Characterization of coproporphyrinogen III oxidase in Plasmodium falciparum cytosol

A unique hybrid pathway has been proposed for de novo heme biosynthesis in Plasmodium falciparum involving three different compartments of the parasite, namely mitochondrion, apicoplast and cytosol. While parasite mitochondrion and apicoplast have been shown to harbor key enzymes of the pathway, there has been no experimental evidence for the involvement of parasite cytosol in heme biosynthesis. In this study, a recombinant P. falciparum coproporphyrinogen III oxidase (rPfCPO) was produced in E. coli and confirmed to be active under aerobic conditions. rPfCPO behaved as a monomer of 61 kDa molecular mass in gel filtration analysis. Immunofluorescence studies using antibodies to rPfCPO suggested that the enzyme was present in the parasite cytosol. These results were confirmed by detection of enzyme activity only in the parasite soluble fraction. Western blot analysis with anti-rPfCPO antibodies also revealed a 58 kDa protein only in this fraction and not in the membrane fraction. The cytosolic presence of PfCPO provides evidence for a hybrid heme-biosynthetic pathway in the malarial parasite.     

Modulatory influence of dietary resveratrol during different phases of 1,2-dimethylhydrazine induced mucosal lipid-peroxidation, antioxidant status and aberrant crypt foci development in rat colon carcinogenesis

To shed light on the association of lipid peroxidation and antioxidant status with the development of aberrant crypt foci (ACF), we studied the modulatory influence of resveratrol, supplemented in three dietary regimens (initiation, post-initiation and entire period) on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. Rats were administered DMH (20 mg/kg body weight, s.c.) for 15 weeks and were supplemented with resveratrol (8 mg/kg body weight, p.o. everyday) in three dietary regimens. Intestines and colons were analyzed for the levels of diene conjugates (DC), lipid hydroperoxides (LOOHs) and thiobarbituric acid reactive substances (TBARS). Enzymic antioxidants (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX; glutathione S-transferase, GST; and glutathione reductase, GR) and non-enzymic reserve (reduced glutathione, GSH; ascorbate; and alpha-tocopherol) were also assessed in the intestine and colon. Unsupplemented DMH exposed rats showed significantly decreased levels/activities of tissue DC, LOOHs, TBARS, SOD, CAT, GSH, GR and significantly elevated (P<0.05) GPX, GST, alpha-tocopherol and ascorbate as compared to control rats. Resveratrol supplementation during the entire period of the study resulted in significant (P<0.01) modulation of lipid peroxidation markers and antioxidants status, which were paralleled with ACF suppression, as compared to DMH-alone treated rats. These results indicate that resveratrol effectively inhibits DMH-induced ACF and colonic tumor development.     

L-deprenyl protects against rotenone-induced, oxidative stress-mediated dopaminergic neurodegeneration in rats

The present study investigated oxidative damage and neuroprotective effect of the antiparkinsonian drug, L-deprenyl in neuronal death produced by intranigral infusion of a potent mitochondrial complex-I inhibitor, rotenone in rats. Unilateral stereotaxic intranigral infusion of rotenone caused significant decrease of striatal dopamine levels as measured employing HPLC-electrochemistry, and loss of tyrosine hydroxylase immunoreactivity in the perikarya of ipsilateral substantia nigra (SN) neurons and their terminals in the striatum. Rotenone-induced increases in the salicylate hydroxylation products, 2,3- and 2,5-dihydroxybenzoic acid indicators of hydroxyl radials in mitochondrial P2 fraction were dose-dependently attenuated by L-deprenyl. L-deprenyl (0.1-10mg/kg; i.p.) treatment dose-dependently attenuated rotenone-induced reductions in complex-I activity and glutathione (GSH) levels in the SN, tyrosine hydroxylase immunoreactivity in the striatum or SN as well as striatal dopamine. Amphetamine-induced stereotypic rotations in these rats were also significantly inhibited by deprenyl administration. The rotenone-induced elevated activities of cytosolic antioxidant enzymes superoxide dismutase and catalase showed further significant increase following L-deprenyl. Our findings suggest that unilateral intranigral infusion of rotenone reproduces neurochemical, neuropathological and behavioral features of PD in rats and L-deprenyl can rescue the dopaminergic neurons from rotenone-mediated neurodegeneration in them. These results not only establish oxidative stress as one of the major causative factors underlying dopaminergic neurodegeneration as observed in Parkinson's disease, but also support the view that deprenyl is a potent free radical scavenger and an antioxidant.     

Analyses of Dynein heavy chain mutations reveal complex interactions between Dynein motor domains and cellular Dynein functions

Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies.     

Recycle in upflow anaerobic sludge blanket reactor on treatment of real textile dye effluent

The discharge of textile wastewater containing dye in the environment is varying for both toxicology and esthetical reasons as dyes impede light penetration, damage the quality of the receiving streams. Upflow anaerobic sludge blanket reactor with anaerobic digester sludge treating starch wastewater has been used to investigate the removal efficiency of chemical oxygen demand (COD) and colour of textile dye wastewater. In this study, the starch and textile dye wastewater was mixed at 70 and 30%, respectively, and the experiments were carried out with recycle of treated wastewater at different percentage as 10, 20, 30 and 40. Maximum removal of COD and colour was 96% and 93.3%, respectively, at 30% recycle. At various OLR and HRT, the maximum removal of COD, colour was 95.9%, 93% at 6.81 kg COD/m³d and 96%, 93% with 24 h of HRT. The maximum production of biogas at 24 h of HRT with 30% recycle was about 355 l/d. The Volatile fatty acid/Alkalinity ratio of methanogenic reactor was found to be 0.049–0.053. The result provided evidence, the starch and dye wastewater have wide variation in their characteristics was treated on combination, this new technology supports the effective utilization of starch waste in destruction of dye.     

5-Nitro-2-furoic acid hydrazones: Design,synthesis and in vitro antimycobacterial evaluation against log and starved phase cultures

Various 5-nitro-2-furoic acid hydrazones were synthesized and evaluated for in vitro activities against log and starved phase culture of two mycobacterial species and Mycobacterium tuberculosis (MTB) isocitrate lyase (ICL) enzyme inhibition studies. Among twenty one compounds, 5-nitro-N′-[(5-nitro-2-furyl)methylidene]-2-furohydrazide (4o) was found to be the most active compound in vitro with MICs of 2.65 and 10.64 μM against log- and starved-phase culture of MTB. Compound 4o also showed good enzyme inhibition of MTB ICL at 10 μM. The docking studies also confirmed the binding potential of the compounds at the ICL active site.     

Mechanism of resistance induced by plant activators against Colletotrichum falcatum in sugarcane

Abstract

A search for plant activators capable of inducing systemic resistance in sugarcane showed that plants pre-treated with synthetic signal inducers confer a high degree of resistance to Colletotrichum falcatum – the red rot pathogen. Among the various treatments, Acibenzolar S- methyl (ASM) was found to be very effective in restricting the pathogen colonization inside the inoculated cane stalk tissues. The induction of resistance was accompanied by a significant increase in peroxidases and polyphenoloxidases activities. A considerable decrease of pathogen titre in the pre-treated tissues as determined by ELISA, clearly demonstrated the restriction of pathogen colonization and proliferation in the sensitized cane stalks. Specific induction of new isoforms of peroxidases and polyphenoloxidases in C. falcatum elicitor treatment indicates the pathogen elicitor induced specific cellular response of sugarcane suspension-cultured cells.     

Structural distortions due to missense mutations in human formylglycine-generating enzyme leading to multiple sulfatase deficiency

The major candidate for multiple sulfatase deficiency is a defective formylglycine-generating enzyme (FGE). Though adequately produced, mutations in FGE stall the activation of sulfatases and prevent their activity. Missense mutations, viz. E130D, S155P, A177P, W179S, C218Y, R224W, N259I, P266L, A279V, C336R, R345C, A348P, R349Q and R349W associated with multiple sulfatase deficiency are yet to be computationally studied. Aforementioned mutants were initially screened through ws-SNPs&GO^3D program. Mutant R345C acquired the highest score, and hence was studied in detail. Discrete molecular dynamics explored structural distortions due to amino acid substitution. Therein, comparative analyses of wild type and mutant were carried out. Changes in structural contours were observed between wild type and mutant. Mutant had low conformational fluctuation, high atomic mobility and more compactness than wild type. Moreover, free energy landscape showed mutant to vary in terms of its conformational space as compared to wild type. Subsequently, wild type and mutant were subjected to single-model analyses. Mutant had lesser intra molecular interactions than wild type suggesting variations pertaining to its secondary structure. Furthermore, simulated thermal denaturation showed dissimilar pattern of hydrogen bond dilution. Effects of these variations were observed as changes in elements of secondary structure. Docking studies of mutant revealed less favourable binding energy towards its substrate as compared to wild type. Therefore, theoretical explanations for structural distortions of mutant R345C leading to multiple sulfatase deficiency were revealed. The protocol of the study could be useful to examine the effectiveness of pharmacological chaperones prior to experimental studies.