Biomedical subjects

M F Wiser

Publications and source records attributed to M F Wiser.

35 records · Page 2Linked to original sources

PubMed1991

Malarial proteins that interact with the erythrocyte membrane and cytoskeleton.

Several distinct classes of Plasmodium proteins have been proposed to interact with the submembrane skeleton of the erythrocyte based upon differential solubility and subcellular localization studies. That the parasite affects the erythrocyte membrane by interacting with the submembrane skeleton is an attractive hypothesis since the membrane skeleton likely regulates many aspects of membrane topography and function. The precise interactions between host and parasite proteins at the molecular level and how the parasite proteins are transported to the erythrocyte membrane are not completely understood. Experiments addressing these questions are under way, and such studies will provide valuable information about the host-parasite interface. In addition, the characterization of the interaction of Plasmodium proteins with the host erythrocyte membrane may also provide new insight into the structure and function of the erythrocyte membrane or membranes in general.

Animals↗

PubMed1990

Association of Plasmodium berghei proteins with the host erythrocyte membrane: binding to inside-out vesicles.

Two acidic phosphoproteins of Plasmodium berghei origin, of 65 and 46 kDa, are associated with the plasma membrane of the host mouse erythrocyte. The 65-kDa protein partitions between a soluble and particulate phase upon host cell lysis, whereas the 46-kDa protein is localized exclusively in the particulate fraction. Both proteins bind to inside-out vesicles derived from erythrocyte ghosts and the conditions of the reassociation reaction indicate that the binding is specific and that the proteins interact only with the cytoplasmic face of the erythrocyte membrane. The 65-kDa protein appears to exist in two membrane-associated states; one loosely bound, which readily dissociates from the membrane, and a more tightly associated state, which does not dissociate under non-denaturing conditions. The 46-kDa protein is tightly bound to the host erythrocyte membrane and does not dissociate. Cross-linking studies suggest that both of these parasite proteins interact with the submembrane cytoskeleton of the erythrocyte, and that the 65-kDa protein also appears to interact simultaneously with the lipid bilayer and erythrocyte membrane proteins. However, direct interaction between the malarial proteins and distinct erythrocyte membrane proteins could not be demonstrated. In summary, these findings indicate that the acidic phosphoproteins of the malarial parasite interact with the cytoplasmic face of the erythrocyte membrane both in vivo and in vitro.

Actins↗

PubMed1989

Plasmodium antigens external to the parasite but with the infected erythrocyte.

Three Plasmodium berghei exoantigens with apparent mol. wt. of 120, 31, and 13 kDa, found in infected erythrocytes by immunofluorescence, are further characterized. These antigens, synthesized in the late trophozoite and schizont stages, were released into the culture medium after schizont-infected erythrocytes were placed in culture; however, they were not found in the sera of infected animals. The 120-kDa antigen proved to be somewhat heat-stable, whereas the other two did not. A monoclonal antibody (MAb) recognizing the 13-kDa antigen cross-reacted with proteins from P. chabaudi and P. yoelii, whereas MAbs against the other two antigens reacted only with proteins from strains of P. berghei.

Animals↗

PubMed1989

Contrasts in antigen expression in the erythrocytic and exoerythrocytic stages of rodent malaria.

The time and site of expression of five antigens, recognized by monoclonal antibodies raised against blood-stage parasites, were studied in the exoerythrocytic stage of Plasmodium berghei using indirect immunofluorescent antibody staining. Two monoclonal antibodies (W 3.5, I 2.6), which stain the cytoplasm of infected erythrocytes, did not stain the cytoplasm of the infected liver cell but stained the parasite itself suggesting a difference in the antigenic architecture of the erythrocytic and exoerythrocytic parasites. Another antibody (17.6.1) revealed a further difference in the antigenic composition of the blood and liver-stage parasites being expressed almost exclusively in the former. Two others (C139 and 17.3.9) showed broadly similar patterns of expression in these two stages of the malarial life-cycle.

Animals↗

PubMed1989

Expression of the precursor of the major merozoite surface antigens during the hepatic stage of malaria.

The precursor of major merozoite surface antigens (PMMSA) and its proteolytic products are candidates for an asexual blood stage vaccine. Previous authors have shown that PMMSA epitopes are expressed in the liver or exoerythrocytic (EE) stage of malaria. Using Plasmodium berghei, we show that the molecular weight of the liver stage PMMSA is similar to that of the blood stage and that both EE and blood stage proteins are similarly processed. In the EE stage, it was synthesized toward the end of schizogony and appeared first to localize to the rough endoplasmic reticulum and then, as the cytomeres began to form, to the parasite plasmalemma. The EE and blood stage merozoites expressed similar amounts of this antigen as determined by indirect immunofluorescence.

Animals↗

PubMed1988

Acidic phosphoproteins associated with the host erythrocyte membrane of erythrocytes infected with Plasmodium berghei and P. chabaudi.

New phosphoproteins appear on the host erythrocyte membrane during Plasmodium berghei and P. chabaudi infection. Distinct proteins having similar properties and all distinguished by isoelectric points of less than 4.0 are identified. Associated with the erythrocyte membranes of P. berghei infected erythrocytes are two proteins with molecular masses of 65 and 46 kDa, whereas 93, 90 and 76 kDa proteins are observed during P. chabaudi infection. These new erythrocyte membrane associated proteins are all of parasite origin as indicated by metabolic labeling with proline and are synthesized during the ring stage of the asexual replicative cycle. Three of these proteins, the 93 kDa P. chabaudi protein and both P. berghei proteins, have been purified and the amino acid composition determined. All three are characterized by a relatively high proportion of aspartate and glutamate residues. Mono-and polyclonal antibodies were also raised against the same three purified proteins. No cross reactivity between these three proteins is observed, but one monoclonal antibody against the 65 kDa P. berghei crossreacts with a 27 kDa mouse erythrocyte protein. Immunofluorescence using the antibodies in combination with subcellular fractionation studies clearly shows that these phosphoproteins are associated with the host erythrocyte membrane and not the parasite.

Animals↗

PubMed1988

Phosphorylation of Plasmodium berghei derived phosphoproteins associated with the host erythrocyte membrane by the spectrin kinase.

Plasmodium berghei derived phosphoproteins are associated with the host erythrocyte membrane. Effectors of the phosphorylation reaction regulate the phosphorylation of the P. berghei derived proteins and spectrin in a similar manner. The spectrin kinase also phosphorylates the P. berghei phosphoproteins in a reconstituted reaction at the same site(s) as the endogenously phosphorylated proteins. These results indicate that a host protein kinase may regulate parasite phosphoproteins during malaria.

Animals↗

PubMed1988

Expression of the parasite protein Pc90 in plasma membranes of erythrocytes infected with Plasmodium chabaudi.

Erythrocytes infected with the malaria parasite Plasmodium chabaudi contain the neo-protein Pc90 in their plasma membrane. We investigate origin, membrane disposition, and intraerythrocytic traffic of this Pc90. Metabolic labeling of P.-infected erythrocytes, combined with cell fractionation as well as Western blot analysis and immunoprecipitation using a Pc90-recognizing monoclonal antibody, show that Pc90 is synthesized by early to mid trophozoites and is transported without any apparent processing steps to the erythrocyte membrane. Based upon the inaccessibility of Pc90 from the outside in intact erythrocytes and the water solubility of membrane-associated Pc90, it is concluded that Pc90 is localized on the cytoplasmic face of the host erythrocyte membrane. Immunoelectron microscopy using a Pc90-specific monoclonal antibody and the occurrence of soluble Pc90 in host cell cytosol indicate that the Pc90 is transported in both a 'vesicle-bound' and a 'free' form through the erythrocyte cytoplasm.

Animals↗

PubMed1987

Plasmodium berghei, P. chabaudi, and P. falciparum: similarities in phosphoproteins and protein kinase activities and their stage specific expression.

Phosphoproteins from Plasmodium berghei, P. chabaudi, and P. falciparum are compared. A major phosphoprotein of 46 kDa is found in all three species. Peptide mapping indicates that this protein is indeed the same in all three cases and is phosphorylated at similar sites in all three species. Monoclonal antibodies were raised against three other P. berghei phosphoproteins. All three monoclonal antibodies recognize both P. berghei and P. chabaudi proteins. Only one of the monoclonal antibodies crossreacts with a P. falciparum protein of 36 kDa, whereas the equivalent P. berghei and P. chabaudi proteins are 34 and 32 kDa, respectively. The highest rate of synthesis of the phosphoproteins is observed during the early trophozoite stage, whereas the highest rate of phosphorylation is observed during the late trophozoite stage.

Animals↗

PubMed1986

Increased sensitivity in antigen detection during immunoblot analysis resulting from antigen enrichment via immunoprecipitation.

The sensitivity in antigen detection during immunoblot analysis is greatly increased if the antigen is first immunoprecipitated from the crude extract before electrophoresis and transfer to nitrocellulose. Not only does the method allow detection of antigens which are minor components of crude mixtures or antigens which cannot be radiolabeled, but the method also resolves problems, such as high background, which are often associated with immunoprecipitation. Also, by modifying the method, whether or not monoclonal antibodies recognize the same or different antigens and/or epitopes can be easily determined.

Animals↗

PubMed1986

Characterization of monoclonal antibodies directed against erythrocytic stage antigens of Plasmodium berghei.

Monoclonal antibodies recognizing various facets of the malaria parasite Plasmodium berghei and of the infected erythrocyte were obtained after generation of hybridomas between spleen cells from immunized mice and myeloma cells. The monoclonal antibodies were characterized by enzyme-linked immunosorbent assay, indirect immunofluorescence, immunoprecipitation of [35S]methionine-labeled proteins and immunoblotting. The most readily identified antigen was a parasite surface-associated protein of 230 kDa which is similar to the polymorphic schizont antigen described in a number of malarial species. In addition, three distinct antigens of 13, 31 and 120 kDa, which are external to the parasite, but within the infected erythrocyte were identified.

Animals↗

PubMed1985

Cytosolic protein kinase activity associated with the maturation of the malaria parasite Plasmodium berghei.

Seven cytosolic phosphoproteins with relative molecular masses of 110, 58, 52, 46, 38, 36 and 34kDa and isoelectric points between 4.2 and 5.0 are identified from the rodent malaria parasite Plasmodium berghei. Similar patterns of phosphorylated proteins are obtained from parasite cytosol after incubation of intact infected erythrocytes with [32P]orthophosphate, or from parasite cytosol incubated with [gamma-32P]ATP. The characteristics of the phosphorylation reaction are similar to the previously described Plasmodium protein kinase [Wiser, M.F., Eaton, J.W. and Sheppard, J.R. (1983) J. Cell. Biochem. 21, 305-314], suggesting that the same protein kinase is involved. More protein phosphorylation activity is associated with the mature parasites than the immature forms, suggesting that these phosphoproteins may play some role in the parasite's erythrocytic stage cycle.

Animals↗

PubMed1983

A plasmodium protein kinase that is developmentally regulated, stimulated by spermine, and inhibited by quercetin.

Plasmodium berghei-infected murine red cells possess protein kinase activity that is associated with the isolated parasites. Schizonts contain significantly higher levels of this protein kinase than the more immature forms, suggesting a relationship between this enzyme activity and parasite development. Partially purified protein kinase has a Km for ATP of approximately 30 microMs, whereas the Km for GTP is approximately 300 microMs and the substrate preference is phosvitin greater than casein much greater than histone greater than protamine. The Mg2+ optimum is 10-20 mM, and the protein kinase activity is stimulated by the polyamines spermine and spermidine. The flavone, quercetin, inhibits the protein kinase activity in a competitive manner with respect to ATP (Ki approximately 3 microMs), and P chabaudi also has a very similarly regulated protein kinase. Protein kinases from both species are very similar to the type I casein kinase.

Animals↗

PubMed1983

Membrane-associated phosphoproteins in Plasmodium berghei-infected murine erythrocytes.

Normal and Plasmodium berghei (NYU-2 strain)-infected murine erythrocytes display substantially different patterns of plasma membrane phosphoproteins phosphorylation. Intact erythrocytes (normal and parasite infected) incubated with 32Pi and isolated washed erythrocyte plasma membranes incubated with gamma-32P-ATP were analyzed for phosphoproteins by SDS PAGE and autoradiography. Two new phosphoproteins of molecular weight 45,000 (pp45) and 68,000 (pp68), which are absent in normal erythrocyte membranes, are associated with the membranes of infected erythrocytes subjected to both intact-cell and isolated-membrane phosphorylation conditions. Two-dimensional gel electrophoresis indicates that pp45 and pp68 are of parasite origin. Partial or complete proteolytic digestion reveals that pp45 is phosphorylated at similar amino acid residues both in intact cells and in isolated membranes. The pp45 phosphoprotein can be detected at as low as 3% parasitemia and its phosphorylation is not affected by 10 microM cAMP, 1 mM Ca2+, or 5 mM EGTA. Extraction of isolated washed plasma membranes with 0.5% Triton X-100 or 0.1 M NaOH indicates that pp45 is detergent insoluble and only partially extractable with NaOH, suggesting that pp45 is closely associated with the host erythrocyte plasma membrane.

Animals↗

PubMed1981

Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines.

Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding.

Affinity Labels↗

PubMedDate not supplied

Proteolysis of a 34 kDa phosphoprotein coincident with a decrease in protein kinase activity during the erythrocytic schizont stage of the malaria parasite.

Protein phosphorylation events may play important roles in the replication and differentiation of the malarial parasite. Investigations into the lability of a Plasmodium protein kinase revealed that a 34 kDa parasite phosphoprotein is rapidly converted into a 19 kDa fragment. Coincident with this conversion is a nearly total loss of a protein kinase activity, as determined from the phosphorylation of endogenous protein substrates. Both the conversion of the 34 kDa protein to the 19 kDa protein and the loss of protein kinase activity are inhibited by thio-protease inhibitors. The presence of low levels of the intact 34 kDa protein restores the protein kinase activity to almost maximum levels. However, it was not possible to demonstrate protein kinase activity associated with the 34 kDa protein, thus suggesting that the 34 kDa protein is probably an activator or regulator of the protein kinase activity and not a protein kinase. The conversion to the 19 kDa fragment also occurs in vivo and only during the schizont stage prior to the appearance of ring forms. During this same period the protein kinase activity decreases suggesting that the proteolytic processing of the 34 kDa protein may be a physiological regulator of the protein kinase.

Animals↗

PubMedDate not supplied

Molecular characterization of a Plasmodium chabaudi erythrocyte membrane-associated protein with glutamate-rich tandem repeats.

The malarial parasite dramatically affects the structure and function of the erythrocyte membrane by exporting proteins that specifically interact with the host membrane. This report describes the complete sequence and some biochemical properties of a 93-kDa Plasmodium chabaudi chabaudi protein that interacts with the host erythrocyte membrane. Approximately 40% of the deduced protein sequence consists of tandem repeats of 14 amino acids that are rich in glutamic acid residues. Comparison of the repeat sequences from two different P. c. chabaudi strains derived from the same initial isolate revealed an exact duplication of 294 nucleotides suggesting a recent unequal crossing-over event. However, in spite of this potentially high level of intragenic recombination activity, the repeat sequences from P. c. adami are rather conserved suggesting structural or functional constraints on the protein and tandem repeats. The 93-kDa protein exists in an oligomeric form as revealed by gel filtration chromatography and non-denaturing gel electrophoresis. A predominantly alpha-helical predicted secondary structure and a discrepancy between the estimated molecular sizes determined from non-denaturing gel electrophoresis and gel filtration chromatography suggest that the protein is a long rod-shaped or fibrillar, protein. Attributes shared between the 93-kDa protein, some P. falciparum proteins with glutamate-rich tandem repeats, and cytoskeletal proteins suggest that these parasite proteins function as cytoskeletal proteins that possibly stabilize the erythrocyte membrane.

Amino Acid Sequence↗