Matrix metalloprotease 2-mediated activation of Ca²⁺-ATPase by superoxide radical (O₂^.-) in plasma membrane of bovine pulmonary vascular smooth muscle

390-396The role of the matrix metalloprotease-2 (MMP-2) in regulating Ca2+-ATPase activity in bovine pulmonary artery smooth muscle plasma membranes during treatment with the O2.-generating system, hypoxanthine (HPX) plus xanthine oxidase (XO) has been studied. The smooth muscle membranes possess matrix metalloprotease (MMP) activity in gelatin Zymogram, having an apparent molecular mass of 72 kDa; the activity is inhibited by the tissue inhibitor of metalloprotease-2 (TIMP-2). Since both protease and MMP-2 have same molecular mass and are inhibited by TIMP-2, it may, therefore, be suggested that the protease is the MMP-2. Treatment of the smooth muscle membrane suspension with the O2.- generating system stimulates MMP-2 activity, as evidenced by an apparent increase in the intensity of the protease activity. O2.- also enhances [14C]-gelatin degradation and Ca2+-ATPase activity. The increase in MMP activity, assessed by [14C] -gelatin degradation and Ca2+- ATPase activity are inhibited upon pretreatment with superoxide dismutase (SOD). The O2.- triggered MMP and Ca2+-ATPase activities in the membrane are found to be inhibited by TIMP-2 . The stimulation of the MMP and Ca2+-ATPase activities remain unaffected by the inhibitors of serine, thiol and cysteine groups of proteases such as phenylmethylsulfonylfluo ride (PMSF). Bowman Birk inhibitor (BBI), chymostatin, N-ethylmaleimide, leupeptin. antipain and pepstatin. Adding pure bovine MMP-2 to the smooth muscle membrane suspension causes an increase in Ca2+-ATPase activity, but the pretreatment with TIMP-2 inhibits the increase in the enzyme activity

<i style="mso-bidi-font-style:normal">Leishmania donovani</i> secretory serine protease alters macrophage inflammatory response via COX-2 mediated PGE-2 production

542-551Leishmania parasites determine the outcome of the infection by inducing inflammatory response that suppresses macrophage’s activation. Defense against <i style="mso-bidi-font-style: normal">Leishmania is dependent on Th1 inflammatory response by turning off macrophages’ microbicidal property by upregulation of COX-2, as well as immunosuppressive PGE-2 production. To understand the role of L. donovani secretory serine protease (pSP) in these phenomena, pSP was inhibited by its antibody and serine protease inhibitor, aprotinin. Western blot and TAME assay demonstrated that pSP antibody and aprotinin significantly inhibited protease activity in the live Leishmania cells and reduced infection index of L. donovani-infected macrophages. Additionally, ELISA and RT-PCR analysis showed that treatment with pSP antibody or aprotinin hold back COX-2-mediated immunosuppressive PGE-2 secretion with enhancement of Th1 cytokine like IL-12 expression. This was also supported in Griess test and NBT assay, where inhibition of pSP with its inhibitors elevated ROS and NO production. Overall, our study implies the pSP is involved in down-regulation of macrophage microbicidal activity by inducing host inflammatory responses in terms of COX-2-mediated PGE-2 release with diminished reactive oxygen species generation and thus suggests its importance as a novel drug target of visceral leishmaniasis

Angiotensin II inhibits Na⁺/K⁺ATPase activity in pulmonary artery smooth muscle cells via glutathionylation and with the involvement of a 15.6 kDa inhibitor protein

119-124The role of angiotensin II in regulating Na+/K+-ATPase activity has been investigated in bovine pulmonary artery smooth muscle cells (BPASMCs). Our study reveals that angiotensin II inhibits the Na+/K+ATPase activity via glutathionylation of the pump with the involvement of an increase in NADPH oxidase-derived O2.-. Additionally, angiotensin II treatment to the cells increases the inhibitory potency of the 15.6 kDa inhibitor towards the Na+/K+ATPase activity. </span

Implications of Calpains in Health and Diseases

316-328The number of mammalian calpain protease family members has grown as many as 15 till recent count. Although initially described as a cytosolic protease, calpains have now been found in almost all subcellular locations i.e., from mitochondria to endoplasmic reticulum and from caveolae to Golgi bodies. Importantly, some calpains do not possess the 28 kDa regulatory subunit and have only the 80 kDa catalytic subunit. In some instances, the 80 kDa subunit by itself confers the calpain proteolytic activity. Calpains have been shown to be involved in a number of physiological processes such as cell cycle progression, remodeling of cytoskeletal-cell membrane attachments, signal transduction, gene expression and apoptosis. Recent studies have linked calpain deficiencies or it’s over production with a variety of diseases, such as muscular dystrophies, gastropathy, diabetes, Alzheimer’s and Parkinson’s diseases, atherosclerosis and pulmonary hypertension. Herein, we present a brief overview on some implications of calpains on human health and some diseases

Defining the Role of Protein Kinase c in Calcium-ionophore-(A23187)-Mediated Activation of Phospholipase A2 in Pulmonary Endothelium

We sought to investigate the mechanisms by which the calcium ionophore A23187 triggers arachidonic acid release in bovine pulmonary endothelial cells and to test the hypothesis that protein kinase C is involved in this process. Our results indicate that the mechanism by which A23187 increases phospholipase A2 activity and arachidonic acid release in bovine pulmonary arterial endothelial cells depends upon the concentration studied. At concentrations of 1 pM and 2.5 pM, A23187 increases phospholipase A2 activity and arachidonic acid release without stimulating protein kinase C. At membrane-bound protein kinase C. To test the hypothesis that these doses of A23187 increase phospholipase A2 activity by stimulating protein kinase C, we studied the effect of prior treatment with the protein kinase C inhibitor sphingosine. Sphingosine inhibits the increase in phospholipase A2 activity and arachidonic acid release caused by A23187 over the range 5 - 12.5 pM. To investigate further the potential role of protein kinase C, we studied the effects of the inactive phorbol ester 4a-phorbol 12P-myristate 13aacetate (4a-PMA) and an active phorbol ester 4P-phorbol 12P-myristate 13a-acetate (48 PMA). concentrations of 5 - 12.5 pM, A23187 increases arachidonic acid release and phospholipase A2 activity in conjunction with a dose-dependent activation of Neither 4a-PMA nor 4P-PMA affected basal arachidonic acid release. 4a-PMA also did not augment the effects of A23187. In contrast, 4P-PMA significantly augments the increase in phospholipase A2 activity and arachidonic acid release caused by lower doses of A231 87. Under these conditions,sphingosine completely inhibits the stimulatory effects of 4P-PMA on protein kinase C translocation,phospholipase A2 and arachidonic acid release. Thus, at low doses (1 pM and 2.5 pM) A23187 increases phospholipase A, activity and arachidonic acid release by a mechanism that does not involve protein kinase C. At these A23187 doses, activating membrane-bound protein kinase C with 4P-PMA causes a synergistic increase in phospholipase Az activity and arachdonic acid release. At higher doses (5 - 12.5 pM), A23187 acts in large part by stimulating protein kinase C translocation. Overall,our results indicate that activating membrane-bound protein kinase C by itself is an insufficient stimulus to increase phospholipase A, activity and arachidonic acid release in pulmonary endothelial cells, but activating protein kinase C can substantially augment the increase in phospholipase A2 activity and arachidonic acid caused by a small increase in intracellular calcium

115 kDa serine protease confers sustained protection to visceral leishmaniasis caused by Leishmania donovani via IFN-�Y induced down-regulation of TNF-� mediated MMP-9 activity

Visceral leishmaniasis caused by the intracellular parasite Leishmania donovani is a major public health problem in the developing world. The emergence of increasing number of L. donovani strains resistance to antimonial drugs recommended worldwide requires the intervention of effective vaccine strategy for treatment of VL. In the present study L. donovani culture derived, soluble, secretory serine protease (pSP) has been shown to be vaccine target of VL. Protection from VL could be achieved by the use of safer vaccine which generally requires an adjuvant for induction of strong Th1 response. To assess the safety, immunogenicity and efficacy of pSP as vaccine candidate in mouse model we used IL-12 as adjuvant. BALB/c mice immunized with pSP+IL-12 were protected significantly from challenged infection even after four months by reducing the parasite load in liver and spleen and suppressed the development of the disease along with an increase in IgG2a antibody level in serum, enhanced delayed type hypersensitivity and strong T-cell proliferation. Groups receiving pSP+IL-12 had an augmented pSP antigen specific Th1 cytokines like IFN-� and TNF-� response with concomitant decrease of Th2 cytokines IL-4 and IL-10 after vaccination. In this study the vaccine efficacy of pSP was further assessed for its prophylactic potential by enumerating matrix metalloprotease-9 (MMP-9) profile which has been implicated in various diseases. MMP-9 associated with different microbial infections is controlled by their natural inhibitors (TIMPS) and by some cytokines. In this study pSP was found to regulate excessive inflammation by modulating the balance between MMP-9 and TIMP-1 expression. This modulatory effect has also been demonstrated by IFN-� mediated down regulation of TNF-� induced MMP-9 expression in activated murine macrophages. This is the first report where a secretory L. donovani serine protease (pSP) adjuvanted with IL-12 could also act as protective imunogen by modifying cytokine mediated MMP-9 expression in experimental VL. These findings elucidate the mechanisms of regulation of MMP-9 following infection of L. donovani in vaccinated animals and thus pave the way for developing new immunotherapeutic interventions for VL

Immunolocalization and Characterization of Two Novel Proteases in Leishmania Donovani: Putative Roles in Host Invasion and Parasite Development

Two novel intracellular proteases having identical molecular mass (58 kDa) were purified from virulent Indian strain of Leishmania donovani by a combination of aprotinin-agarose affinity chromatography, ion exchange chromatography and finally continuous elution electrophoresis. Both of these proteases migrate in SDS-PAGE as a single homogeneous bands suggesting monomeric nature of these proteases. The enzyme activity of one of the proteases was inhibited by serine protease inhibitor aprotinin and another one was inhibited by metalloprotease inhibitor 1, 10 phenanthroline. The purified enzymes were thus of serine protease (SP-Ld) and metalloprotease (MP-Ld) type. The optimal pH for protease activity is 8.0 and 7.5 for SP-Ld and MP-Ld respectively. The temperature optimum for SP-Ld is 28 �C and for MP-Ld is 37 �C showing their thermostability upto 60 �C. Broad substrate (both natural and synthetic) specificity and the effect of Ca2þ upon these enzymes suggested novelty of these proteases. Kinetic data indicate that SP-Ld is of trypsin like as BAPNA appears to be the best substrate and MP-Ld seems to be collagenase type as it degrades azocoll with maximum efficiency. Both immunofluorescence and immune-gold electron microscopy studies revealed that the SP-Ld is localized in the flagellar pocket as well as at the surface of the parasite, whereas MP-Ld is located extensively near the flagellar pocket region. This work also suggests that the uses of anti SP-Ld and anti MP-Ld antibodies are quite significant in interfering with the process of parasite invasion and multiplication respectively. Thus the major role of SP-Ld could be predicted in invasion process as it down regulates the phagocytic activity of macrophages, and MP-Ld appears to play important roles in parasitic development

Effect of different serine protease inhibitors in validating the 115 kDa <i style="mso-bidi-font-style:normal">Leishmania donovani </i>secretory serine protease as chemotherapeutic target

14-22Proteases have been considered as an important group of targets for development of antiprotozoal drugs due to their essential roles in host-parasite interactions, parasite immune evasion, life cycle transition and pathogenesis of parasitic diseases. The development of potent and selective serine protease inhibitors targeting L. donovani secretory serine protease (pSP) could pave the way to the discovery of potential antileishmanial drugs. Here, we employed different classical serine protease inhibitors (SPIs), such as aprotinin, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), N-tosyl-lysine chloromethyl ketone (TLCK), benzamidine (Bza) and pSP-antibody to determine the role of the protease in parasitic survival, growth and infectivity. Among the different classical SPIs, aprotinin appeared to be more potent in arresting L. donovani promastigotes growth with significant morphological alterations. Furthermore, aprotinin and anti-pSP treated parasites significantly decreased the intracellular parasites and percentage of infected macrophages. These results suggest that SPIs may reduce the infectivity by targeting the serine protease activity and may prove useful to elucidate defined molecular mechanisms of pSP, as well as for the development of novel antileishmanial drugs in future