Anti-MAPK3 monoclonal antibody

Protozoan parasites of the Leishmania genus have evolved unique signaling pathways that can sense various environmental changes and trigger stage differentiation for survival and host infectivity. MAP kinase (MAPK) plays a critical role in various cellular activities like cell differentiation, proliferation, stress regulation, and apoptosis. The Leishmania donovani MAPK3 (LdMAPK3) is involved in the regulation of flagella length and hence plays an important role in disease transmission. Here, we reported the gene cloning, protein expression, biochemical characterizations, inhibition studies and cell proliferation assay of LdMAPK3. The recombinant purified LdMAPK3 enzyme obeys the Michaelis-Menten equation with K-m and V-max of LdMAPK3 was found to be 20.23 nM and 38.77 +/- 0.71 nmoles ATP consumed/mg LdMAPK3/min respectively. The maximum kinase activity of LdMAPK3 was recorded at 35 degrees C and pH 7. The in-vitro inhibition studies with two natural inhibitors genistein (GEN) and chrysin (CHY) was evaluated against LdMAPK3. The K-i value for GEN and CHY were found to be 3.76 +/- 0.28 mu M and K-i = 8.75 +/- 0.11 mu M respectively. The IC50 value for the compounds, GEN and CHY against L. donovani promastigotes were calculated as 9.9 mu g/mL and 13 mu g/mL respectively. Our study, therefore, reports LdMAPK3 as a new target for therapeutic approach against leishmaniasis.

Egg activation at fertilization in mammals is initiated by prolonged Ca2(+) oscillations that trigger the completion of meiosis and formation of pronuclei. A fall in mitogen-activated protein kinase (MAPK) activity is essential for pronuclear formation, but the precise timing and mechanism of decline are unknown. Here, we have measured the dynamics of MAPK pathway inactivation during fertilization of mouse eggs using novel chemiluminescent MAPK activity reporters. This reveals that the MAPK activity decrease begins during the Ca2(+) oscillations, but MAPK does not completely inactivate until after pronuclear formation. The MAPKs present in eggs are Mos, MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) and MAPK3 and MAPK1 (ERK1 and ERK2, respectively). Notably, the MAPK activity decline at fertilization is not explained by upstream destruction of Mos, because a decrease in the signal from a Mosluciferase reporter is not associated with egg activation. Furthermore, Mos overexpression does not affect the timing of MAPK inactivation or pronuclear formation. However, the late decrease in MAPK could be rapidly reversed by the protein phosphatase inhibitor, okadaic acid. These data suggest that the completion of meiosis in mouse zygotes is driven by an increased phosphatase activity and not by a decline in Mos levels or MEK activity.