Biomedical subjects

M L Rhoads

Publications and source records attributed to M L Rhoads.

At least 37 records · Page 2Linked to original sources

PubMed1997

Extracellular matrix: A tool for defining the extracorporeal function of parasite proteases.

The significance of cysteine protease activity present in excretory/secretory products of the feeding stages of Haemonchus contortus is discussed here by Marcia Rhoads and Raymond Fetterer. Based, in part, on the in vitro degradation and uptake of extracellular matrix components by live parasites, they argue that the cysteine proteases have an essential extracorporeal function in the digestion of host tissues. They also outline the merits of the extracellular matrix model, which mimics the in vivo structure of connective tissue and basement membranes, in analyzing host-parasite interactions and (possibly) parasite developmental processes.

Journal Article↗

PubMed1997

The in vitro uptake and incorporation of hemoglobin by adult Haemonchus-contortus.

The incorporation of radioactivity from [3H]leucine-labeled hemoglobin (Hb) into adult Haemonchus contortus proteins was investigated. Further, the role of previously described cysteine proteases present in intestinal tissue and excretory/secretory products of H. contortus was assessed in the breakdown of Hb. A cell lysate preparation (predominantly Hb) was obtained from reticulocytes metabolically labeled, in vitro, with [3H]leucine. Following 24-h incubation in the presence of [3H]Hb, adult H. contortus incorporated radioactivity. The presence of the protein synthesis inhibitor puromycin (200 micrograms ml-1) reduced incorporation by 72%, indicating that this process was dependent on protein synthesis. The specific cysteine protease inhibitor Z-phe-ala-FMK (PAF) at 0.1 mM had no effect on incorporation of radioactivity; however, the breakdown of Hbg in the culture medium was reduced by 50%. In contrast, PAF at 1.0 mM caused a 78% reduction in incorporated radioactivity. Parasite viability was also decreased by 1.0 mM PAF, and thus the reduction of incorporation of radioactivity may not be due to specific enzyme inhibition. The serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) at 1.0 mM caused a 40% reduction in incorporation of radioactivity; the aspartic protease inhibitor pepstatin (1 mM) was without effect. Adult H. contortus also incorporated radioactivity from [3H]leucine-labeled intact reticulocytes. This incorporation was inhibited-by 1.0 mM PAF and AEBSF in a manner similar to that for the cell lysate preparation. These data indicate that adult H. contortus degrade Hbg and incorporate the radioactivity into their macromolecules. The specific action of the endogenous cysteine protease in the digestion of Hbg could not be demonstrated unequivocally. However, the hypothesis that the secreted cysteine protease functions in extracorporeal digestion was supported.

Animals↗

PubMed1997

Neonatal hypothyroidism permanently alters follicle-stimulating hormone and luteinizing hormone production in the male rat.

Transient neonatal hypothyroidism, induced with the goitrogen 6-n-propyl-2-thiouracil (PTU), results in dramatic increases in both testis size and sperm production in the adult rat. The observed increases in testis size and function occur in the presence of normal circulating testosterone levels. However, circulating gonadotropin levels are chronically reduced by 30-50% at all times in treated males. To better understand the permanent reduction in serum gonadotropin levels following transient neonatal hypothyroidism, we conducted a series of experiments to evaluate pituitary and hypothalamic function in the adult male PTU-treated rat. PTU treatment led to a significant reduction in GnRH-stimulated LH production. Castration resulted in 3.9- to 8.5-fold increases in circulating gonadotropin levels in both treated and control males; however, the absolute increases were significantly reduced in treated males. In contrast to circulating levels, pituitary gonadotropin contents did not increase in treated males after castration. PTU treatment did not lead to a reduction in the density of either luteotropes or folliculotropes, and both cell types increased in size and density after castration. The relative concentrations of both gonadotropin beta-subunit messenger RNAs increased more slowly in treated males than in controls after castration. Thus, although treated rats have the intrinsic ability to produce normal circulating levels of LH and FSH, gonadal feedback and an overall reduction in gonadotrope synthetic ability combine to produce the chronically reduced circulating levels of these hormones.

Animals↗

PubMed1997

Secretion of an aminopeptidase during transition of third- to fourth-stage larvae of Ascaris suum.

Protease activity was identified in culture fluids collected during in vitro development of L3 to L4 larval stages of Ascaris suum. Fluorogenic peptide substrates with unblocked N-termini were specifically hydrolyzed indicating aminopeptidase activity; a terminal arginyl residue was preferred. Culture fluids did not hydrolyze fluorogenic peptide substrates with blocked N-termini (endopeptidase substrates). The aminopeptidase activity was inhibited by 1,10-phenanthroline (metalloprotease inhibitor) and by amastatin and bestatin (aminopeptidase inhibitors); AEBSF (serine protease inhibitor), Z-phe-ala-FMK and E-64 (cysteine protease inhibitors), and pepstatin A (aspartyl protease inhibitor) had little effect on activity. The apparent molecular weight of the aminopeptidase was estimated by sucrose density gradient centrifugation at 293 kDa. The aminopeptidase displayed an acidic isoelectric point of 4.7. The peak secretion of the aminopeptidase was temporally associated with molting and suggests a function for the protease in this complex process.

Aminopeptidases↗

PubMed1996

The role of the sheath in resistance of Haemonchus contortus infective-stage larvae to proteolytic digestion.

Surface iodinated larvae of Haemonchus contortus were incubated in the presence of the fungal protease, proteinase K, and proteolysis quantified by scintillation counting of released radioactivity. No radioactivity was released from live ensheathed infective-stage larvae (L3(2M)). In contrast, 58% of the radioactivity was released from ecdysed, second molt (2M) cuticles (sheaths) of L3(2M) and 48% from live exsheathed third-stage larvae (L3). When L3(2M) larvae were killed by heat (80 degrees C for 10 min) prior to proteinase K incubation, 61% of the radioactivity was released, whereas less than 7% was released from larvae killed by the metabolic inhibitors NaN3 or KCN. Proteinase K released 44% of the radioactivity from live L3(2M) larvae which had been preincubated with 1% sodium dodecylsulfate (SDS), whereas no radioactivity was released from L3(2M) larvae preincubated with either 1% Triton X-100, 0.2% CTAB, 50% methanol, 50% ethanol, or water. Following incubation with proteinase K, only L3(2M) larvae which had been heat-killed or preincubated with SDS showed visible damage to the sheath. Material released from L3(2M) larvae by exposure to either heat or SDS contained a 98,000 M(r) protein by SDS-PAGE autoradiography. These results indicate that viable L3(2M) larvae are resistant to attack by proteinase K and that this resistance is dependent on structural properties of the sheath.

Animals↗

PubMed1996

Extracellular matrix degradation by Haemonchus contortus.

To better understand the in vivo function of secreted cysteine proteases of Haemonchus contortus, the ability of live parasites to degrade connective tissue was investigated using [3H]proline-labeled extracellular matrix produced by smooth-muscle cells (R22). The matrix was composed of glycoprotein(s) (34%), elastin (49%), and collagen (15%) in an insoluble, multilayered, cross-linked structure. No degradation of the extracellular matrix by third-stage larvae (L3) (10,000/ml) occurred during 24-hr in vitro incubation. In contrast, fourth-stage larvae (L4) (1,000/ml) degraded 42% of the matrix, whereas adults (100/ml) degraded the entire matrix. The presence of Z-phe-ala-FMK (100 microM), a specific cysteine protease inhibitor, during incubation of adults, reduced matrix degradation to 30% without affecting parasite motility. Isolated adult excretory/secretory products (ESP) (0.1 mg protein/ml) degraded 64% of the total matrix; specific degradation consisted of 80.3% of the glycoprotein, 67.1% of the elastin, and 27.6% of the collagen matrix components. Degradation of the matrix by ESP was stimulated by dithiothreitol (2 mM) and inhibited by Z-phe-ala-FMK. Thus, the secretory cysteine proteases of H. contortus are active under physiological conditions and able to degrade the major components of connective tissue in an in vitro model system that simulates their structure in vivo. These data strengthen the proposed role of these enzymes in the breakdown of host tissue.

Animals↗

PubMed1995

Developmentally regulated secretion of cathepsin L-like cysteine proteases by Haemonchus contortus.

Cysteine protease activity was present in media collected after 24 hr in vitro culture of adult Haemonchus contortus. The released cysteine protease hydrolyzed the fluorogenic 7-amino-4-trifluoromethyl coumarin (AFC)-substituted synthetic peptides Z-phe-arg-AFC and Z-ala-arg-arg-AFC, but not Z-arg-arg-AFC or Z-arg-AFC, characterizing this activity as cathepsin L-like. Within the parasite, cysteine protease activity was highest in extracts of intestinal tissue. Secreted cysteine protease inhibited the clotting of sheep blood and hydrolyzed hemoglobin, fibrinogen, collagen, and IgG; the IgG hydrolysis site was within the hinge region. Four proteases with M(r) values of 30, 34, 37, and 41 kDa were identified with biotinylated-phenylalanine-arginine-fluoromethyl ketone, a specific probe that binds to active cysteine proteases. Adult parasites cultivated in the presence of 0.1 mM levamisole released 50% less protease activity compared to control cultures; in the presence of rafoxanide (0.1 mM), protease was not detected. Cathepsin L-like cysteine protease activity was released also by L4, but not the L3 larval stage. The active and developmentally regulated release of cysteine proteases by H. contortus may have a critical function in worm nutrition, immune evasion, or both.

Amino Acid Sequence↗

PubMed1994

A Taenia crassiceps cDNA sequence encoding a putative immunodiagnostic antigen for bovine cysticercosis.

A cDNA expression library was constructed in lambda gt11 using poly A mRNA from the metacestode stage of Taenia crassiceps. The library was screened with rabbit antiserum to a previously defined protein fraction from Taenia hydatigena immunodiagnostic for bovine cysticercosis and with sera from cattle with experimentally induced cysticercosis. One clone (lambda TcA2) containing a 279-bp cDNA insert, reacted strongly with both antisera. A second clone (lambda TcA5.5) revealed the full-length cDNA sequence to be 361 bp. Data from Southern blots and enzymatically amplified genomic DNA segments were consistent with multiple copies or a gene family within the genome. The lambda TcA2 cDNA insert was subcloned into the plasmid pPR987 which generated a 47-kDa maltose-binding fusion protein (TcA2-MBP). Affinity-purified TcA2-MBP antigen reacted positively by ELISA with sera from cattle with experimentally induced T. saginata infections but not with sera from cattle with Fasciola hepatica or common gastrointestinal parasite infections. Rabbit polyclonal, monospecific antisera to TcA2-MBP recognized a 10-kDa protein in the cyst fluid, body wall and excretory/secretory products of the metacestode stage of T. crassiceps and immonolocalized this protein to organelles within the matrix of the cyst wall.

Animals↗

PubMed1994

Purification and characterization of surface-associated proteins from adult Haemonchus contortus.

Extrinsic radioiodination experiments have shown that male and female adults of Haemonchus contortus (BPL strain) express a stage-specific set of surface-associated proteins with apparent molecular mass values of 30, 58, 81, and 143 kDa. A quantitatively different pattern of iodinated surface proteins is expressed by adults of the PPR strain of H. contortus, whereas the pattern of iodinated proteins expressed by Haemonchus similis is qualitatively distinct (38, 68, and 121 kDa). The 58-, 81-, and 143-kDa proteins of the BPL strain are glycosylated, whereas the 30-kDa protein is not. The binding of wheat germ agglutinin to the surface glycoproteins was inhibited by the trimer of N-acetylglucosamine (N,N,N-triacetylchitotroise) but not by the monosaccharide, indicating the presence of chitin-like homopolymers. The carbohydrate portion of the 58-kDa protein is N-linked and accounts for 30% of its apparent mass. Under nonreducing conditions, the 58-kDa glycoprotein forms a high molecular mass polymer that is unable to penetrate a 10% acrylamide gel. The 143- and 81-kDa surface glycoproteins were not hydrolyzed by either N- or O-glycanase, indicating unusual modifications to the saccharide-linkage and rendering it resistant to glycosidase digestion. The 30-, 58-, and 143-kDa purified surface proteins produced distinct peptide maps with Staphylococcus aureus V8 protease.

Abomasum↗

PubMed1993

Trichuris suis: a zinc metalloprotease from culture fluids of adult parasites.

A zinc metalloendoprotease has been isolated from in vitro culture fluids of Trichuris suis adults. The protease was purified from total culture fluids by passage through a cation exchange high-pressure liquid chromatography column. The 45-kDa protease has a pH optimum of 7.0 and an isoelectric point of 8.0 and was localized to the stichosome of the parasites using immunohistochemistry techniques.

Animals↗

PubMed1993

Biochemistry of the nematode cuticle: relevance to parasitic nematodes of livestock.

The cuticle of nematodes is a thin, flexible outer covering composed primarily of protein with trace amounts of lipid and carbohydrate. There has been considerable recent interest in the biochemistry, immunology and molecular biology of the cuticle of parasitic nematodes because of its role as an interface between parasite and host. The cuticle consists of: (1) collagen-like proteins that form the medial and basal layers; (2) non-collagen proteins that form the epicuticular and external cortical regions; (3) non-structural proteins associated with the external surface. The collagen-like proteins are solubilized by reducing agents, have molecular weights of 30-120 kDa and exhibit stage and species variations. Nematode collagen genes, however, code only for proteins with molecular weights of 30 kDa. The non-collagenous proteins, referred to as cuticlin, exhibit unusual chemical properties as indicated by their resistance to solubilization even under strongly denaturing conditions. Recent studies of Ascaris suum have demonstrated the presence of tyrosine-derived cross-links, dityrosine and isotrityrosine, that may form the linkage between subunits in assemblage of the collagenous and noncollagenous structural components of the cuticle. A peroxidase enzyme has been implicated in the synthesis of these cross-links. Recent 125I labeling studies of Haemonchus contortus have identified and characterized stage-specific proteins on the cuticular surface.

Animals↗

PubMed1993

Synthesis of tyrosine-derived cross-links in Ascaris suum cuticular proteins.

Tritiated dityrosine and isotrityrosine were detected by high performance liquid chromatography (HPLC) of acid hydrolysates of cuticular proteins from larval Ascaris suum following their 96-hr in vitro incubation in [3H]tyrosine. Sixty percent of the HPLC-recovered radiolabel was present as tyrosine, 20% as dityrosine, and 6% as isotrityrosine. Approximately 13% of radioactivity was associated with several unidentified peaks. A similar distribution of radioactivity was observed in acid hydrolysates of cuticular proteins from young adults of A. suum following 48 hr in vitro incubation with [3H]tyrosine. The 2-mercaptoethanol (2ME)-insoluble cuticular protein from the larval stages had a higher rate of synthesis of [3H]dityrosine than did the 2ME-soluble cuticular proteins, whereas the 2ME-soluble cuticular proteins had higher rates of synthesis of [3H]isotrityrosine. Pulse-chase studies of A. suum larvae demonstrated a relatively low rate of synthesis of both dityrosine and isotrityrosine. The addition to the culture media of the peroxidase inhibitors, phenylhydrazine (PHEN), 3-amino-1,2,4-triazole (AT), and N-acetyltyrosine (NAT) reduced the amount of [3H]tyrosine synthesized into both dityrosine and isotrityrosine. In a cell-free system, soluble extracts of A. suum larvae also converted radiolabeled tyrosine to dityrosine; isotrityrosine was produced by some extracts. The rate of conversion correlated with time of incubation and the volume of added extract and was inhibited by AT, NAT, and PHEN, with PHEN being the most potent inhibitor. The results of the present study suggest that the tyrosine residues of the cuticular proteins are posttranslationally modified by the formation of dityrosine and isotrityrosine cross-links. This modification is most likely mediated by a peroxidase.

Animals↗

PubMed1992

Assessment of genetic diversity and population structure of Xanthomonas oryzae pv. oryzae with a repetitive DNA element.

A repetitive DNA element cloned from Xanthomonas oryzae pv. oryzae was used to assess the population structure and genetic diversity of 98 strains of X. oryzae pv. oryzae collected between 1972 and 1988 from the Philippine Islands. Genomic DNA from X. oryzae pv. oryzae was digested with EcoRI and analyzed for restriction fragment length polymorphisms (RFLPs) with repetitive DNA element as a probe. Twenty-seven RFLP types were identified; there was no overlap of RFLP types among the six races from the Philippines. Most variability (20 RFLP types) was found in strains of races 1, 2, and 3, which were isolated from tropical lowland areas. Four RFLP types (all race 5) were found among strains isolated from cultivars grown in the temperate highlands. The genetic diversity of the total population of X. oryzae pv. oryzae was 0.93, of which 42% was due to genetic differentiation between races. The genetic diversities of strains collected in 1972 to 1976, 1977 to 1981, and 1982 to 1986, were 0.89, 0.90, and 0.92, respectively, suggesting a consistently high level of variability in the pathogen population over the past 15 years. Cluster analysis based on RFLP banding patterns showed five groupings at 85% similarity. The majority of strains from a given race were contained within one cluster, except for race 3 strains, which were distributed in three of the five clusters.

DNA, Bacterial↗

PubMed1991

Efficacy of the immunoblot assay for cysticercosis in pigs and modulated expression of distinct IgM/IgG activities to Taenia solium antigens in experimental infections.

A recently invented immunoblot assay for human cysticercosis was evaluated for efficacy in pigs. The test population consists of 45 pigs with parasitologically confirmed cysticercosis, 47 with heterologous infections, 45 SPF or concrete raised control animals. With this group of 137 animals the test performance was 100% sensitive and 100% specific. The antigen-specific responses of immunoglobulin A (IgA), IgG and IgM in four pigs infected with Taenia solium eggs derived from a human were quantified by immunoblot. Antigen-specific activities were observed as early as 1 week postinfection. The first antigen-specific isotypic response was IgM antibodies directed against a glycoprotein at 97 KD (GP97). This activity generally disappeared between the sixth and ninth week postinfection. Between Weeks 5 and 8, IgG activity rose as IgM activity fell. The IgG activity, however, was directed mostly towards GP50 and GP42 antigens. If the same response occurs in people with cysticercosis, identifying specific isotype activity may help to distinguish new infection from old.

Animals↗

PubMed1991

A recombinant immunodiagnostic antigen for bovine cysticercosis.

The 70% ammonium sulfate-soluble fraction of the cyst fluid of Taenia hydatigena (designated ThFAS) was previously shown to have potential as an immunodiagnostic reagent for bovine cysticercosis. Western blot analysis indicated that the specific reactivity with antibodies in sera of T. saginata-infected cattle was associated with a 10 kDa component. Rabbit antiserum to ThFAS identified a homologous antigenic protein from the cestode Taenia crassiceps. Consequently, a cDNA expression library was constructed in lambda gt11 using poly A mRNA purified from T. crassiceps metacestodes and screened with rabbit antiserum to ThFAS. One strongly reactive clone (designated lambda TCA-2) produced a 123 kDa beta-galactosidase fusion protein which reacted in Western blot with sera from calves experimentally-infected with T. saginata and did not react with sera from uninfected calves or from cattle infected with Fasciola hepatica or with common gastrointestinal cattle parasites.

Animals↗

PubMed1991

Development of a serologic assay for cysticercosis, using an antigen isolated from Taenia spp cyst fluid.

An ammonium sulfate-soluble fraction of Taenia hydatigena cyst fluid (ThFAS) was further evaluated for use in the immunodiagnosis of cysticercosis. Analysis of ThFAS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblot analysis confirmed earlier reports of a highly specific, low molecular weight antigen in this preparation; in contrast, other components of ThFAS were shown to react nonspecifically. Antibodies against the less than 12-kD diagnostic antigen were detected in sera from 10 cattle and 4 swine inoculated with metacestodes of T saginata and T solium, respectively, but not in animals inoculated with Fasciola hepatica, Trichinella spiralis, Brucella abortus, or Toxoplasma gondii, or in noninoculated controls. Isolation and immobilization of the less than 12-kD antigen on a hydrophobic transfer membrane resulted in development of an unambiguous dipstick assay capable of correctly identifying fully developed (10-week) experimentally induced infections in cattle and swine. In addition, the dipstick assay was highly specific for diagnosis of the disease in human beings, and offers the potential of distinguishing between human clinical cases of cysticercosis and taeniasis. A similar reactive antigen of diagnostic potential was also identified and isolated from T crassiceps and T taeniaeformis cyst fluids.

Animals↗

PubMed1990

Tyrosine-derived cross-linking amino acids in the sheath of Haemonchus contortus infective larvae.

The sheath or second-molt cuticle (2M) was isolated from in vitro exsheathed Haemonchus contortus infective larvae (L3[2M]). Acid hydrolysates of 2-mercaptoethanol (2ME)-soluble and 2ME-insoluble cuticular proteins were analyzed by high performance liquid chromatography for tyrosine-derived cross-linking amino acids. Dityrosine and isotrityrosine were identified by their chromatographic behavior, absorbance spectra, and other chemical characteristics in both the 2ME-soluble and 2ME-insoluble fractions. Dityrosine and isotrityrosine were found in greater amounts in the 2ME-insoluble proteins. When intact 2M cuticles were labeled with 125I prior to acid hydrolysis, radiolabel was recovered in tyrosine but not dityrosine or isotrityrosine indicating that the tyrosine cross-links are not susceptible to iodination in the intact protein. The results are consistent with a hypothesis that tyrosine-derived cross-links are important components of H. contortus 2M cuticular proteins.

Animals↗

PubMed1989

The serological response of pigs experimentally infected with a species of Taenia from Taiwan.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody in pigs infected with a possibly new species of Taenia isolated in Taiwan is described. The test antigen ThFAS was fractionated from the cyst fluid of a heterologous cestode Taenia hydatigena. In lightly infected pigs (less than or equal to 4 recovered cysts at necropsy 17 weeks post-inoculation), antibody was detected as early as 3 weeks post-inoculation. In more heavily infected pigs (6-72 recovered cysts at necropsy 32 weeks post-inoculation), antibody was still detectable at the time of necropsy. Cysticerci were found only in the livers of the infected pigs. This ELISA should be highly useful for detecting infection of pigs with this larval cestode in regions where the presence of Taenia solium is unlikely.

Animals↗