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D M Dwyer

Publications and source records attributed to D M Dwyer.

At least 73 records · Page 4Linked to original sources
PubMed1991

Leishmania donovani: long-term culture of axenic amastigotes at 37 degrees C.

L. donovani promastigotes were subjected to heat treatment yielding an axenic amastigote stage which was long-term cultured at 37 degrees C. No differences were observed between the growth rates of axenic amastigotes and promastigotes. Flow cytometry-derived DNA histograms of axenic amastigotes and promastigotes were typical of exponentially growing cell populations. Moreover, axenic amastigotes were metabolically active as evidenced by the release of an immunoprecipitable extracellular acid phosphatase (SAcP) into their culture supernatant. Cell transformation was confirmed by transmission electronmicroscopic examination of thin sections and extended by fracture-flip survey which allowed differentiation of cell membranes. The ultrastructure and nanoanatomy of axenic amastigotes was identical to that of intracellular amastigotes. The production of large amounts of heat-shock axenic amastigotes suitable for biochemical and biological studies of differentiation in Leishmania donovani may have important implications in the development of prevention and/or treatment strategies.

Acid Phosphatase↗
PubMed1990

Golgi-mediated post-translational processing of secretory acid phosphatase by Leishmania donovani promastigotes.

Monensin, an inhibitor of Golgi function, was used to investigate the role of this cell compartment in the glycosylation of Leishmania donovani promastigote secretory acid phosphatase (EC 3.1.3.2). Monensin-treated cells demonstrated morphological changes in the Golgi complex and secreted enzyme with an altered electrophoretic mobility: two discrete bands of approximately 95 and 110 kDa were found, as compared to the heterodisperse nature of the enzyme from untreated controls. Chemical deglycosylation by mild acid hydrolysis resulted in a similar effect on the electrophoretic mobility of purified extracellular enzyme. Acid phosphatase was also treated with N-glycosidase F (EC 3.5.1.52) to remove N-linked oligosaccharides. The altered lectin-binding properties of the enzyme after these two treatments demonstrated that an unusual type of galactose-containing acid-labile carbohydrate was present in secretory acid phosphatase in addition to the N-linked oligosaccharides. Further, experiments with 32P-labelled enzyme indicated that phosphodiester bonds were the structural component responsible for the sensitivity of this carbohydrate to mild acid hydrolysis. Cumulatively, these results demonstrated that a novel form of Golgi-mediated posttranslational modification had occurred to the secretory acid phosphatase presumably by the addition of an acid-labile phosphoglycan.

Acid Phosphatase↗
PubMed1989

Binding and release of C3 from Leishmania donovani promastigotes during incubation in normal human serum.

We have examined the nature and extent of C3 deposition on Leishmania donovani, strain 1S, clone 2D, promastigotes. Total molecules of C3 bound/parasite after 60 min was similar for parasites incubated in normal human serum, normal human serum adsorbed to remove natural antibody, or either serum source chelated with Mg-EGTA to limit activation to the alternative pathway. A comparison of parasites grown to early, mid, late-log or stationary phases revealed no difference in the extent and kinetics of C3 binding. C3 bound covalently to the parasite primarily through a hydroxylamine resistant (putatively amide) linkage. Of the bound C3, 75% was present as hemolytically inactive iC3b. Nearly 50% of the bound C3 was spontaneously released within 30 min at 37 degrees C. This spontaneous release was due to an unusual proteolytic cleavage event that released C3 from the C3 acceptor on the parasite surface. These results define and characterize the unusual features of C3 binding to L. donovani promastigotes during incubation in serum.

Animals↗
PubMed1989

Leishmania donovani: immunochemical localization and secretory mechanism of soluble acid phosphatase.

Monoclonal antibodies specific for the soluble, secreted acid phosphatase (EC 3.1.3.2) of Leishmania donovani were used to investigate the localization of this enzyme in extracellular promastigotes and intracellular amastigotes. Indirect immunofluorescence showed a weak general staining in the promastigote cytoplasm, together with strong fluorescence in the flagellar reservoir. Immunofluorescence studies on U937 cells infected in vitro with L. donovani showed that the pathogenic amastigote stage also produced soluble acid phosphatase. Metabolic labeling experiments using promastigotes indicated that the intracellular enzyme was soluble prior to secretion and no evidence was found for the association of secretory acid phosphatase with cell membranes after protein synthesis. The rapid release of acid phosphatase from the flagellar reservoir was energy dependent and may be coupled to beating of the flagellum. The results demonstrated that acid phosphatase was secreted into the flagellar reservoir by Leishmania promastigotes using a conventional constitutive secretory mechanism, and subsequently released from the reservoir into the extracellular medium.

Acid Phosphatase↗
PubMed1989

Preventive care card for general practice.

A new preventive care card for use in general practice has been designed and produced by a working party of general practitioners from the local medical committees of Devon and Cornwall and the Tamar faculty of the Royal College of General Practitioners. The new card, which is described in this paper, is proposed as a replacement for forms FP7A and FP8A in general practice records.

Family Practice↗
PubMed1989

Adverse events following DTP immunization in Maryland, 1987.

Between January 1, 1987 and December 31, 1987, a total of 184 adverse events were reported to have occurred within 28 days following diphtheria, tetanus, pertussis (DTP) immunization in Maryland. More than half the reports (54 percent) were of serious or major vaccine reactions. Screaming episodes were the most frequently reported serious reaction, having occurred in 64 (35 percent) of the reports.

Child↗
PubMed1988

Identification of a surface membrane proton-translocating ATPase in promastigotes of the parasitic protozoan Leishmania donovani.

ATPase activities were measured in surface membranes and mitochondria isolated from promastigotes of the parasitic protozoan Leishmania donovani. The two enzymes were differentiated on the basis of pH optima, inhibitor sensitivity and by immunochemical methods. The surface-membrane (SM-) ATPase had an activity of 100 nmol/min per mg of protein, which was optimal at pH 6.5. The enzyme was Mg2+-dependent, partially inhibited by Ca2+, and unaffected by Na+ or K+. The SM-ATPase was inhibited by orthovanadate, NN'-dicyclohexylcarbodi-imide, and N-ethylmaleimide [IC50 (concentration causing half-maximal inhibition) 7.5, 25 and 520 microM respectively]; however, it was unaffected by ouabain, azide or oligomycin. The SM-ATPase demonstrated a Km of 1.05 mM and a Vmax. of 225 nmol/min per mg of protein. Moreover, fine-structure cytochemical results demonstrated that the SM-ATPase was localized to the cytoplasmic lamina of the parasite SM. A method was devised for the isolation of SM-derived vesicles. These were used to demonstrate the proton-pumping capacity of the SM-ATPase. Cumulatively, these results constitute the first demonstration of a surface-membrane proton-translocating ATPase in a parasitic protozoan.

Animals↗
PubMed1988

Leishmania donovani: identification of glycoproteins released by promastigotes during growth in vitro.

Culture supernatants of metabolically labeled Leishmania donovani promastigotes were shown to contain approximately 40 electrophoretically distinct released protein compounds. Of these, approximately 20 were glycoproteins which contained terminal mannose residues, as judged by their specific binding to concanavalin A-agarose beads. Smaller subsets of the released glycoproteins were bound by agarose-conjugated Lens culinaris, Ricinus communis, and peanut lectins. Promastigote mannose-containing released glycoproteins were isolated by concanavalin A affinity chromatography and used to immunize a rabbit. This antiserum recognized the parasite-released mannose-containing glycoproteins, including the soluble acid phosphatase, both by immunoprecipitation from solution and in immunoblot analyses. In an antibody bridged enzyme assay this polyspecific serum was also capable of binding native acid phosphatase out of solution and bridging it to the denatured enzyme on SDS-PAGE transblots. Although this antiserum was raised against all 20 released glycoproteins, in agarose gels its major precipitin activity was against the secreted soluble acid phosphatase.

Acid Phosphatase↗
PubMed1987

Preexposure immunization with intradermal human diploid cell rabies vaccine. Risks and benefits of primary and booster vaccination.

Intradermal human diploid cell rabies vaccine (ID HDCV) was licensed for preexposure use in the United States on May 30, 1986. We studied the safety and efficacy of this newly approved route and dose of administration. Serologic results were available from 112 (90%) of the 124 persons who participated in an HDCV low-dose preexposure study in which five different ID, intramuscular (IM), and subcutaneous primary immunization regimens were administered. Three 1.0-mL IM doses of vaccine resulted in titers similar to those from three 0.1-mL ID doses when compared 49 days, one year, and two years after primary immunization. Uniformly high postbooster titers occurred in all five groups when ID boosters were administered at one year or two years. Adverse reactions were similar following both ID and IM vaccination. Although ID HDCV can be a cost-effective substitute for IM vaccine, excessive use of unnecessary preexposure booster doses by any route may be inadvisable because of systemic allergic reactions. In addition, poor immune responses to HDCV have been documented in persons vaccinated in some developing countries. This may limit the use of low-dose regimens in some places.

Antibodies, Viral↗
PubMed1987

Leishmania donovani: generation of monospecific antibody reagents to soluble acid phosphatase.

Four monoclonal antibodies (McAbs) were generated against the soluble extracellular acid phosphatase (EC 3.1.3.2) (S-AcP) of Leishmania donovani. These were detected in the primary screen using an ELISA with promastigote culture supernatants as antigen. Three of the McAbs demonstrated bound S-AcP from such culture supernatants in an enzyme activity binding assay. All immunoprecipitated metabolically labeled S-AcP but none showed any binding to the promastigote surface by indirect immunofluorescence. Moreover, none reacted with Triton X-100 solubilized plasma membranes by immunoprecipitation or Western blotting. These results demonstrated that the McAbs did not recognize the surface membrane bound acid phosphatase, but were specific for the extracellular soluble enzyme. Further, none of the antibodies immunoprecipitated any of the five human acid phosphatase isozymes or reacted with them in Western blots or the enzyme activity binding assay. Therefore, they are specific for the parasite-derived enzyme. One of these was used to affinity purify sufficient L. donovani S-AcP to immunize a rabbit and generate a specific, polyvalent antiserum. This polyvalent antibody immunoprecipitated S-AcP activity but did not cross-react with the surface membrane acid phosphatase, indicating that these two parasite enzymes are separate gene products.

Acid Phosphatase↗
PubMed1987

Biosynthesis and secretion of acid phosphatase by Leishmania donovani promastigotes.

Metabolic labeling and immunoprecipitation experiments demonstrated that soluble acid phosphatase (EC 3.1.3.2) was rapidly synthesized and released into culture medium by Leishmania donovani promastigotes. The kinetics of release indicated a constitutive secretory process (t 1/2 = 45 min). Moreover, acid phosphatase was the major secretory protein. The extracellular enzyme is composed of two heterodisperse bands of approximately 110 and 130 kDa in sodium dodecyl sulphate-polyacrylamide gels. It is synthesized as two intracellular precursors of 92.5 and 107 kDa which acquire the heterodisperse form characteristic of the mature extracellular enzyme during biosynthesis. Labeling in the presence of tunicamycin altered the electrophoretic mobility of the acid phosphatase, indicating the presence of several N-linked oligosaccharides on the mature enzyme. However, tunicamycin did not block secretion of the enzyme or its processing to the heterodisperse form. The biosynthetic effect of tunicamycin was mimicked by N-glycosidase F treatment of acid phosphatase immunoprecipitates. In contrast to tunicamycin, labeling in the presence of monensin inhibited processing of the phosphatase to its heterodisperse form. This indicates that Golgi processing, probably glycosylation, is responsible for the heterodispersity of the mature enzyme in sodium dodecyl sulphate-polyacrylamide gels. As with tunicamycin, monensin treatment did not prevent secretion of the acid phosphatase. These cumulative results demonstrate that release of this enzyme by L. donovani promastigotes occurs via a secretory pathway.

Acid Phosphatase↗
PubMed1987

Identification and characterization of a ribose transport system in Leishmania donovani promastigotes.

A transport system for ribose in Leishmania donovani promastigotes was identified and characterized by measuring the uptake of radioisotope-labeled ribose. The pentoses arabinose, 2-deoxyribose and xylose inhibited ribose uptake, whereas hexoses (glucose, alpha-methylglucoside, thioglucose, galactose, lactose, maltose, mannose), adenosine, and proline did not inhibit uptake, indicating that the transporter exhibited substrate specificity. Intracellular ribose exchanged with 2-deoxyribose. Uptake of ribose showed saturation kinetics with an apparent Km = 2 mM and Vmax = 11 nmol (mg protein)-1 min-1. Both N-ethylmaleimide and p-hydroxymercuribenzoate inhibited ribose uptake which was prevented by dithiothreitol. The uncoupling agents 2,4-dinitrophenol and carbonylcyanide p-(trifluoromethoxy)phenylhydrazone and a variety of inhibitors of energy-driven transport had no significant effect on ribose uptake. Following transport, the intracellular ribose pool contained two-thirds of the sugar in the phosphorylated form and one-third in the neutral form. These cumulative results indicate that a specific carrier mediates ribose uptake via a facilitated diffusion system in L. donovani promastigotes.

Animals↗
PubMed1987

Salmonella newport infections transmitted by fiberoptic colonoscopy.

During a 2-week period following the colonoscopy and biopsy of a patient with acute Salmonella newport gastroenteritis, S. newport was recovered from colonic aspirates or fecal specimens of eight of 28 patients from whom specimens were cultured during or after colonoscopy. Two of the eight persons from whom S. newport was isolated developed acute gastroenteritis, two had asymptomatic infections, and four had positive aspirates collected through a colonoscope but did not become infected. Although S. newport was never recovered from the four colonoscopes used during the outbreak, cultures of one of the colonic biopsy forceps grew S. newport. Contamination of the equipment most likely occurred during colonoscopy of the index patient. Inadequate disinfection of the equipment allowed the organism to survive and possibly to cross-contaminate other colonoscopes, and the organism was then transmitted to other patients by use of the contaminated colonoscopes or the contaminated biopsy forceps. Implemented control measures terminated the outbreak.

Adult↗
PubMed1987

Measles spread in medical settings: an important focus of disease transmission?

During the period September 1981 to August 1985, we investigated every reported case of measles in Oklahoma to confirm the diagnosis, to determine the source, and to identify contacts to prevent spread of the disease. During this time, 33 serologically and/or epidemiologically confirmed cases were investigated. Nine (27%) persons acquired measles in a medical office or clinic waiting area. Eight of these recalled direct face-to-face contact with a source. An additional six (18%) cases were associated with exposure to these medically acquired cases, for a total of 45% that were the direct or indirect result of exposures in medical waiting rooms. The medical waiting room is a location where a reservoir of susceptible individuals may congregate, allowing for potential exposures to measles and other infectious diseases. Because many persons in these settings are too young to have received routine measles vaccination, other measures to decrease exposures in this setting may be necessary to achieve the goal of measles elimination in the United States.

Ambulatory Care Facilities↗
PubMed1986

Identification and biochemical characterization of the plasma membrane glucose transporter of Leishmania donovani.

The plasma membrane glucose transporter of Leishmania donovani, an obligate intracellular protozoan parasite of humans, was specifically labeled, identified, and biochemically characterized. Cytochalasin B, a known inhibitor of D-glucose transport in mammalian cells, but not cytochalasin E inhibited the transport of 2-deoxy-D-glucose in the extracellular promastigote form of this organism. Hydroxysuccinimidyl-4-azido-benzoate was used to photochemically cross-link [3H] cytochalasin B to the glucose transporter in isolated surface membranes and plasma membrane vesicles of L. donovani promastigotes. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the covalently labeled glucose transporter migrated as a 20-30-kDa protein band. This band was eluted from SDS-PAGE gels and subsequently analyzed by isoelectric focusing. The latter revealed two major peaks focusing at pH 6.8 and 6.6 [3H]Cytochalasin B-labeled membrane activity was detergent-solubilized, bound to concanavalin A-agarose beads, and specifically eluted with alpha-methyl mannoside. Analysis of the eluted material by SDS-PAGE revealed a D-glucose-inhibitable cytochalasin B peak with an apparent Mr approximately 20,000. The cumulative results indicate that the glucose transporter of L. donovani promastigotes is a glycoprotein which contains mannose as its major carbohydrate constituent.

Animals↗