Parimal C. Sen

Biochemical characterization of a calcium ion stimulated-ATPase from goat spermatozoa

The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (C12E8) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca2+ only. The membrane fraction has also found to contain Mg2+-dependent Ca2+-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca2+ required for maximum activity is 3 mM for both Mg2+-dependent and Mg2+-independent Ca2+-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca2+-uptake study shows that the uptake in the presence of Ca2+ and ATP is higher than in the presence of Mg2+, Ca2+ and ATP. The findings lead us to believe that the Mg2+-independent Ca2+-ATPase has some role in Ca2+ transport like Mg2+-dependent enzyme.

Immunosuppression in Hamsters with Progressive Visceral Leishmaniasis Is Associated with an Impairment of Protein Kinase C Activity in Their Lymphocytes That Can Be Partially Reversed by Okadaic Acid or Anti-Transforming Growth Factor β Antibody

ABSTRACT Progressive visceral infection of golden hamsters by Leishmania donovani amastigotes led to gradual impairment of the proliferative responses of their splenic or peripheral blood mononuclear cells (SPMC or PBMC, respectively) to in vitro stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io). Removal of macrophage-like adherent cells from SPMC or PBMC of infected animals (I-SPMC or I-PBMC) was earlier shown to restore almost completely their lymphoproliferative responses to PMA plus Io. The present study was directed to evaluate the status of protein kinase C (PKC), a molecule(s) known to play a key role in the lymphoproliferative process. Our results demonstrate that PKC activities (Ca2+, phosphatidyl serine, and diacyl glycerol dependent) in the cytosolic fraction of untreated nonadherent I-SPMC or I-PBMC as well as in the membrane fraction of PMA-treated cells were decreased significantly relative to those for normal controls. However, removal of adherent cells from I-SPMC or I-PBMC and subsequent overnight in vitro cultivation of nonadherent cells (lymphocytes) resulted in significant restoration of PKC activity in the cytosolic or membrane fraction of untreated or PMA-treated cells, respectively. Partial, though significant, restoration of PKC activity could also be achieved in the membrane fraction of PMA-treated cells following overnight in vitro treatment of I-SPMC or I-PBMC with the Ser/Thr phosphatase inhibitor okadaic acid (OA) or an anti-transforming growth factor β (anti-TGF-β) neutralizing antibody. These results correlated well with the ability of OA or the anti-TGF-β antibody to restore the lymphoproliferative response of I-SPMC or I-PBMC following stimulation with PMA plus Io. Interestingly enough, immunoblotting experiments failed to show any reduction in the level or translocation (following PMA treatment) of conventional PKC isoforms in the SPMC or PBMC of infected animals compared to those of normal controls. The results presented in this study suggest that the adherent cells generated in the SPMC or PBMC of infected animals exert a suppressive effect on the proliferative response of nonadherent cells (lymphocytes) which is likely to be mediated through the downregulation of the activation pathway involving PKC and its downstream molecules such as mitogen-activated protein kinases. Further, the observed suppression of PKC activity and subsequent lymphoproliferative responses can be attributed to alternations in the intracellular phosphorylation-dephosphorylation events. The relevance of these results is discussed in relation to the role of TGF-β, levels of which are known to be elevated in visceral leishmaniasis.

Identification and characterization of a Mg+2-dependent and an independent Ca+2-ATPase in microsomal membranes of rat testis

Rat testicular microsomal membrane fraction contains both Mg+2-dependent and Mg+2-independent Ca+2-ATPase activity. The latter activity is about two times higher than the former. Calcium ion required for maximum activation of Mg+2-independent Ca+2-ATPase in 3.0 mM, whereas for the dependent one it is 2.5 mM. Both the enzymes are resistant to cold shock upto seven days. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities, respectively. The pH optima for dependent one is 7.5, whereas for the independent one it is 8.5. Temperature optima for the former is 37°C and for latter one it is 40°C. Among all the nuclestides tested, ATP is found to be the best substrate for both the enzymes. The optimum concentration of ATP for dependent and independent enzyme activities are 3.0 mM and 1.5 mM respectively. Divalent metal ions like Zn+2, Ba+2 and Mn+2 have been found to inhibit Mg+2-dependent Ca+2-ATPase activity whereas Mg+2-independent Ca+2-ATPase activity is inhibited by the divalent ions except zinc which is found to stimulate the enzyme activity. Both the enzymes are inhibited by vanadata, EDTA and EGTA. I50, for vanadate is 0.05 and 0.125 mM for dependent and independent activities, respectively. Sulfhydral groups modifying agents e.g., NEM, DTNB and chlorpromazine are found to affect the enzyme activities in different ways. Thus NEM and chlorpromazine are found to inhibit and DTNB stimulate the enzyme activities in both the cases.

The in vivo inhibition of transport enzyme activities by chloroquine in different organs of rat is reversible

The antimalarial drug chloroquine is found to inhibit Na+, K+-ATPase, Ca2+, Mg2+-ATPase, Ca2+-ATPase, pNPPase and acetylcholinesterase activities in different organs of rat in vivo when injected for a certain periods of time. The inhibition seems to be due to the changes in the level of phospholipid, cholesterol and the fatty acid of the lipid and the alteration of the fluidity of the microsomal membranes. However, the enzyme activities return to the normal level in about 2–3 weeks after the discontinuation of the drug suggesting that the drug effect is reversible.

Lymph node cells from BALB/c mice with chronic visceral leishmaniasis exhibiting cellular anergy and apoptosis: Involvement of Ser/Thr phosphatase

Visceral leishmaniasis (VL) produced in BALB/c mice through intracardial administration of Leishmania donovani amastigotes was accompanied by hepatosplenomegaly with high organ parasite load and lymphadenopathy when followed up to 4-months or so. To elucidate the mechanism of immunosuppression associated with VL, we report here progressive impairment of the proliferative response of lymph node cells (lymphocytes) from infected animals (I-LNC) to in vitro stimulation with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) that could be related to the downregulation of PKC and MAP kinase (ERK 1/2) activation process. Further, pretreatment of I-LNC with the protein phosphatase inhibitor okadaic acid (OA), but not with calyculin A or sodium orthovanadate, significantly restored their proliferative response as well as PMA-induced activation of PKC. A population of LNC (primarily T-lymphocytes) from chronically infected animals was shown to undergo apoptosis, the number of which increased considerably following PMA+ Io stimulation. The apoptotic pathway, which was followed through binding of cells to Annexin V, activation of caspase-3 and fragmentation of DNA, involved destabilization of mitochondria, probably as a result of downregulation of PKC and Bcl-2. Interestingly, prior incubation of I-LNC with OA reversed the state of cell cycle arrest (anergy) and apoptosis through progression of cells from G0/G1 to S and G2/M phases with transcriptional activation of IL-2 and IL-2R genes. Our results suggest that the cellular (immune) dysfunction in VL could be attributed to dephosphorylation of key molecules in the T-lymphocyte signaling pathway by Ser/Thr phosphatase leading to their inactivation.

The chlorpromazine inhibition of transport ATPase and acetylcholinesterase activities in the microsomal membranes of rat in vitro and in vivo

Chlorpromazine, an antipsychotic drug, is found to inhibit Na+,K+-ATPase activity in rat brain microsomal membranes in vitro in concentration and time dependent manner but some inconsistency is observed when the effect was studied with respect to different temperatures. Various ligands and/or substrate affect the inhibition by chlorpromazine in different ways. The in vivo study with this drug shows that the activities of Na+,K+-ATPase, Ca−2-ATPase and acetylcholinesterase in the microsomal membranes of different organs are inhibit with increases in concentration or lengths of time of treatment and then levels off.

In vitro synthesis of phosphatidylinositol and phosphatidylcholine by phospholipase D

Abstract Phosphatidylinositol (PI) was prepared from egg lecithin by a one-step transphosphatidylation reaction catalysed by phospholipase D in the presence of myo-inositol. Similarly phosphatidylcholine (PC) has been synthesized by the same technique from egg phosphatidylethanolamine using phospholipase D and choline chloride.The yield of PI was ca 25 % and that of PC ca 28 %. The transphosphatidylase function of phospholipase D offers a useful route for the synthesis of different classes of phospholipids.

Resveratrol ameliorates benzo(a)pyrene-induced testicular dysfunction and apoptosis: involvement of p38 MAPK/ATF2/iNOS signaling

Benzo(a)pyrene [B(a)P] is an environmental toxicant that alters the steroidogenic profile of testis and induces testicular dysfunction. In the present study, we have investigated the molecular signaling of B(a)P and the ameliorative potential of the natural aryl hydrocarbon receptor (AhR) antagonist and antioxidant, resveratrol, on B(a)P-induced male reproductive toxicity. Studies showed that B(a)P treatment resulted in p38 MAPK activation and increased inducible nitric oxide synthase (iNOS) production along with testicular apoptosis and steroidogenic dysfunction. Resveratrol cotreatment maintained testicular redox potential, increased serum testosterone level and enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3βHSD, 17βHSD) and prevented subsequent onset of apoptosis. Resveratrol cotreatment resulted inhibition of testicular cytochrome P4501A1 (CYP1A1) expression, which is the major B(a)P metabolizing agent for BPDE-DNA adduct formation. Resveratrol also significantly decreased the B(a)P-induced AhR protein level, its nuclear translocation and subsequent promoter activation, thereby decreased the expression of CYP1A1. Resveratrol also down-regulated B(a)P-induced testicular iNOS production through suppressing the activation of p38 MAPK and ATF2, thus improved the oxidative status of the testis and prevented apoptosis. Our findings cumulatively suggest that resveratrol inhibits conversion of B(a)P into BPDE by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.

THE EFFECT OF BINDING OF CHLORPROMAZINE AND CHLOROQUINE TO ION TRANSPORTING ATPASES

The inhibition of ion transporting ATPases (Na+,K+-ATPase, Ca2+,Mg2+- and Ca2+-ATPase) by two amphiphilic drugs e.g. chlorpromazine (antipsychotic) and chloroquine (antimalarial) are found to be competitive in nature in vitro with respect to the substrate. Two binding sites - high and low affinity are found to exist on all the three ATPases toward these drugs as evident from the plot of F/F0 vs. different drug concentrations of tryptophan fluorescence of the enzymes. Circular dichroism analysis suggest that binding of drugs to the high affinity site does not involve any change in conformation of ATPase molecules which occur only when drug binds to the low affinity sites. The drug binding sites and possible effect on conformational change of ATPase molecules of these two drugs have been described in this report.

Nifetepimine, a Dihydropyrimidone, Ensures CD4+ T Cell Survival in a Tumor Microenvironment by Maneuvering Sarco(endo)plasmic Reticulum Ca2+ ATPase (SERCA)

Background: Tumor-induced SERCA3 up-regulation is a major cause of death of CD4+T lymphocytes leading to immune suppression in cancer bearers. Results: Nifetepimine down-modulates SERCA3 expression and thereby protects the lymphocytes from tumor-induced apoptosis. Conclusion: The present finding strongly suggests nifetepimine as a potent immuno-restoring agent that protects T lymphocytes from tumor insult. Significance: The results suggest that nifetepimine may be developed into a potent immuno-restoring agent in tumor-bearers. Multiple mechanisms have been proposed by which tumors induce T cell apoptosis to circumvent tumor immune-surveillance. Although sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) have long been known to regulate intracellular Ca2+ homeostasis, few studies have examined the role of SERCA in processes of T lymphocyte survival and activation. In this context it remains largely unexplored as to how tumors jeopardize SERCA function to disable T cell-mediated anti-tumor immunity. Here, we show that human CD4+ T cells in the presence of tumor conditions manifested an up-regulation of SERCA3 expression that resulted in development of endoplasmic reticulum stress leading to CD4+ T cell apoptosis. Prostaglandin E2 produced by the tumor cell plays a critical role in up-regulating SERCA3 by enhancing the binding of its transcription factor Sp1. Gene manipulation and pharmacological approaches further established that an increase in SERCA expression also resulted in subsequent inhibition of PKCα and -θ and retention of NFκB in the cytosol; however, down-modulation of SERCA3 expression by a dihydropyrimidone derivative, ethyl-4-(3-nitro)-phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate (nifetepimine), protected the CD4+ T cells from tumor-induced apoptosis. In fact, nifetepimine-mediated restoration of PKC activity resulted in nuclear translocation of p65NFκB, thereby ensuring its survival. Studies further undertaken in a tumor-bearing mice model revalidated the immunoprotective role of nifetepimine. Our present study thus strongly suggests that imbalance in cellular calcium homeostasis is an important factor leading to CD4+ T cell death during cancer and holds promise that nifetepimine may have the potential to be used as an immunorestoring agent in cancer bearers.