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D M Dwyer

Publications and source records attributed to D M Dwyer.

At least 91 records · Page 5Linked to original sources
PubMed1986

Cloning of Leishmania donovani genes encoding antigens recognized during human visceral leishmaniasis.

In this report we describe the construction and analysis of a genomic library of Leishmania donovani gene segments in the bacteriophage vector lambda gt11. This cloning vector permits the expression of parasite polypeptides as fusion products with Escherichia coli beta-galactosidase. A group of 90 clones which express L. donovani antigens have been isolated from this library using various polyvalent antisera. Many of these clones appear to encode parasite membrane antigens some of which are recognized by sera from patients with visceral leishmaniasis (kala azar). Some clones reacted with specific subsets of kala azar sera while others reacted with all kala azar sera tested. In addition, some of the clones appear to encode conserved antigens that are recognized by sera from mice immunized with L. major. Preliminary characterization of five clones indicated that each one contains a distinct genetic element and that at least four encode different fusion peptides.

Animals↗
PubMed1986

Purification and characterization of the extracellular acid phosphatase of Leishmania donovani.

A tartrate sensitive acid phosphatase activity was purified from culture supernatants of Leishmania donovani promastigotes grown in a macromolecule-free defined medium. Purification was accomplished by ultrafiltration, lentil lectin affinity chromatography, and sucrose density gradient ultracentrifugation. This enzyme was determined to be an acid glycoprotein containing 0.37 mg hexose per mg protein. A molecular weight of 134 000 by sucrose density gradient centrifugation was observed, although on molecular sieve chromatography the enzyme eluted with an apparent molecular weight of greater than 700 000. The specific activity of the purified enzyme was greater than 200 mumol min-1(mg protein)-1 when assayed with 4-methylumbelliferylphosphate as the substrate. In addition to various hexose phosphates, the enzyme hydrolyzed phosphorylated amino acids, in particular phosphotyrosine. The purified enzyme was heterodisperse with respect to both protein and activity staining patterns upon polyacrylamide gel electrophoresis.

Acid Phosphatase↗
PubMed1986

3'-nucleotidase activity in procyclic and bloodstream stages of Trypanosoma rhodesiense.

Both procyclic and bloodstream-derived trypanosomes of Trypanosoma rhodesiense exhibit a nucleotidase activity which is capable of hydrolyzing 3'-ribonucleotides. Nucleotidase activity in assays employing 5'-nucleotide substrates is not detectable in preparations of either phenotype. The 3'-nucleotidase activity in lysates of procyclic trypanosomes is pelletable at 100,000 g, whereas that activity from bloodstream forms is operationally soluble under the conditions employed. The 3'-nucleotidase activities of both stages share the following characteristics: pH optimum of 8.5, substrate preference for purine ribonucleotides, resistance to commonly used phosphatase inhibitors, including fluoride, tartrate and molybdate ions. Both activities are reversibly inhibited by EDTA and reactivated by Co2+ ions. As visualized by fine-structure cytochemical methods, 3'-nucleotidase activity was distributed over the entire surface of procyclic trypanosomes, but not bloodstream parasites bearing an intact surface coat.

Animals↗
PubMed1985

Lipid analyses of isolated surface membranes of Leishmania donovani promastigotes.

Constituent lipids of surface membranes (SM) isolated from Leishmania donovani promastigotes were analyzed and compared with those obtained from whole cells and an isolated kinetoplast-mitochondrion fraction (KM). On a dry weight basis, the total extractable lipids constituted approximately 47%, 12% and 24% of the SM, cells and KM, respectively. The total lipids of SM, cells and KM all were composed of approximately 70% phospholipids (PL), 20-25% neutral lipids and 5-10% glycolipids. Sterols and diglycerides composed 60% and 30%, respectively, of the various neutral lipid fractions. Several mannose- and galactose-containing glycolipids were fractionated but not identified. The glycolipid fractions from cells and SM had demonstrable antigenic activities with rabbit anti-SM sera. Striking quantitative differences were apparent between the PL profiles of the 3 cellular components examined. The PL of SM, whole cells and KM, respectively, were composed of: 15%, 51% and 24% phosphatidylcholine; 37%, 13% and 11% phosphatidylethanolamine (PE); 18%, 10% and 14% phosphatidylinositol; 10%, 1% and 4% phosphatidylserine and traces of cardiolipin, phosphatidylglycerol and phosphatidic acid. An unknown PL containing sphingosine, choline and vicinal hydroxyl groups but no free amino moieties made up approximately 19% of the PL of SM and whole cells, but it constituted approximately 27% of the PL of KM. The PL side chain constituents of whole cells and SM were composed mainly of longchain fatty acids (C18-20). Further, over 50% of the PE of SM was in the alkyl and alK-1-enyl ether forms. These SM properties might contribute to the organism's resistance to digestion in the hydrolytic environs of both its insect vector and mammalian hosts.

Animals↗
PubMed1985

Protonmotive force-driven active transport of D-glucose and L-proline in the protozoan parasite Leishmania donovani.

Midlogarithmic phase Leishmania donovani promastigotes accumulate 2-deoxy-D-glucose (2-dGlc) and L-proline, maintaining concentration gradient factors across the surface membrane of 78.7 and 60, respectively. Cyanide (1 mM) and iodoacetate (0.5 mM) inhibited the transport of both substrates. L-proline uptake was also inhibited by 2-dGlc (10 mM). Transport of neither substrate was affected by Na+, phlorizin, or ouabain, indicating the sodium-independent transport of both systems. However, N',N'-dicyclohexylcarbodiimide (DCCD; 20 microM) significantly inhibited the transport of both 2-dGlc and L-proline (70% and 90%, respectively). The ionophores valinomycin (1 microM) and nigericin (5 microM) each partially inhibited the uptake of both substrates. In parallel experiments, nigericin and valinomycin were added concomitantly to promastigotes, each at a concentration that individually inhibited the transport of 2-dGlc and L-proline by less than 30%. Under such conditions, the transport of 2-dGlc and L-proline was inhibited by 69% and 78%, respectively. However, these ionophores had no significant effect on the promastigotes cellular ATP level. Carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 1 microM) inhibited 2-dGlc (79%) and L-proline (85%) transport, whereas ATP levels of such cells were diminished by only 20%. Symport of D-glucose/H+ and L-proline/H+ was measured directly in cells pretreated with KCN and DCCD. Upon addition of D-glucose to such cells, a rapid movement of protons into the organisms occurred and was reversed upon addition of FCCP. Conversely, no proton movement was observed when L-glucose was added to such cells. L-proline, as D-glucose, caused a rapid influx of protons into the promastigotes, indicating that both substrates were cotransported with protons. We conclude that transport of D-glucose and L-proline in L. donovani promastigotes is protonmotive force-driven and is coupled to both delta pH and delta psi.

Animals↗
PubMed1985

Outbreaks of group A streptococcal abscesses following diphtheria-tetanus toxoid-pertussis vaccination.

Two outbreaks of group A streptococcal abscesses following receipt of diphtheria-tetanus toxoid-pertussis (DTP) vaccine from different manufacturers were reported to the Centers for Disease Control (CDC) in 1982. The clustering of the immunization times of cases, the isolation of the same serotype of Streptococcus from all cases in each outbreak, and the absence of reported abscesses associated with receipt of the same lots of vaccine in other regions of the country, suggest that each outbreak was probably caused by contamination of a single 15-dose vial of vaccine. The preservative thimerosal was present within acceptable limits in unopened vials from the same lot of DTP vaccine in each outbreak. Challenge studies indicate that a strain of Streptococcus from one of the patients can survive up to 15 days in DTP vaccine at 4 degrees C. Contamination of vials during manufacturing would have required survival of streptococci for a minimum of 8 months. Preservatives in multidose vaccine vials do not prevent short-term bacterial contamination. Options to prevent further clusters of streptococcal abscesses are discussed. The only feasible and cost-effective preventive measure now available is careful attention to sterile technique when administering vaccine from multidose vials.

Abscess↗
PubMed1984

Antidepressants cause lethal disruption of membrane function in the human protozoan parasite Leishmania.

The antidepressant compounds clomipramine and nitroimipramine were cidal to extracellular promastigotes of both human protozoan parasites Leishmania donovani and Leishmania major. Clomipramine also killed amastigotes of both species within murine macrophages with no apparent toxicity to the host cells. Further, amastigotes were more sensitive than promastigotes to clomipramine. Clomipramine (100 micromoles per liter or 0.2 nanomole per 1 X 10(6) cells) inhibited L-proline transport in promastigotes. Synergistic inhibition of L-proline transport was observed with clomipramine after addition of either of the ionophores valinomycin or nigericin. These observations suggest that the cytotoxic effects of clomipramine result from its disruption of the proton electrochemical gradient of the parasite surface membrane.

Animals↗
PubMed1984

Mortality among diabetic patients using continuous subcutaneous insulin-infusion pumps.

Because of concern about deaths among diabetic patients using continuous subcutaneous insulin-infusion pumps, we conducted an investigation to determine whether the number of such deaths was excessive. By October 1982 we had identified 35 deaths among the estimated 3500 diabetic patients using pumps in the United States. The observed number of deaths was not greater than the expected number calculated from age-specific death rates for conventionally treated patients with Type I diabetes. The causes of death were not unusual, with two exceptions. One death was associated with malfunction of the device, and the other was attributed to acute bacterial endocarditis that arose from an abscess at the catheter-insertion site. Patients who died had high rates of autonomic neuropathy (66 per cent) and renal disease (39 per cent with serum creatinine levels above 5 mg per deciliter). We conclude that the use of continuous subcutaneous insulin-infusion pumps is not associated with excess mortality. However, certain types of patients may be at greater risk for death, and physicians should take great care in selecting patients for pump therapy.

Adolescent↗
PubMed1984

Surface membrane localization of 3'- and 5'-nucleotidase activities in Leishmania donovani promastigotes.

Distinct 3'- and 5'-nucleotidase activities were localized to the surface membrane of Leishmania donovani promastigotes using enzymatic reactions with live intact cells and fine structure enzyme-mediated cytochemical reactions with intact cells, cell homogenates, and isolated surface membranes. The results indicated that virtually all activity of both enzymes was restricted to the parasite surface membrane. Cytochemical localization results demonstrated that both activities were externally disposed on the intact organism, and were restricted to the external face of the isolated parasite surface membrane. The two activities were cytochemically differentiated by their substrate specificities and sensitivity to several inhibitors. On the basis of their cytochemical sensitivity to glutaraldehyde treatment, the two nucleotidase enzymes were distinguished from another external surface membrane enzyme, a nonspecific acid phosphatase. Results of the cytochemical assays further demonstrated the biochemical and structural asymmetry of the isolated parasite surface membrane with regard to the localization and distribution of the active sites of these two enzymes.

5'-Nucleotidase↗
PubMed1984

Glucose transport in Leishmania donovani promastigotes.

Mid-log phase Leishmania donovani promastigotes accumulated 2-deoxy-D-glucose (2-DOG) via a carrier mediated transport system, maintaining an apparent Km of 24.4 microM and a Vmax of 3.12 nmol mg-1 protein min-1. D-Glucose but not L-glucose competitively inhibited the 2-DOG transport with an apparent Ki of 18.7 microM. Transport of 2-DOG was inhibited by 2,4-dinitrophenol and NaN3. The parasites maintained a 2-DOG gradient of at least 79 fold across the surface membrane, demonstrating the active nature of the transport system.

Animals↗
PubMed1984

Identification of cross-reactive promastigote cell surface antigens of some leishmanial stocks by 125I labeling and immunoprecipitation.

Externally oriented surface membrane constituents of promastigotes from several Leishmania species were radiolabeled with 125I. Autoradiographs of cell surface-labeled and sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the stocks revealed distinctive patterns of bands in the molecular weight range of 6,000 to 240,000. Immunoprecipitation of detergent extracts of the labeled promastigote stocks with anti-Leishmania donovani membrane serum demonstrated that each of the stocks contained some antigenically cross-reactive determinants. The electrophoretic patterns of these determinants serve both to distinguish the parasite stocks (by unique, species-specific patterns) and to indicate antigenic similarities in stocks thought to be different by other biochemical criteria. At least 12 cross-reactive cell surface antigens in two New World leishmanias are recognized by polyvalent anti-L. donovani serum, suggesting that these common leishmanial antigens may account for the documented serological cross-reactivities among various Leishmania species. In all stocks tested, an iodinated protein was identified which had a relative molecular weight of 65,000 under reducing conditions but which demonstrated an increase in relative mobility in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Distinctive patterns of the antigens common to the several stocks were also demonstrated with the use of monoclonal antibodies.

Animals↗
PubMed1983

Whirlpool-associated Pseudomonas aeruginosa urinary tract infections.

Three previously healthy outpatients had urinary tract infection caused by Pseudomonas aeruginosa. Investigation disclosed that all three patients had been exposed to whirlpool baths within 48 hours before the onset of symptoms. Pseudomonas aeruginosa was isolated from the whirlpool bath associated with each case. Whirlpool baths and spas are commonly contaminated with Pseudomonas and have in the past been associated with rash illness and folliculitis. In addition to these skin infections, our results indicate that whirlpool bath or spa exposure may be an emerging risk factor associated with Pseudomonas urinary tract infections among outpatients.

Acute Disease↗
PubMed1983

Evidence for distinct 5'- and 3'-nucleotidase activities in the surface membrane fraction of Leishmania donovani promastigotes.

A surface membrane fraction isolated from Leishmania donovani promastigotes contained distinct 5'- and 3'-nucleotidase activities. These were distinguished from each other, and from a previously described surface membrane nonspecific acid phosphomonoesterase, on the basis of several properties. The 5'- and 3'-nucleotidases had p' optima of 6.5 and 8.5, respectively. In contrast to the 3'-nucleotidase, the 5'-nucleotidase was inhibited by both ammonium molybdate and fluoride ions; the latter inducing a biphasic response. Neither divalent cations nor chelators affected the 5'-enzyme activity whereas the 3'-enzyme was inactivated by EDTA. This inactivation was fully reversed following removal of the chelator, either by resuspension of the membranes in EDTA free medium or by addition of certain divalent cations in excess; Co2+ being the most effective. The 5'-nucleotidase had activity with both ribo- and deoxyribonucleotide substrates, whereas the 3'-nucleotidase did not hydrolyse deoxyribonucleotides.

5'-Nucleotidase↗
PubMed1983

Radioiodination and identification of externally disposed membrane components of Leishmania tropica.

Two methods, one involving a lactoperoxidase-glucose oxidase coupled reaction and the other employing the insoluble catalyst 1,3,4,6-tetrachloro-3 alpha, 6-alpha-diphenyl-glycoluril (Iodo-Gen), were used to label the surface membrane of promastigotes of Leishmania tropica major. Both methods labelled approximately 20 proteins or glycoproteins (apparent size range 10 to 110 kDa) in a qualitatively similar manner, however the lactoperoxidase method labelled one additional constituent (260 kDa). By omission of both enzymes, or of Iodo-Gen; by comparison of radioactivity incorporated by particulate and soluble cell fractions; and through the action of proteases on live, labelled promastigotes, the surface-labelling specificity of both procedures was confirmed. Immunoprecipitation of Triton X-100 extracts of labelled cells with rabbit antisera revealed a minimum of twelve (seven major) protein antigens in the homologous system and different but cross-reactive protein species from two other isolates of L. tropica. Lectin precipitation of radiolabelled surface components was possible with concanavalin A (but not with other lectins tested) identifying a minimum of 12 glycoproteins. Two of these glycoproteins (210 and 88 kDa) were not recognized by rabbit antiserum.

Animals↗
PubMed1983

Recognition of Leishmania donovani antigens by murine T lymphocyte lines and clones. Species cross-reactivity, functional correlates of cell-mediated immunity, and antigen characterization.

Studies in man and experimental animals suggest that cell-mediated immunity is of primary importance in limiting the pathogenesis of cutaneous and visceral leishmaniasis. In an attempt to determine, more directly, the role of T lymphocytes and the nature of the antigens that activate them, we have propagated antigen-specific murine T lymphocyte lines and clones that proliferate in response to antigens present on the membrane of intact Leishmania donovani promastigotes. One such line cross-reacts with membrane antigens on seven other Leishmania species and, to a lesser extent, with antigens on African procyclic trypanosomes. T lymphocyte clones that also exhibited a broad range of species cross-reactivity were isolated. About 40% of these clones had highly restricted specificity, whereas 60% were more extensively cross-reactive. The parent line and some clones passively transferred footpad DTH when injected locally, and some secreted a lymphokine activity that elicited intracellular killing of amastigotes within infected macrophages. Although the proliferative response of most clones was H-2 restricted, two clones appeared to be reactive in the presence of allogeneic antigen presenting cells. The majority of the clones appeared to recognize carbohydrate containing antigens, and absorption with solid substrate-bound lectins indicated that these antigens contained both mannose and galactose ligands. The antigenic activity was also absorbed using either of two extensively cross-reactive anti-parasite monoclonal antibodies.

Animals↗