Characterization of a novel microsomal glutathione S-transferase produced by Aspergillus ochraceus TS

Purification and characterization of microsomal glutathione S-transferase produced by Aspergillus ochraceus TS are reported. The isozymes are located in microsomes and were active against 1-chloro-2,4-dinitrobenzene, ethacrynic acid, 1,2-dichloro-4-nitrobenzene, trans-4- phenyl-3-buten-2-one,p-nitrobenzyl chloride and bromosulphophthalein. They were inhibited by N-ethylmaleimide and bromosulphophthalein. The GST isozymes produced by Aspergillus ochraceus TS are indistinguishable in respect of their molecular mass both in native and denatured state. The subunit of the purified protein had an apparent M_r of 11 kDa while molecular mass of the native protein is around 56 kDa. The substrate specificity and pl values of the isozymes were different. The GSTs produced by Aspergillus ochraceus TS fairly share functional properties with mammalian cytosolic isozymes.     

High level expression of human tissue-type plasminogen activator gene in mouse C127 cell.

We have expressed human tissue plasminogen activator (t-PA) gene at high levels in a mouse cell line. The t-PA cDNA with deletion of the long 3' untranslated region was inserted into a bovine papilloma virus (BPV) derived vector under the control of a mouse metallothionein promoter. The mouse metallothionein (mMT) gene also provided signals for splicing and polyadenylation. Mouse C127 cells transfected with this construct secreted t-PA at high levels into the cell culture medium. When an SV40 polyadenylation signal was inserted between the t-PA cDNA and the mMT splicing signals, the expression level increased by several fold. The expression levels did not increase further upon either introduction of Rous sarcoma virus LTR into the plasmid or mutation of the translation initiation context sequence to conform with the consensus one. Most of the plasmid appears to be integrated into the host chromosome. Cells producing high levels of t-PA tend to detach from the dish in a few days after passage. When grown on porous microcarriers, however, such cells can be maintained in culture for months and t-PA can be harvested continuously.     

Effects of exogenous 1,3-diaminopropane and spermidine on senescence of oat leaves : I. Inhibition of protease activity, ethylene production, and chlorophyll loss as related to polyamine content

Excision and dark incubation of oat (Avena sativa L., var. Victory) leaves cause a sharp increase in protease activity, which precedes Chl loss. Both these senescence processes are inhibited by exogenously applied 1,3-diaminopropane (Dap), which occurs naturally in leaf segments. The inhibition of protease activity is much greater in vivo than in vitro, suggesting inhibition of protease synthesis as well as protease action by Dap. Chl breakdown in leaves of radish and broccoli, which also senesce rapidly in the dark, is only slightly inhibited by DaP. These differences between cereal and dicotyledonous plants are correlated with the natural occurrence of Dap in cereals. In the light, Dap promotes, rather than retards, the loss of Chl in oat leaves. This resembles previously described effects of other polyamines. Addition of Mg²⁺ to the medium does not antagonize this effect. In the dark, the accumulated Dap also inhibits ethylene production and decreases titer of other polyamines. Addition of Ca²⁺ to the incubation medium containing Dap competitively reduces the effects of Dap. Thus, Dap, like other polyamines, seems to require an initial attachment to a membrane site shared with Ca²⁺ before exerting its antisenescence action.     

Synthesis of (2'-5')(A)n from ATP. Characteristics of the reaction catalyzed by (2'-5')(A)n synthetase purified from mouse Ehrlich ascites tumor cells treated with interferon

The treatment of Ehrlich ascites tumor cells with mouse interferon increases the level of the latent enzyme (2'-5')(A)n synthetase. If activated by double-stranded RNA, this catalyzes the synthesis from ATP of a series of 2'-5'-oligoadenylates: (2'-5')(A)n where n extends from 2 to about 15. We isolated (2'-5')(A)n synthetase in a homogeneous state. In the presence of double-stranded RNA, the purified enzyme can convert the large majority (about 97%) of the ATP into (2'-5')(A)n and pyrophosphate, although it does not cleave the pyrophosphate. The stoichiometry of the reaction can be formulated as: (n + I) ATP leads to (2'-5') pppA(pA)n + n pyrophosphate. Added pyrophosphate does not inhibit the synthesis of (2'-5')(A)n. The extent of the reverse reaction, i.e. the pyrophosphorolysis of (2'-5')(A)n, was below the level of detection under our conditions. The affinity of the enzyme for ATP is low: the rate of the reaction increases by about 10% when the concentration of ATP is increased from 5 mM to 10 mM. The optimal concentration of double-stranded RNA increases with the concentration of the enzyme. As tested at 0.4, 2, and 10 micrograms/ml of enzyme concentrations, close to maximal (2'-5')(A)n synthesis can be obtained if reovirus double-stranded RNA or poly(I) . poly(C) are used at about half the concentration (in w/v) of the enzyme. The plot of the reaction rate versus enzyme concentration is sigmoidal. It remains to be seen if this reflects on a cooperative behavior of the enzyme.     

Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site-specific mutagenesis--distal effects on dimer stability

Cys126 is a completely conserved residue in triosephosphate isomerase that is proximal to the active site but has been ascribed no specific role in catalysis. A previous study of the C126S and C126A mutants of yeast TIM reported substantial catalytic activity for the mutant enzymes, leading to the suggestion that this residue is implicated in folding and stability [Gonzalez-Mondragon E et al. (2004) Biochemistry 43, 3255-3263]. We re-examined the role of Cys126 with the Plasmodium falciparum enzyme as a model. Five mutants, C126S, C126A, C126V, C126M, and C126T, were characterized. Crystal structures of the 3-phosphoglycolate-bound C126S mutant and the unliganded forms of the C126S and C126A mutants were determined at a resolution of 1.7-2.1 ?. Kinetic studies revealed an approximately five-fold drop in k(cat) for the C126S and C126A mutants, whereas an approximately 10-fold drop was observed for the other three mutants. At ambient temperature, the wild-type enzyme and all five mutants showed no concentration dependence of activity. At higher temperatures (> 40 °C), the mutants showed a significant concentration dependence, with a dramatic loss in activity below 15 μM. The mutants also had diminished thermal stability at low concentration, as monitored by far-UV CD. These results suggest that Cys126 contributes to the stability of the dimer interface through a network of interactions involving His95, Glu97, and Arg98, which form direct contacts across the dimer interface.     

Protection of mouse jejunum against lethal irradiation by Podophyllum hexandrum

Radiation induced gastrointestinal damage occurs due to the destruction of the clonogenic crypt cells and eventual depopulation and denudation of the villi. P. hexandrum, a plant, known for its antitumour activity, has been shown to protect the mice against whole body lethal (10 Gy) irradiation. Present study was undertaken to investigate the radioprotective effect of P. hexandrum on jejunal villi cells, crypt cells, their proliferative capacity and mitigation of apoptosis.

In an in vivo micro colony survival assay, pre-irradiation administration of P. hexandrum (–2 h) increased the number of surviving crypts in the jejunum by a factor of 3.0 (P < 0.05) and villi cellularity by 2.7 (P < 0.05) fold in comparison to irradiated control. Pre-irradiation administration of P. hexandrum reduced the incidence of apoptotic bodies in the crypts (P < 0.05) in a time dependent manner and depicted a mitotic arrest till the 24 h. However, after 84 h the percentage of mitosis was observed to be nearly similar to that of unirradiated control.

This study suggests that arrest of cell division may help in protecting the clonogenic cells against radiation. It would be interesting to investigate further the role of P. hexandrum in influencing various cell cycle regulators like bcl-2, TGF-β, Cyclin-E etc.     

Application of particle swarm optimization and proximal support vector machines for fault detection

This paper presents a novel application of particle swarm optimization (PSO) in combination with another computational intelligence (CI) technique, namely, proximal support vector machine (PSVM) for machinery fault detection. Both real-valued and binary PSO algorithms have been considered along with linear and nonlinear versions of PSVM. The time domain vibration signals of a rotating machine with normal and defective bearings are processed for feature extraction. The features extracted from original and preprocessed signals are used as inputs to the classifiers (PSVM) for detection of machine condition. Input features are selected using a PSO algorithm. The classifiers are trained with a subset of experimental data for known machine conditions and are tested using the remaining data. The procedure is illustrated using the experimental vibration data of a rotating machine. The influences of the number of features, PSO algorithms and type of classifiers (linear or nonlinear PSVM) on the detection success are investigated. Results are compared with a genetic algorithm (GA) and principal component analysis (PCA). The PSO based approach gave test classification success above 90% which were comparable with the GA and much better than PCA. The results show the effectiveness of the selected features and classifiers in detection of machine condition.     

Molecular characterization and expression analysis of two crucial MAPKs- jnk1 and erk1 as cellular signal transducers in Labeo rohita in response to PAMPs stimulation and pathogenic invasion

Mitogen-activated protein kinases (MAPKs) are crucial Ser/Thr protein kinases that play important roles in innate immunity by converting extracellular stimuli into a wide range of cellular responses, including the production of cytokines. In this study, two MAPK genes, jnk1 and erk1, were cloned and characterized in rohu (Labeo rohita), a commercially important freshwater fish species in the Indian subcontinent. In healthy rohu, both jnk1 and erk1 gene expressions were highest in the spleen as compared to gill, liver, blood and kidney tissues. In vitro stimulation of the L. rohita gill (LRG) cell line with γ-D-glutamyl-meso-diaminopimelic acid, muramyl dipeptide and polyinosinic: polycytidylic acid (poly I:C) resulted in significantly enhanced expressions of jnk1 and erk1 genes. In the in vivo experiments, jnk1 and erk1 gene expressions were also enhanced in lipopolysaccharides and poly I:C-treatment. Infection of rohu fingerlings with Aeromonas hydrophila and Bacillus subtilis revealed significantly enhanced expressions of the jnk1 and erk1 genes in all of the tested organs/tissues. Together these results imply the important role of jnk1 and erk1 genes in fish during pathogenic invasion and diseases.