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Biomedical subjects

R M Franklin

Publications and source records attributed to R M Franklin.

At least 37 records · Page 2Linked to original sources
PubMed1992

An 88-kDa protein of Plasmodium falciparum is related to the band-3-binding domain of human erythrocyte ankyrin.

Three tryptic-peptide sequences of an 88-kDa pair of phosphoproteins of the malaria parasite Plasmodium falciparum were determined. They exhibit a striking similarity to corresponding sequences of the 89-kDa domain of human erythrocyte ankyrin. [35S]Methionine labeling of the two proteins demonstrated their parasitic origin. Using an appropriate oligonucleotide probe, Southern-blot analysis of genomic malaria DNA and Northern-blot analysis of malaria RNA suggest the existence of ankyrin-related sequences in the parasite genome and the presence of an ankyrin-related transcript of about 3.2 kb. Our studies provide further evidence of malaria-specific analogues of host-cell proteins, implying an unusual kind of parasite/host interaction.

Amino Acid Sequence↗
PubMed1992

Molecular cloning, stage-specific expression and cellular distribution of a putative protein kinase from Plasmodium falciparum.

A putative protein kinase gene (PfPK2) has been isolated from the human parasite Plasmodium falciparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serine/threonine protein kinases. The complete nucleotide sequence of 5 kb suggests the existence of a second transcriptional unit besides that of the PfPK2 gene, separated by a highly (A+T)-rich region and transcribed in a different orientation. No intron sequence exists in PfPK2. The predicted amino acid sequence of PfPK2 contains features characteristic of eukaryotic serine/threonine protein kinases. Within its putative catalytic domain it shares 33%, 30%, and 28% amino acid identities with rat calcium-calmodulin-dependent protein kinase, human protein kinase C, and bovine cAMP-dependent protein kinase, respectively. Outside the catalytic domain, however, PfPK2 has no homology with regulatory domains of other protein kinases, indicating PfPK2 might be modulated by signals different from those of higher eukaryotes or might be associated with other regulatory subunits. Using a specific antiserum raised in rabbits against a recombinant fragment of the protein expressed in Escherichia coli, PfPK2 was found to be expressed in a stage-specific fashion and mainly localized in the parasitic membrane.

Amino Acid Sequence↗
PubMed1991

Hybridization arrest of cell-free translation of the malarial dihydrofolate reductase/thymidylate synthase mRNA by anti-sense oligodeoxyribonucleotides.

In order to inhibit the in vitro translation of Plasmodium falciparum mRNA coding for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS), oligodeoxynucleotides (ODNs) were directed against the translation initiation site or a site in the TS-coding region. In both cases considerable hybridization arrest, i.e. greater than 50% inhibition, was only achieved if the lengths of the ODNs to the two regions were 30 and 39 nucleotides, respectively, or longer. The ODN with the highest efficiency was a 49-mer directed against the TS-coding region (OTS49); 45 microM was sufficient to inhibit the expression of DHFR-TS by almost 90%. In this case the synthesis of DHFR-TS was interrupted at the binding site of OTS49 by a RNase H-independent mechanism. The resulting polypeptide was smaller (55 kDa) than one subunit of the native protein (71 kDa) and lacked TS activity.

Animals↗
PubMed1991

Localization and stage specific phosphorylation of Plasmodium falciparum phosphoproteins during the intraerythrocytic cycle.

Fifty-nine Plasmodium falciparum specific phosphoproteins with molecular weights between 15,000 and 192,000 were analyzed by SDS-PAGE and two-dimensional gel electrophoresis. 40 phosphoproteins were identified by [gamma-32P]ATP labeling of cell lysates, 19 by [32P]orthophosphate labeling of parasitic cultures in vivo. Changes in the phosphorylation pattern during the infectious erythrocytic cycle were determined for all proteins. In parallel, cell fractionation studies were done to follow up possible changes in the cellular distribution of these proteins. Several phosphoproteins are associated with the membrane fraction of infected erythrocytes. One pair of proteins of approx. 88 kDa and a pI of about 5.0 was further characterized. Both proteins are located in the parasitic fractions as well as in the membrane of infected erythrocytes during the entire cycle. Phosphorylation of these proteins, however, is restricted to the trophozoite and schizont stages. Peptide mapping studies demonstrated that both proteins are identical with the exception of minor modifications which are probably not the result of differences in phosphorylation.

Animals↗
PubMed1991

The use of antisense oligonucleotides as chemotherapeutic agents for parasites.

Although several approaches to the control of human parasites are possible, the prevention and therapy of the corresponding diseases still remain a difficult task. The development of vaccines has been hampered by the poor immunological response to or the high variability of parasitic antigens. Problems also arise for chemotherapy where differences in the biochemistry of host and parasite must be exploited. The increasingly difficult search for new drugs is always challenged by the appearance of resistance.

Journal Article↗
PubMed1990

Mode of action of tubulozoles against Plasmodium falciparum in vitro.

The mode of action of the tubulozole isomers, recently recognized as a new class of potential antimalarial agents, was investigated. Whereas neither glycolysis, protease activity, or nucleic acid biosynthesis was primarily affected, protein biosynthesis decreased soon after addition of the drug. Inhibitors of protein biosynthesis, however, did not show synergistic activity with tubulozole. Colcemid, on the other hand, had an effect on protein synthesis similar to that seen with the tubulozoles. Furthermore, combinations of the tubulozole isomers with compounds known to interact with tubulin inhibited malaria in a synergistic or antagonistic fashion. Therefore, the inhibition might be elicited by interaction with tubulin or some other component of the microtubules. This is remarkable insofar as only one of the tubulozole isomers affects mammalian cells by binding to tubulin.

Animals↗
PubMed1989

Compounds binding to cytoskeletal proteins are active against Plasmodium falciparum in vitro.

The effects of nine different tubulin-binding substances and of one actin-binding compound, cytochalasin B, upon the intraerythrocytic development of the malaria parasite Plasmodium falciparum were investigated in vitro. From the data obtained, plasmodial tubulins seem to be quite different from the mammalian proteins on the molecular level. Tubulozole-T, a substance which is inactive in mammalian systems, appears to be a promising novel antimalarial drug.

Actins↗
PubMed1989

Compounds binding to cytoskeletal proteins are active against Plasmodium falciparum in vitro.

The effects of nine different tubulin-binding substances and of one actin-binding compound, cytochalasin B, upon the intraerythrocytic development of the malaria parasite Plasmodium falciparum were investigated in vitro. From the data obtained, plasmodial tubulins seem to be quite different from the mammalian proteins on the molecular level. Tubulozole-T, a substance which is inactive in mammalian systems, appears to be a promising novel antimalarial drug.

Actins↗
PubMed1989

The ocular secretory immune system: a review.

The majority of immunoglobulin in tears is of the immunoglobulin A (IgA) isotype, which is produced mainly by plasma cells of the lacrimal gland. The mechanism responsible for the lodging of the IgA cells in this gland is unknown and probably not dependent on direct glandular encounter with antigen. Previous experiments have suggested that a Th cell from the lacrimal gland can influence B lymphocytes to differentiate into IgA plasma cells. Mechanisms responsible for the IgA-specific Th cell accumulation in the LG could be related to nervous system influences, possibly neuropeptides. These effects on Th cells could be mediated either directly or through lacrimal gland epithelial cells. Such a mechanism could explain the presence of IgA-producing plasma cells in the lacrimal gland, and would allow strategies for manipulation of this important first line defense immune system.

B-Lymphocytes↗
PubMed1988

HSV-specific IgA from tears blocks virus attachment to the cell membrane.

The results of this investigation demonstrate that the IgA antibody in tears inhibits the attachment of herpes simplex virus (HSV) to cells and implicate immunoglobulin A (IgA) isotype antibody in the protection of the ocular surface. Tears were collected from the eyes of infected and uninfected rabbits and tested for the presence of infectious virus, for viral neutralization titer, and in an enzyme-linked immunoassay (ELISA) for antibody to HSV. Neither the tears from the uninfected rabbits nor the tears from the infected rabbits produced viral cytopathic effects on indicator cells in vitro. Tears from uninfected rabbits had no virus neutralization capacity while the tears from infected rabbits had titers of 1:10 to 1:40. Tears collected from HSV-1 infected rabbits, but not uninfected rabbits, had anti-HSV antibody titers as determined by the enzyme-linked immunosorbent assay. Lectin affinity column enrichment of IgA from the tears of uninfected and infected rabbits was examined for the capacity to block viral adsorption to cells using 32P-labeled virus. It was found that exposure of the radio-actively-labeled virus to the purified IgA fraction of tears from infected rabbits reduced the amount of virus binding to Vero cells, whereas the purified IgA obtained from uninfected tears had minimal activity. These results may imply a role for the IgA-containing fraction of tears from HSV-infected animals in preventing viral attachment to cells and suggest that antibody of this isotype plays a protective role at the ocular surface.

Animals↗
PubMed1988

Characterization of a specific inhibitor of IL2-mediated proliferation from serum of Plasmodium berghei infected mice.

Serum pools from mice undergoing lethal infection with Plasmodium berghei inhibit the growth of an IL2-dependent mouse cytotoxic T cell line (CTLL). A partially purified preparation of the inhibitory factor specifically inhibited IL2-mediated events such as IL2-dependent CTLL growth and the Con A mitogenic response of normal mouse spleen cells. Production of and response to IL1, as well as growth of myeloma lines, was not affected. Administration of the partially purified preparation to normal mice resulted in a significant depression in IL2 production, thereby indicating a role for the inhibitory factor in maintaining immune depression in malaria-infected mice.

Animals↗
PubMed1987

Mott cells: a model to study immunoglobulin secretion.

Mott cells are plasma cells defective in immunoglobulin (Ig) secretion. They display this defect by accumulating Ig in the rough endoplasmic reticulum, detectable by Ig+ intracellular inclusion. We have previously produced hybridoma cell lines (Alanen, A. et al., Eur. J. Immunol. 1985. 15: 235) in which this phenotype is preserved, and shown the inability of these cells to secrete the Ig. In order to study this defect further, we fused these hybridoma cells with a kappa-secreting hybridoma cell line, Sp1, and, using double selection with hypoxanthine, aminopterin, thymidine and ouabain, obtained hybrid cell lines expressing various combinations of the three Ig chains involved (Mott gamma 1, Mott kappa and Sp1 kappa chains). We studied the presence of Ig+ inclusions in these cells as well as Ig secretion by metabolic labeling and immunoprecipitation. All inclusion-positive clones expressed both Mott heavy and Mott light chains with or without the Sp1 light chain, whereas none of the inclusion-negative clones produced both Mott-derived Ig chains. In all of the clones, even those with inclusions, the Ig secretion was at least partially rescued by the fusion. This occurred also in an inclusion-positive clone which maintained the original Ig status of the Mott without Sp1 kappa chain, indicating a complementation by the cell fusion of some cellular factor involved in Ig secretion. Furthermore, we injected Xenopus oocytes with mRNA isolated from three different original Mott cell hybridomas and could show secretion of the Ig, which is not secreted from the original Mott cells, from the oocytes.

Animals↗
PubMed1987

Polyclonal B-cell activation and autoantibody formation during the course of mosquito-transmitted Plasmodium berghei infection in mice.

The time course of polyclonal B cell activation, as measured by titers of antibodies to DNP, FITC, and haemocyanin, as well as the time course of autoantibody formation, was followed in mice infected with Plasmodium berghei via Anopheles stephensi. IgM class antibodies to DNP, FITC, and haemocyanin appeared earlier than IgG class antibodies and persisted until death. Only IgM class anti-DNP peaked, the others remained high until death. Although IgM class autoantibodies also appeared earlier than IgG class, they peaked sharply at days 14 to 15, as did antibodies to mouse RBC. Polyclonal B cell activation, as measured by spleen plaque-forming cells (PFC) using SRBC and TNP-SRBC in the direct test, peaked at day 13. This latter event could be correlated with the time course of hypergammaglobulinemia.

Animals↗
PubMed1986

Microscopic and flow cytophotometric analysis of parasitemia in cultures of Plasmodium falciparum vitally stained with Hoechst 33342--application to studies of antimalarial agents.

Conditions for rapid vital staining of Plasmodium falciparum infected human erythrocytes were 1 microgram/ml of the dye Hoechst 33342 for 15 min in the standard culture medium at 37 degrees C. Fixed and stained cultures were analyzed by fluorescence microscopy and flow cytophotometry. The usefulness of this type of analysis for in vitro studies of antimalarial agents was demonstrated using three such agents--cyclosporin A, chloroquine, and pyrimethamine.

Antimalarials↗
PubMed1986

Chicken strain G-B1 exhibits a relative resistance to avian osteopetrosis.

The disease induced by the avian myeloblastosis associated virus MAV-2-O in the susceptible chicken strains Brown Leghorn (BLH) and Prague CB (CB) was compared with that induced in the resistant G-B1 strain. Osteopetrosis, stunting and lymphoid organ atrophy were more severe in BLH than in CB chickens. G-B1 animals remained superficially normal until the end of the experiment. In contrast to the other two strains, the histopathological changes were very mild and there was no sign of immunosuppression. After 4 months, however, nephroblastomas could be detected in more than 50 per cent of the infected G-B1 chickens. Similar tumors were also found in CB birds kept for up to 5 months. Antibodies against MAV-2-O specific viral proteins were detected in plasma from infected G-B1 chickens but the titers were less than in plasma of convalescent birds. Virus could be demonstrated in peripheral blood until the end of the experiment (at 8 weeks). Therefore the resistance of the G-B1 strain is due neither to a restriction at the receptor level nor the result of a humoral immune reaction, but represents a new type of relative resistance at the cellular level. From (CC X G-B1)F1 and (CC X G-B1)F2 crosses the resistant phenotype is determined by a single genetic factor. This gene is not linked to the major histocompatibility complex. There is also a sex-dependent factor, possibly hormonal, involved in the resistant phenotype.

Anemia↗
PubMed1986

MAV-2-O replicates at a reduced rate in cells from the osteopetrosis resistant G-B1 chicken.

The replication of the avian osteopetrosis virus MAV-2-O was compared in chick embryo fibroblasts from two strains of chicken. These were G-B1 which is relatively resistant to MAV-2-O and CB which is susceptible. The production of MAV-2-O was delayed in G-B1 cells (compared with CB cells). The same result was observed after infection with Rous sarcoma viruses of subgroups B, C, and D. In addition, the transforming viruses induced foci on G-B1 fibroblasts 24 to 48 hours later than on CB fibroblasts. In G-B1 cells there was also a delayed kinetics of intracellular viral RNA production. Integrated and linear unintegrated MAV-2-O DNA species were also present in lower amounts in G-B1 than in CB fibroblasts at 3 days postinfection. In vivo studies confirmed the in vitro situation. There was a marked difference in the amount of virus present in the osteoid bone matrix and the osteocytic lacunae of osteopetrotic bones from susceptible and G-B1 chickens. In contrast to the bone lesions from susceptible animals, budding virus particles were not detectable in lesions from G-B1 chickens. There was no difference in the amount of virus in osteopetrotic and non-osteopetrotic bone of susceptible chickens suggesting that virus replication alone is not sufficient for induction of osteopetrosis and that an additional specific virus-cell interaction is required. The relative resistance of strain G-B1 may therefore, be a consequence of a reduced frequency of this interaction. Its basis may be the lower amount of integrated, as well as unintegrated, viral DNA.

Animals↗