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Related post: equivalent in extracting the "^ 40 representative PM petide constituents. Rabbit anti-PM sera were reacted against the various PM detergent soluble fractions in gel diffusion and Immunoelectrophoresis (lEP) assays. Results of these assays indicated that Tx=OG>NP>r)G>LB>ZG for retaining the anti- genic reactivity of the extracted PM components. The PM-Triton extract (PM-TX) was used in all subsequent immunologic assays. Results of lEP and 2-dimensional lEP demonstrated that Neoral Cost the isolated PM contained a minimum of 19-21 major antigenic components. In gel diffusion reactions, sera from patients with visceral leishmaniasis gave numerous strong precipitin lines against PM-TX indicating that these membrane antigens are recognized by the host's immune system Neoral 100 during the course of infection. Moreover, these results demonstrate that certain membrane antigens are common between the amastigote and promastigote developmental stages (i.e. PM-TX Cyclosporine Neoral was derived from promastigotes) of the parasite. Further, sera from L,. donovani terminally infected hamsters gave a precipitin line of identity with Kala-azar patients' sera indicating that the infected animal recognizes the same parasite PM antigen as the infected patient. Sera from patients with a diffuse cutaneous form of leishmaniasis (i.e. from the Dominican Republic, Leishmania sp.?) also gave a single specific immunoprecipitin with PM-TX. Immunoprecipitates from the latter patients' sera and from I., donovani infected hamsters with PM-TX were analyzed via SDS-PAGE. A single parasite Buy Neoral PM anti;?en was identified in such gels. It was a glyco- protein of '^' MW of 8X10^ daltons and was localized on the PM outer lamina. 2. Cross -reactive surface membrane carbohydrate antigens of I.. donovani and Trypanosoma cruzi (Dwyer and Gottlieb) . Antiserum (Tc-CHO-As) against a Trypanosoma cruzi surface membrane polysaccharide antigen (Tc-CHO) specifically agglutinated Leishmania donovani promastigotes (Ld-P) . No agglutination occurred with Tc-CHO-As following its absorption with Tc-CHO, Ld-P, surface membranes (Ld-PM)- or polysaccharide (Ld-CHO) isolated from Ld-P. Specific binding of Tc-CHO-As on the Ld-P surface membrane was demonstrated by light and electron microscopy using fluorescein and ferritin conjugated antibody methods. As ascertained by indirect immunofluorescence, binding of Tc-CHO-As 25-59 Serial No. ZOl Neoral 125 Mg AI 00162-04 LPD to living Ld-P at 0-4°C induced the movement (i.e., "patching" and "capping") of the cross-reactive Ld-P surface membrane ligands. Briefly fixed Ld-P treated identically gave similar, albeit less extensive, surface Neoral Sandimmune "patching" indicating the polysaccharide-like nature and apparent lipid linkage of the Ld-P surface ligands. Antigenic identity between Tc-CHO, Ld-CHO, and a Triton X-100 extract of Ld-PM (Ld-PMTX) was demon- strated in gel diffusion reactions against Tc-CHO-As . In reciprocal reactions, antiserum (Ld-PM-As) made against Ld-PM gave precipitin lines of antigenic identity between Ld-PM-TX, Ld-CHO, Neoral Coupon and Tc-CHO demonstrating the surface membrane origin of the Ld-P cross-reactive polysaccharide antigens. The Ld-PM-As also gave precipitin lines of identity between Ld-PM-TX, Ld-CHO, and polysaccharide fractions isolated from Leishmania tropica (Lt-CHO) and L. braziliensis (Lb-CHO) . Further, sera from several Kala-azar patients gave similar precipitin lines of identity among Ld-PM-Tx, Ld-CHO, Lt-CHO, Lb-CHO, and Tc-CHO suggesting the clinical significance of these cross-reactive antigens. These results indicate that a common or cross-reactive polysaccharide-like antigen exists in the surface membrane of the various species studied. This antigen(s) may be analogous to those group-type antigens which have been used for the clinical serodiagnosis of Neoral 50 Mg various bacterial genera. 3. Localization and characterization of L. donovani Pellicular membrane enzymes (Gottlieb and Dwyer) Studies have continued concerning the enzymes characteristic of the L. donovani surface membrane. Acid phosphatase (E.C. 22.214.171.124.) was characterized enzymically (pH optimum=5, fluoride sensitive, and substrate specificity determined) . A 5 fold enrichment of the enzyme was obtained with isolated PM versus whole cell homogenates. The enzyme was localized in intact cells to the surface membrane and to lysosome-like vesicles intracellularly. Cytochemical enzyme reaction product was restricted only to the external lamina of isolated PM. A 5' and 3' nucleotidase were demonstrated enzymically on the surface membranes of living promastigotes. The specific activity of the 3' enzyme was 20 times greater than the 5' activity in isolated PM fractions. The pH optima for the 3' and 5' enzymes were 8.5 and 7.0, respectively. Only the 5' enzyme was sensitive to fluoride inhibition. The 3' enzyme had an absolute divalent cation requirement for activity and the following ions were effective: Co''"2> Ca"*""^ >Mg"'''^ >Mn"'"'^ . Substrate specificity of the 3' enzyme Sandimmune Neoral was demonstrated: 3'AMP>3'UMP>3'GMP>3'CMP; no 3' deoxyribonucleo tides were hydrolyzed. Cytochemically , both the 5' and 3' enzymes were uniformly Neoral 25 Mg distributed on the surface of intact cells and were localized Neoral 75 Mg to the outer lamina of isolated PM. These results suggest that the 2 nucleotidase activities are important in salvaging purine bases as the parasites require exogenous sources Neoral Price of these bases for growth. Further, in light of the fact that vertebrate host cells do not possess any 3' nucleotidase activity, it seems possible that toxic 3' nucleotide analogs or drugs conjugated to 3' nucleotides might prove to be highly specific leishmanicidal agents for the chemotherapy of this disease Buy Neoral Online in the future. A Mg+2 stimulated 25-60 Serial No. ZOl AI 00162-04 LPD ATPase and adenyl cyclase have Neoral 100 Mg been localized cytochemically to the inner lamina (i.e. cytoplasmic surface) in isolated PM. Results of these studies further demonstrate both the biochemical and physiologic asymmetry of the parasite surface membrane. 4. Freeze-fracture and -etching of 1.. donovani PM Neoral Cyclosporine (Pinto DaSilva and Dwyer) . Fine structure freeze-fracture and -etching studies of intact and isolated _L, donovani PM were continued to further discern the supramolecular structure of these membranes. Intramembranous particles (IMP) in the membrane "P"-face appeared to be aligned with respect to the subtending
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