-modified proteins in Fe-NTA-mediated carcinogenesis.     

Improved growth and essential oil yield and quality in Foeniculum vulgare mill on mycorrhizal inoculation supplemented with P-fertilizer

Two arbuscular mycorrhizal (AM) fungi Glomus macrocarpum and Glomus fasciculatum significantly improved growth and essential oil concentration of Foeniculum vulgare Mill. However, AM inoculation of plants along with phosphorus fertilization significantly enhanced growth, P-uptake and essential oil content of plants compared to either of the components applied separately. Among the two fungal inoculants, G. fasciculatum registered the highest growth at both levels of phosphorus used with up to 78% increase in essential oil concentration of fennel seeds over non-mycorrhizal control. The essential oil characterization by gas liquid chromatography revealed that the level of anethol was significantly enhanced on mycorrhization.     

A comparative study on phyllosphere nitrogen fixation by newly isolated Corynebacterium sp. & Flavobacterium sp. and their potentialities as biofertilizer

A number of nitrogen fixing bacteria has been isolated from forest phyllosphere on the basis of nitrogenase activity. Among them two best isolates are selected and identified as Corynebacterium sp. AN1 & Flavobacterium sp. TK2 able to reduce 88 and 132 n mol of acetylene (10(8)cells(-1)h(-1)) respectively. They were grown in large amount and sprayed on the phyllosphere of maize plants as a substitute for nitrogenous fertilizer. Marked improvements in growth and total nitrogen content of the plant have been observed by the application of these nitrogen-fixing bacteria. An average 30-37% increase in yield was obtained, which is nearer to chemical fertilizer treatment. Comparatively better effect was obtained by application of Flavobacterium sp.     

Terpenoid metabolism in wild-type and transgenic Arabidopsis plants

Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants.     

Effect of endothelial cell polarity on beta-amyloid-induced migration of monocytes across normal and AD endothelium

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.     

Mutagenic effects of carbosulfan,a carbamate pesticide

The genotoxic effects of carbosulfan were evaluated using chromosome aberration (CA), bone marrow micronucleus (MN) and sperm abnormality assays in mice. All the three acute doses (1.25, 2.5 and 5mg/kg) of carbosulfan induced significant dose-dependent increase in the frequency of CA (P<0.02), micronucleated polychromatic erythrocytes (PCEs) (P<0.05) and sperm head abnormalities (P<0.05) but did not affect the total sperm count. The highest acute dose of carbosulfan induced >7-fold increase in the frequency of CA, >3.5-fold increase in the frequency of micronucleated PCEs and >4.6-fold increase in the frequency of sperms with abnormal head morphology following intraperitoneal exposure as compared to the untreated controls. The present findings suggest that carbosulfan is a potent genotoxic agent and may be regarded as a potential germ cell mutagen also.     

Genotoxic effects of malathion: an organophosphorus insecticide, using three mammalian bioassays in vivo

The genotoxic effects of malathion was evaluated using chromosome aberration, sister chromatid exchange (SCE) and sperm abnormality assays in mice. All the three acute doses (2.5, 5 and 10mg/kg) of malathion tested in the present study, induced significant dose-dependent increase in the frequency of chromosome aberrations and sperm abnormalities, but did not affect the total sperm count. The highest acute dose induced a >12-fold increase in the frequency of chromosome aberrations, two-fold increase in the frequency of SCEs and four-fold increase in the frequency of sperms with abnormal head morphology following intraperitoneal (i.p.) exposure. Further, a significant increase in the frequency of SCEs was observed, but the increase was not dose-dependent. At higher doses, malathion induced a moderate delay in cell cycle as evident from the increase in average generation time (AGT). The present findings suggest that technical grade malathion is a potent genotoxic agent and may be regarded as a potential germ cell mutagen also.     

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.     

Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines: negative regulation of IKKalpha

The present study describes the role of RhoA as a negative regulator of iNOS expression via the inactivation of NF-kappaB in transformed brain cell lines [C(6) glioma, human astrocytoma (T98G, A172), neuroblastoma (NEB), and immortal rat astrocytes]. Treatment with lovastatin resulted in the induction of LPS/IFN-gamma-mediated iNOS mRNA and increased nitric oxide (NO) production. The addition of mevalonate and geranylgeranylpyrophosphate (GGPP) reversed the lovastatin-mediated effect, whereas FPP had no effect. An inhibitor of geranylgeranyltransferase inhibitor (GGTI 298) further induced the cytokine and lovastatin-mediated iNOS expression, suggesting the involvement of geranylgeranylated proteins in the regulation of iNOS. Bacterial toxin B (inactivates RhoA, B, and C; CDC42; Rac proteins), C3 ADP-ribosyltransferase (C3) toxin from C. botulinum (inactivates RhoA, B, and C proteins), and Y-27632 (selective inhibitor of Rho-associated kinases) increased the LPS/IFN-gamma-mediated iNOS expression. Lovastatin treatment induced NO by increasing NF-kappaB translocation and its association with the CREB-binding protein (CBP/p300) via the downregulation of RhoA. Inhibition of RhoA resulted in increased activation of IKKalpha. Cotransfection studies with dominant-negative form of RhoA and iNOS-luciferase or NF-kappaB-luciferase reporter constructs further support these observations. Taken together, these studies show that downregulation of RhoA by lovastatin resulted in increased iNOS expression via the activation of NF-kappaB-CBP/p300 pathway in transformed brain cells.