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M L Rhoads

Publications and source records attributed to M L Rhoads.

49 records · Page 3Linked to original sources
PubMed1988

Purification, characterization, and immunochemical studies of beta-N-acetyl-D-hexosaminidase from the parasitic nematode Trichinella spiralis.

The exoglycosidase, beta-N-acetyl-D-hexosaminidase was purified 600-fold from the muscle-stage larvae (L1) of Trichinella spiralis. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the purified enzyme-active fraction contained 4 polypeptides with apparent molecular weights of 100,000, 68,000, 58,000 and 54,000. The beta-N-acetyl-D-hexosaminidase corresponds to the Mr 100,000 polypeptide as demonstrated by SDS-PAGE analysis of the enzyme-stained region isolated from a non-denaturing polyacrylamide gel. In addition, rabbit antiserum to a homogeneous preparation of the Mr 100,000 polypeptide (isolated by electroelution from an SDS-PAGE gel) specifically immunoprecipitated beta-N-acetyl-D-hexosaminidase activity from an extract of L1. Isoelectrofocusing (pH 3-10) resolved 4 isoenzymes of T. spiralis beta-N-acetyl-D-hexosaminidase with isoelectric points (pI) of 5.35, 5.49, 5.63 and 5.79. The T. spiralis beta-N-acetyl-D-hexosaminidase is a glycoprotein based on its binding to lentil-lectin Sepharose affinity column and its specific binding of concanavalin A on Western blots. The IgG fraction of T. spiralis-infected mouse serum specifically immunoprecipitated T. spiralis beta-N-acetyl-D-hexosaminidase. The removal of carbohydrate from T. spiralis beta-N-acetyl-D-hexosaminidase significantly reduced its antigenicity. Immunocytochemical analysis of L1 tissue sections with polyclonal rabbit antisera to the homogeneous beta-N-acetyl-D-hexosaminidase enzyme indicated localization on cell membranes and the epicuticle.

Animals↗
PubMed1988

Application of biotechnology methods to the study of cestodes.

The advent of biotechnology has invigorated research on the control of cestode diseases, especially cysticercosis infections in man and animals. The utilization of hybridoma technology to produce antigen-specific monoclonal antibodies has resulted in great strides towards obtaining pure antigens relevant for immunodiagnostic purposes and for research on vaccines. However, the isolation and identification of antigens is only the initial step in the development of such reagents. Production of antigens in quantities sufficient for research/development and commercialization is hampered by the scarcity of viable parasite material for extraction. Expectations are that this problem can be surmounted by application of recombinant DNA methods to produce cloned genes for antigen expression in cultured microorganisms or cells. Remarkable progress has been made recently in isolating and cloning genes from several important cestode species and antigens have been expressed in vitro with genes cloned from Taenia taeniaformis and T. hydatigena. Although these early efforts have not as yet resulted in practical antigen production, the prospects for doing so appear good. The complex epidemiology of cestode diseases is another research subject that has benefited from the successful application of the tools of biotechnology. For example, the greater precision in typing biological variants afforded by DNA analysis has led to important revisions of the understanding of hydatid disease. DNA probes are now available for Echinococcus spp, which are effective for typing isolates. These probes may also find use as reagents for distinguishing eggs of Echinococcus from other taeniid eggs, a serious difficulty for field investigations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Evaluation of an antigenic fraction of Taenia hydatigena metacestode cyst fluid for immunodiagnosis of bovine cysticercosis.

An antigenic fraction (ThFAS) isolated from Taenia hydatigena metacestode cyst fluid was used in an ELISA to detect antibodies to T saginata in experimentally and naturally infected cattle. In 10 calves given 1,000 to 100,000 T saginata eggs (20% to 60% viability), IgG and IgM antibodies were detected in all the calves by post-inoculation week 3. Immunoglobulin G antibody values remained increased until calves were slaughtered at post-inoculation weeks 13 to 26. Six naturally infected calves (determined by postmortem examination) were considered positive, using the ELISA. Shared antigens were demonstrated between ThFAS and T saginata and T crassiceps; there were no shared antigens between ThFAS and Haemonchus contortus or Fasciola hepatica. Specific lectin binding to ThFAS indicated the presence of glycoconjugates. Immunoblot analysis indicated that a low molecular weight polypeptide (10,000 Mr) bears the immunodiagnostic antigen.

Animals↗
PubMed1985

Glycosidases of Trichinella spiralis.

The exoglycosidases beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-D-galactosaminidase, alpha-1-fucosidase, alpha-D-glucosidase and alpha-D-mannosidase, and a non-specific acid phosphohydrolase are present at high levels in extracts of adult and muscle-stage (L1) Trichinella spiralis and at lower (5-30-fold) levels in extracts of the newborn larvae. The enzyme activities from the L1 extract were characterized. All displayed maximum activity at acid pH. beta-N-acetyl-D-glucosaminidase and beta-N-acetyl-D-galactosaminidase had identical molecular weights (110 000), pH optima (5.0), and isoelectric points (5.7) indicating that both of these substrate specificities reside in the same protein molecule. alpha-1-Fucosidase had a molecular weight of 125 000 and exhibited two pH optima (5.0 and 6.0) and four isoelectric points (5.9, 6.4, 6.7 and 7.1) indicating its presence in multiple molecular forms. alpha-D-Glucosidase had a molecular weight of 85 000, a pH optimum of 6.0 and an isoelectric point of 5.2; alpha-D-mannosidase had a molecular weight of 192 000, a pH optimum of 6.0 and an isoelectric point of 4.5; and acid phosphatase had a molecular weight of 81 000, a pH optimum of 6.0 and two isoelectric points (4.8 and 5.9) indicating its existence in two molecular forms. The same glycosidases and acid phosphatase were detected also in culture fluids collected after 15-20-h incubation of both L1 and adults. As in the worm extracts, beta-N-acetyl-D-glucosaminidase was present in these culture fluids at the highest activity with acid phosphatase present at the next highest activity.

Acetylglucosaminidase↗
PubMed1985

A potential diagnostic reagent for bovine cysticercosis.

A fraction of larval Taenia hydatigena cyst fluid was shown to have high sensitivity and specificity in the enzyme-linked immunosorbent assay (ELISA) for the detection of bovine antibodies to the heterologous parasite Taenia saginata. This antigenically active lipoprotein fraction was isolated by ultracentrifugal density flotation using either ammonium sulfate (specific gravity = 1.231 g per ml) or NaCl/KBr (specific gravity = 1.225 g per ml), followed by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that this fraction was composed of high molecular weight (65,000 to 77,000 Mr) and low molecular weight (9,500 to 16,000 Mr) proteins. Electrophoresis under non-denaturing conditions in either acrylamide (5%) or agarose (1%) resulted in 1 major diffuse band staining for both protein and lipid. The high and low molecular weight proteins observed on SDS-PAGE under reducing conditions could not be resolved by gel filtration chromatography and emerged as a single lipoprotein peak. This T. hydatigena cyst fluid fraction appears promising as a diagnostic reagent in the ELISA for bovine cysticercosis.

Animals↗
PubMed1983

Trichinella spiralis: identification and purification of superoxide dismutase.

A metalloprotein with superoxide dismutase activity was isolated and purified from muscle-stage Trichinella spiralis. The anti-genicity of the purified enzyme was demonstrated by an immunospecific reaction with T. spiralis antiserum in an enzyme-linked immunosorbent assay. In addition to its presence in somatic extracts of T. spiralis, the enzyme was also excreted into culture fluids in which the muscle-stage larvae had been incubated for periods as short as 3 hr and up to 72 hr. The enzyme was characterized as a copper- and zinc-containing, cyanide-sensitive, superoxide dismutase with a molecular weight of 36,000 (estimated by get filtration), consisting of two subunits of 17,000 Mr (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The isoelectric point was 5.6. Muscle-stage T. spiralis contained one molecular form of the enzyme, whereas adult T. spiralis contained two molecular forms. This enzyme may function as an essential defense mechanism against the highly destructive superoxide radical encountered either intracellularly, as a product of biological oxidation, or externally, as a component of the host's immune system.

Animals↗
PubMed1981

Cholinesterase in the parasitic nematode, Stephanurus dentatus. Characterization and sex dependence of a secretory cholinesterase.

An antigenic secretory protein with cholinesterase activity was isolated from the excretory gland cells of Stephanurus dentatus and was purified by gel filtration and ion exchange chromatography. The antigenicity of the cholinesterase was demonstrated by an esterase-active immunoprecipitate formed with S. dentatus antiserum and by the ability of the antiserum to protect the enzyme from heat inactivation. The enzyme was found to be secreted by the adult nematodes during in vitro cultivation. The level of cholinesterase activity and its release from the excretory gland cells of the parasite were 27-fold greater in the male than in the female. Ninety per cent of the enzyme activity was localized in the soluble fraction of the gland cells. The molecular weight of the enzyme, estimated by sucrose density gradient centrifugation, was 100,000. Two molecular forms were separated by isoelectrofocusing, with isoelectric points of 7.0 and 6.9. At optimum substrate concentrations, the rate of hydrolysis of acetylthiocholine was 8 times greater than that of butyrylthiocholine; the Michaelis constants were 560 microM and 81 microM for acetylthiocholine and butyrylthiocholine, respectively. The enzyme exhibited substrate inhibition at substrate concentrations greater than 10 mM and was inhibited by eserine sulfate, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, Tris, and acetone. The enzyme was highly unstable in dilute protein solutions.

Animals↗
PubMed1978

Proteinase inhibitors from the excretory gland cells of Stephanurus dentatus. Purification and properties of three secretory proteinase inhibitors.

Three proteinase inhibitors designated as I, II, and III were isolated from the excretory gland cells of the swine kidney worm, Stephanurus dentatus. The inhibitors, which were trichloroacetic acid-soluble, were purified by affinity chromatography and ion exchange chromatography. The homogeneity of each inhibitor was shown by polyacrylamide gel electrophoresis and electrofocusing. The molecular weights of the inhibitors estimated by sodium dodecyl sulfate gel electrophoresis fell within a limited range of 9300 to 9700, and the isoelectric points were 6.45, 6.20, and 5.34 for Inhibitors I, II, and III, respectively. The inhibitors formed complexes with trypsin having apparent dissociation constants (Ki) of 2.9 X 10(-11), 7.6 X 10(-11), and 6.4 X 10(-11) M, respectively. Each inhibitor inhibits the esterolytic and proteolytic activities of both trypsin and chymotrypsin. A proteinase inhibitor present in the reproductive organs, intestines, body walls, and esophagi was identical with Inhibitor II found in the excretory gland cells. Culture medium collected after 24-h incubation with adult worms contained the same three inhibitors as the excretory gland cells. These data suggest that the gland cells may secrete the inhibitors internally and externally.

Amino Acids↗
PubMedDate not supplied

Biochemical and immunochemical characterization of 125I-labeled cuticle components of Haemonchus contortus.

Live Haemonchus contortus developmental stages were radioiodinated and then subjected to a stepwise extraction procedure consisting of a buffer extract (with or without detergent) to solubilize putative surface-associated antigenic macromolecules, followed by a detergent/beta-mercaptoethanol (BME) extract to solubilize putative cuticle collagen proteins. A buffer-extracted iodinated 100-kDa protein was present in the free-living, infective L3(2M) stage. This labeled protein was released during in vitro exsheathment of L3(2M) and was not present in the ecdysed second molt (2M) cuticle. In addition to the 100-kDa protein, exsheathment fluid contained a 70-kDa labeled protein that was not extracted from iodinated L3(2M) with either detergent or BME. The data suggest that these proteins are components of the specialized ring portion of the 2M cuticle that is enzymatically ruptured during ecdysis. The L3(2M) and the exsheathed third-stage larvae (L3) contained 3 labeled, BME-extracted, collagenase-sensitive proteins of 108, 88 and 53 kDa. In contrast, four detergent-extracted, collagenase-insensitive, iodinated proteins (143, 81, 58 and 30 kDa) were present in adult H. contortus. The 143-kDa protein was both glycosylated and immunogenic. All 4 adult cuticle proteins were released from the cuticle surface into culture fluids. Furthermore, a cysteine protease was secreted by adults which apparently hydrolyzed the released 81-, 58- and 30-kDa surface proteins.

Animals↗