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J A Lyon

Publications and source records attributed to J A Lyon.

At least 37 records · Page 2Linked to original sources
PubMed1988

Two approximately 300 kilodalton Plasmodium falciparum proteins at the surface membrane of infected erythrocytes.

Two very large Plasmodium falciparum proteins are identified as constituents of the infected erythrocyte membrane. Sera were obtained from Aotus monkeys that had been repeatedly infected with asexual P. falciparum from one of four strains. The capacity of these sera to block in vitro cytoadherence of infected erythrocytes and agglutinate intact infected cells was determined. The sera were also used to immunoprecipitate protein antigens from detergent extracts of 125I-surface labeled or biosynthetically radiolabeled infected erythrocytes. For each serum/antigen combination, precipitation of only one protein correlated with the ability of the serum to interfere with cytoadherence and agglutinate infected cells. This malarial protein, denoted Pf EMP 1 (P. falciparum-erythrocyte-membrane-protein 1) bore strain-specific epitope(s) on the cell surface and displayed size heterogeneity (Mr approximately 220,000-350,000). Pf EMP 1 was strongly labeled by cell-surface radioiodination but was a quantitatively very minor malarial protein. Pf EMP 1 was distinguished by its size, surface accessibility and antigenic properties from a more predominant malarial protein in the same size range (Pf EMP 2) that is under the infected erythrocyte membrane at knobs. Monoclonal antibodies and rabbit antisera raised against Pf EMP 2 were used to show that this size heterogeneous antigen was indistinguishable from the previously described MESA (mature parasite infected erythrocyte surface antigen), identified by precipitation with rabbit antisera raised against the MESA hexapeptide repeats. Antibodies raised against Pf EMP 2/MESA did not precipitate Pf EMP 1. We conclude that Pf EMP 1 is either directly responsible for the cytoadherence phenomenon, or is very closely associated with another as yet unidentified functional molecule. Pf EMP 2/MESA must have a structural property/function that is important under the host cell membrane.

Animals↗
PubMed1988

Allelic forms of gp195, a major blood-stage antigen of Plasmodium falciparum, are expressed in liver stages.

Mature exoerythrocytic (EE) forms of two cloned lines (3D7 and HB3) of Plasmodium falciparum were obtained in the livers of splenectomized chimpanzees. Sectioned preparations were examined by immunofluorescence (IFA) using mAbs that distinguished allelic variants of the blood-form antigen gp195 and mAbs that recognized multiple conserved epitopes of gp195. EE forms and blood schizonts exhibited identical IFA reactions for each respective clone, showing that the antigen was expressed identically in liver and blood-stage parasites. A third chimpanzee was infected with sporozoites derived from a mixture of 3D7 and HB3 gametocytes that had undergone cross-fertilization in the mosquitoes. IFAs on the EE forms in this animal showed that segregation of each gp195 allele had occurred earlier in the life cycle, providing evidence that the parasite is haploid for the whole of its mammalian development.

Alleles↗
PubMed1988

Mature liver stages of cloned Plasmodium falciparum share epitopes with proteins from sporozoites and asexual blood stages.

The liver merozoites of malaria parasites are of paramount importance, as they initiate the parasite invasion of red blood cells and start the cycle associated with the clinical features of malaria. Investigating liver merozoite antigen is difficult because of the lack of a rodent model of human malaria. In addition, only a low proportion of cells are obtained in vivo, the parasites from Cebus and Aotus monkeys are immature, and in-vitro experiments with liver cells are often confounded by contamination with the natural mosquito flora copurified with the sporozoites used for seeding the liver cultures. In our study, mature liver schizonts were shown to possess many of the antigenic determinants recognized by MoAbs and sera specific for defined sporozoite and blood-stage antigens. We employed an immunofluorescence procedure based on evaluating parasites in cryosections prepared from infected chimpanzee liver. Sufficient numbers of sectioned parasites were evaluated with each antibody to assure the reproducibility of the results, and the fixation procedure used was sufficiently non-destructive to parasite antigens so that clear differences between reactions of specific antibodies and negative controls were observed. Our evidence for sharing of epitopes by liver merozoites and sporozoites or by liver merozoites and asexual blood-stage parasites raises the possibility that immune responses elicited against sporozoites or asexual stage antigens being considered as vaccine candidates may also act against this important, little-studied stage of the parasite.

Animals↗
PubMed1988

Availability of iron from four prenatal multivitamin/multimineral products.

Iron absorption from four prenatal multivitamin/multimineral preparations was compared in 36 healthy pregnant women in their second or third trimester of pregnancy. The products studied were the currently marketed formulations of Stuartnatal 1 + 1, Stuart Prenatal, Materna, and Natalins Rx. Each fasted subject received each of the four preparations, according to a randomized sequence, at intervals of three to seven days between drug administrations. Stuartnatal 1 + 1 demonstrated the best iron absorption. Total iron absorbed from Stuartnatal 1 + 1 and Stuart Prenatal was well above the additional daily 3.5 mg recommended during pregnancy. Following administration of Materna the amount of iron was only slightly above that recommended, and following Natalins Rx it was well below. All formulations were generally well tolerated by the subjects.

Adolescent↗
PubMed1987

Monoclonal antibody characterization of Plasmodium falciparum antigens in immune complexes formed when schizonts rupture in the presence of immune serum.

When Plasmodium falciparum parasites are cultured with some immune sera, merozoites are agglutinated by antibodies to form immune clusters of merozoites and prevent their invasion into erythrocytes. Within these immune clusters of merozoites, several antigens that are normally found in the soluble fraction after detergent extraction accumulate in relatively insoluble immune complexes. From mice immunized with these immune complexes, we obtained hybridomas secreting monoclonal antibodies (mAb) that react with various immune clusters of merozoites antigens, including mAb 3D5, which recognizes a 101-kDa antigen (p101) and mAb, 5E3, which recognizes a 113-kDa antigen (p113). Both mAb reacted with antigens at the surface of schizonts, in the vacuolar space, and at the surface of merozoites before their release from schizont-infected cells. Both p101 and p113 were synthesized by mature trophozoites and young schizonts. In pulse-chase experiments, p113 was processed to 100-, 70-, 55-, and 50-kDa products. Both p101 and p113 appeared in the culture medium when schizont rupture occurred in normal culture medium but were found in immune complexes when schizont rupture occurred in the presence of immune serum. Antibodies in immune complexes, when dissociated with acid and used to probe immunoblots, reacted with affinity-purified p101 and p113. Antigens such as these, which are accessible at the parasite surface and react with antibodies present in immune serum that inhibits parasite invasion, are logical candidates to study in the search for a vaccine against the erythrocytic stages of malaria.

Animals↗
PubMed1987

Monoclonal antibody characterization of the 195-kilodalton major surface glycoprotein of Plasmodium falciparum malaria schizonts and merozoites: identification of additional processed products and a serotype-restricted repetitive epitope.

The gp195 from Camp strain parasites was characterized with eight monoclonal antibodies (MAb) that recognize different epitopes on gp195 and three of its merozoite-associated processed products. Four MAb (3H7, 3B10, 7F1, and 4G12) reacted with different epitopes on the 45-kDa glycosylated product (gp45), shown by differences in their reactivities with soluble and immunoblotted gp45. One MAb (7H10) reacted with a conformational epitope probably formed as a result of the interaction of gp45 with a nonglycosylated 45-kDa product (p45). Three other MAb (3D3, 7B11, and 7B2) reacted with different epitopes on a nonglycosylated 83-kDa product (p83), shown by differences in their reactivities against various parasite isolates in immunofluorescent antibody assays. Immunoprecipitation of antigens that were pulse-labeled with [3H]isoleucine and chased with cold isoleucine showed that p45 and gp45 were processed products of gp195 and p83 was sequentially processed into smaller fragments of 73 and 67 kDa (p73 and p67). Immunoblots showed that the 7B11 and 7B2 epitopes were present on p83, p73, and p67, but that the 3D3 epitope was present only on p83 and p73. A two-site immunoassay showed the 3D3 epitope to be repetitive. The 3D3 and 7B11 epitopes were serotype restricted (present in seven and 24 of 33 isolates, respectively), but the other five epitopes were common to all isolates tested. The gp195 and its processed products have Mr that are consistent with the Mr of a number of antigens shown previously to be associated with the immune complexes that are formed when merozoites are agglutinated by antibodies contained in some growth inhibitory immune sera.

Animals↗
PubMed1987

Plasmodium berghei: cloning of the circumsporozoite protein gene.

A DNA fragment encoding the carboxy terminal 80% of the Plasmodium berghei circumsporozoite protein was selected from a genomic DNA expression library. Sequencing revealed that the P. berghei circumsporozoite protein was similar in overall structure to circumsporozoite proteins from other malaria species, although the central repeat region was unique in comprising two different blocks of tandem peptide repeats: 11 eight amino acid repeats with predominant sequence DPAPPNAN were followed by 16 two amino repeats, predominantly PQ. The P. berghei circumsporozoite protein exhibited limited, but about equal amino acid homology to circumsporozoite proteins from P. knowlesi, P. vivax, and P. falciparum, indicating that P. berghei is not closely related to any of these other malaria species. Cloning of the P. berghei circumsporozoite protein gene will allow direct testing of sporozoite vaccines in mice.

Amino Acid Sequence↗
PubMed1987

Characterization of gp195 processed products purified from Plasmodium falciparum culture supernates.

Schizonts of the malaria parasite Plasmodium falciparum synthesize a 195 kDa surface glycoprotein (gp195) that is processed into several smaller products including one of 83 kDa, which, in the case of the Camp strain, is sequentially processed into 73 and 67 kDa products. gp195 and its processing intermediates larger than 83 kDa were not precipitated from culture supernates, but the 83 and 73 kDa products were precipitated by three monoclonal antibodies (McAbs). The 83 and 73 kDa products were affinity purified from culture supernates by adsorbing to McAb 7B2 coupled to Affigel 10 and eluting either with 0.2 N acetic acid, pH 2.8, or with 3 M potassium isothiocyanate (KSCN). The epitope recognized by McAb 7B2 was denatured by acid elution but could be regenerated by treating with 8 M urea followed by dialysis. The implications of renaturing antigens to regenerate epitopes should be considered in studies on the purification, function and immunogenicity of malaria antigens.

Animals↗
PubMed1987

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane.

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.

Animals↗
PubMed1987

Serotypic antigens of Plasmodium falciparum recognized by serotype-restricted inhibitory human sera.

Immune sera from some Cambodian refugees contain functional serotypic antibodies that inhibit invasion of erythrocytes by the Camp strain but not by the FCR-3 strain of Plasmodium falciparum. Using a new assay, the "competitive heterologous antigen assay" (CHAA), the serotypic antibodies in a pool of three inhibitory sera were characterized by the antigens they precipitated. In the CHAA, immunoprecipitation of antigens by antibodies to common or cross-reacting antigenic determinants was blocked with excess heterologous unlabeled FCR-3 antigens before 3H-labeled Camp schizont and merozoite antigens were immunoprecipitated. The predominant Camp strain serotypic antigens revealed after electrophoresis and autoradiography were the major 195 Kd glycoprotein surface antigen (gp195) and its processed products at 150, 83, 73, and possibly 45 Kd. Additional serotypic antigens were identified at 180, 130, 65, 50, and 32 Kd. It is likely that one or more of these serotypic antigens is a target for the serotypic antibodies that inhibit invasion.

Animals↗
PubMed1987

Personal computers: Rx for improving productivity.

The purpose of this article is to guide the computer-shy hospital pharmacy manager through the various choices concerning personal computer technology. The author discusses available computer programs that are easy to learn and can help pharmacists manage time and resources more efficiently. The Tandy 1000SX computer with the Deskmate integrated software program has been selected as one system that contains all basic computer systems. It is inexpensive and requires minimal learning time. Mastery of the computer and an integrated software program can facilitate improved productivity for interested pharmacists.

Computers↗
PubMed1986

Variation in the gene encoding a major merozoite surface antigen of the human malaria parasite Plasmodium falciparum.

Plasmodium falciparum merozoites have a variable surface protein of about 195,000 molecular weight which may be involved in strain-specific immunity. We have cloned and sequenced a major portion of the gene encoding this antigen from the CAMP strain and have located sites of preferred mung bean nuclease cleavage around the gene. These sites depend on reaction conditions, but at 40% formamide and 2 units of mung bean nuclease per microgram DNA, the intact gene was excised from the chromosome. Comparison of the CAMP strain gene with the same gene from other strains of P. falciparum by matching available DNA sequences and by DNA hybridization revealed five regions of homology separated by divergent segments. Two of the variable regions encoded three amino acid repeats, predominantly Ser-Gly-Thr and Thr-Glu-Glu. Implications of these findings on the function of the antigen, and possible mechanisms for generation of variants are discussed.

Amino Acid Sequence↗
PubMed1986

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface when schizonts mature in the presence of protease inhibitors.

Schizonts of Plasmodium falciparum were cultured in medium containing a mixture of 10 micrograms/ml each of leupeptin, chymostatin, pepstatin, and antipain. The protease inhibitors did not inhibit macromolecular synthesis but were associated with decreased reinvasion of red cells and the accumulation of well preserved merozoites clustered around pigment granules (PCM, protease inhibitor clusters of merozoites). The parasite pellet from PCM cultures contained increased amounts of merozoite antigens, particularly at Mr 83, 73, 66, 45, and 17 kDa. The increases of the Mr 83, 73, and 45 kDa surface antigens observed in PCM had been observed also in similar merozoite clusters obtained by culturing schizonts in the presence of inhibitory antibodies. These three antigens are processed products of the abundant Mr 195 kDa schizont surface antigen. Liquid-phase double immunofluorescence of PCM demonstrated a residual red cell membrane through which monoclonal antibodies passed and reacted with the Mr 83, 73, and 45 kDa merozoite surface antigens or their precursors. The processes associated with normal reinvasion apparently involve protease(s), which plays a role(s) in the breakdown of the red cell membrane and the shedding of merozoite surface antigens. Interference with these processes by protease inhibitors is useful in increasing recoveries of merozoite antigens, as well as in elucidating mechanisms of reinvasion.

Animals↗
PubMed1986

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum.

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.

Animals↗
PubMed1986

Epitope map and processing scheme for the 195,000-dalton surface glycoprotein of Plasmodium falciparum merozoites deduced from cloned overlapping segments of the gene.

DNA fragments from human malaria parasites were cloned into lambda gt11 to produce a genomic DNA expression library. A pool of monoclonal antibodies (mAbs) recognizing three domains of the 195-kDa major merozoite surface glycoprotein (gp195) reacted with seven clones expressing malaria antigens. mAbs recognizing the 83-kDa product of gp195 reacted with the clones, but mAbs recognizing a glycosylated 45-kDa and a nonglycosylated 45-kDa domain did not. Restriction enzyme mapping revealed that the clones contained overlapping segments encoding about 70% of the gene beginning at the 5' end and ending at an EcoRI restriction enzyme site 3.3 kilobase pairs downstream. The mAbs recognizing the 83-kDa domain reacted differently with the clones, allowing the mapping of three epitopes, one of which was repetitive. Affinity-purified antibodies were selected from immune monkey serum with recombinant expression proteins adsorbed to nitrocellulose filters. When used to probe electrophoretic immunoblots of parasite extracts, these antigen-selected antibodies reacted with specific sets of processed products of gp195, including those associated with the 83- and the nonglycosylated 45-kDa domains. This information, combined with the mAb epitope map, allowed a tentative scheme for processing gp195 from the Camp strain to be proposed.

Animals↗
PubMed1985

Imipenem/cilastatin: the first carbapenem antibiotic.

Imipenem/cilastatin is the first of a new class of beta-lactam antibiotics called carbapenems. The antibacterial spectrum of imipenem exceeds any antibiotic investigated to date and includes gram-positive, gram-negative, and anaerobic organisms. Only methicillin-resistant organisms, Strep. faecium, Pseudomonas cepacia, and Pseudomonas maltophilia have been shown to be resistant. Imipenem is administered in a 1:1 ratio with cilastatin, which inhibits a renal enzyme (dehydropeptidase) and improves urinary recovery of imipenem. The elimination half-life of both compounds is 1.0 hours and recommended doses are 0.25-0.5 g iv q6h. Adverse events are similar in nature and incidence to beta-lactam antibiotics, with phlebitis/thrombophlebitis, diarrhea, nausea, skin rash, and elevations of hepatic enzymes most common. Clinical studies in phase II and III trials have shown imipenem/cilastatin to be effective in soft tissue infections, endocarditis, obstetrics and gynecology, complicated urinary tract infections, mixed anaerobic-aerobic infections, osteomyelitis, bacteremias, and pneumonias. Several comparative clinical trials have shown imipenem/cilastatin to be equal in efficacy to combination therapy. Imipenem/cilastatin may prove to be an alternative to combination antibiotic therapy because of its extremely broad spectrum of activity.

Anti-Bacterial Agents↗
PubMed1984

Phosphate-binding specificities of monoclonal antibodies against phosphoinositides in liposomes.

Monoclonal antibodies against phosphatidylinositol phosphate were produced after injecting a mouse with liposomes containing dimyristoylphosphatidylcholine, cholesterol, phosphatidylinositol phosphate and lipid A. The antibodies raised were IgM (kappa) and their activities were assayed by complement-dependent damage to liposomes lacking lipid A but containing the rest of original immunizing mixture of lipids. Three of the four antibodies selected cross-reacted with liposomes containing phosphatidylinositol instead of phosphatidylinositol phosphate; and two of the antibodies cross-reacted with liposomes containing phosphatidylinositol diphosphate. Each of the antibodies had a phosphate-binding specificity. Each also cross-reacted with liposomes containing sulfogalactosyl ceramide, but not with liposomes containing galactosyl ceramide, or gangliosides or with liposomes containing lipid A but lacking phosphoinositides. Recognition of sulfogalactosyl ceramide probably occurred because the chemical characteristics of the sulfate group were sufficiently similar to those of phosphate to allow recognition by the antibody. The phosphate-binding specificity was further confirmed by inhibition by phosphocholine, inositol hexaphosphate, ATP, AMP and even sodium phosphate, but not by choline or inositol.

Adenosine Monophosphate↗