Seasonal Abundance of Acanthamoeba rhysodes (Singh, 1952) (Protozoa: Gymnamoebia) in a Mangrove Litter-Soil Ecosystem of Gangetic-Estuary,India1,2

Acanthamoeba rhysodes has been found to be a predominant intertidal benthic gymnamoeba in the mangrove ecosystem of Sundarbans of lower deltaic Bengal, facing the Bay. The sampling zones under study were the highest high tide regions, with characteristic mangrove litter-soil, inundated twice per month during the highest ebb of spring tide. Population abundance of this species, both in its trophic and cystic forms in the three distinct seasonal periods of pre-monsoon (March to June), monsoon (July to October), and post-monsoon (November to February) has been surveyed for over two years. These seasonal periods affect the physico-chemical parameters of the habitat substrata, including temperature, pH, and salinity. It has been found that the overall number of organisms per gram of soil attains peak value during the monsoon period. This value comes down in post-monsoon samples and is the least in pre-monsoon ones.     

Effect of short-and long-term exposure to low environmental temperature on brain regional GABA metabolism

Single exposure of adult male rats to low environmental temperature (LET, 12 ± 0.5°C) for 2 h significantly increased the hypothalamic and striatal GABA levels without affecting those in other regions of brain. The activity of glutamate decarboxylase (GAD) was elevated in hypothalamus (H) and corpus striatum (CS) under these conditions. GABA accumulation rate (measured with ethanolamine-O-sulfate, an inhibitor of GABA-transaminase) was also increased in both H and CS of rats exposed to LET for 2 h. Unlike after a single exposure, the repeated exposure (2 h/day) for 7, 15, and 30 onsecutive days did not change the hypothalamic GABA metabolism. No change in GABA metabolism was observed in CS when rats were repeatedly exposed to LET for 7 consecutive days. Prolongation of repeated exposure to LET (2 h/day) for 15 and 30 consecutive days decreased the striatal GABA level and increased the activity of GABA-transaminase, although GAD activity was not altered under these conditions. These results suggest that single exposure to LET accelerates GABA synthesis and may reduce the GABAergic activity in both H and CS; whereas repeated exposure to LET for 15 or 30 consecutive days enhances GABAergic activity with the stimulation of GABA utilization only in CS without affecting its synthesizing process. Thus, it may be suggested that the hypothalamic and striatal GABA system may play a characteristic role in response to short-and long-term exposure to LET.     

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.     

Pentylenetetrazol: Effect on carbaryl-induced changes of serotonin metabolism in pons-medulla of rat brain

Oral administration of carbaryl to adult male albino rats produced a dose dependent increase in the steady state level of 5-hydroxytryptamine (5-HT) at 1.00 h in pons-medulla (PM). 5-Hydroxyindole acetic acid (5-HIAA) concentration was significantly elevated only in response to a higher dose of this pesticide under similar conditions. A time course study with carbaryl and pentylenetetrazol (PTZ) showed a characteristic elevation of the steady state level of 5-HT in PM, but the 5-HIAA level was significantly elevated at 0.5 h only after carbaryl treatment. No significant change of the 5-HIAA level was evident after administration of PTZ alone or in combination with carbaryl. Tryptophan concentration was significantly elevated in PM at 0.5 h after carbaryl treatment and at 1.0 h after carbaryl + PTZ treatment. No significant change of tryptophan concentration was evident after the administration of PTZ alone under similar conditions. Measurement of (1) pargyline induced (a) accumulation of 5-HT and (b) depletion of 5-HIAA levels, and (2) probenecid-induced accumulation of 5-HIAA level in presence and absence of carbaryl and revealed that carbaryl accelerated the synthesis as well as the breakdown of 5-HT, whereas PTZ alone or in combination with carbaryl accelerated the synthesis of 5-HT without affecting its catabolism. The potency of this pesticide in elevating the pargyline-induced accumulation of 5-HT is in the order of carbaryl + PTZ greater than PTZ congruent to carbaryl. These results suggest that the carbaryl-induced increase in the synthesis of 5-HT is potentiated, and the turnover is reduced, in PM when PTZ is administered to the carbaryl-intoxicated rats.     

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.     

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.