Biomedical subjects

M L Rhoads

Publications and source records attributed to M L Rhoads.

At least 19 recordsLinked to original sources

PubMed2005

Detrimental effects of high plasma urea nitrogen levels on viability of embryos from lactating dairy cows.

High plasma urea nitrogen (PUN) concentrations are associated with decreased fertility in lactating dairy cows. Our objective was to evaluate the quality of embryos flushed from superovulated lactating cows having moderate or high PUN concentrations. Subsequent embryo survival was determined after transfer to recipient heifers with either low or high PUN. Lactating Holstein dairy cows (n = 23; 50-120 days in milk) were randomly assigned to one of two diets designed to result in moderate or high PUN concentrations (15.5 +/- 0.7 and 24.4 +/- 1.0 mg/dl, respectively; P < 0.001) and were fed for 30 days before embryo flushing and recovery. Embryos (n = 94) were evaluated morphologically, frozen and subsequently transferred into synchronized virgin heifers that were fed one of two diets designed to result in either low or high PUN concentrations (7.7 +/- 0.9 and 25.2 +/- 1.5 mg/dl, respectively; P < 0.001; 2 x 2 factorial design). The number, quality and stage of development of recovered embryos were similar for cows with moderate or high PUN. Transfer of embryos from moderate PUN donor cows resulted in a higher pregnancy rate (35%; P < 0.02) than the transfer of embryos from high PUN donor cows (11%). Pregnancy rate was not affected by either recipient diet or the interaction of donor and recipient diets (P > 0.05). These results indicate that high PUN concentrations in lactating dairy cows decrease embryo viability through effects exerted on the oocyte or embryo before recovery from the uterus 7 days after insemination.

Animal Feed↗

PubMed2004

Effects of urea infusion on the uterine luminal environment of dairy cows.

Previous research indicates that high plasma urea nitrogen (PUN) concentrations are associated with decreased fertility in lactating dairy cows. The objective of this study was to monitor changes in the uterine environment during acute elevation of PUN. Lactating dairy cows (n = 8) were infused with saline or urea (0.01 g of urea/h per kg of body weight) through jugular vein catheters on d 7 after estrus. After 24 h, cows were switched to the opposite treatment for a second 24-h infusion period. Blood samples were collected every 2 h, and the pH within the lumen of the uterine horn ipsilateral to the corpus luteum was recorded every 6 h. At the end of each 24-h infusion period, 30 mL of sterile saline was flushed into the uterine lumen and immediately retrieved. Mean PUN concentration increased from 16.6 +/- 1.3 mg/dL during saline infusion to 22.6 +/- 1.3 mg/dL during urea infusion. Uterine pH decreased during urea infusion from 7.08 +/- 0.07 at 6 h to 6.88 +/- 0.08 at 18 h, but was unchanged during saline infusion (7.01 +/- 0.08 at 6 h to 7.06 +/- 0.07 at 18 h). Protein concentration, PGF(2alpha), and prostaglandin E(2) concentrations in uterine lavage samples were not different between treatments. The results of this study indicate that a short-term increase in PUN can exert direct effects on the uterine environment by decreasing uterine pH.

Animals↗

PubMed2001

Release of hyaluronidase during in vitro development of Ascaris suum from the third to fourth larval stage.

An enzyme that degraded glycosaminoglycan hyaluronic acid was released during in vitro development of Ascaris suum L3 to L4. The enzyme did not hydrolyze glycosaminoglycan chondroitin sulfate A. One molecular form of hyaluronidase was detected, with a molecular weight estimated at 47.8 +/- 8.6 kDa by sucrose density gradient centrifugation and at 55.0 +/- 1.3 kDa by substrate SDS-PAGE zymography. Activity of the enzyme was optimal between pH 5.0 and 6.0, and was present at neutral pH. Hyaluronidase activity was not affected by 5 mM concentrations of cupric sulfate, zinc chloride, calcium chloride, manganese chloride or EDTA. In addition, NaCl had no effect on enzyme activity at concentrations of 0.2-1.0 M. The highest level of hyaluronidase was present in culture fluid collected between days 4 and 6 of in vitro culture, and this period corresponded with that of the highest rate of increase in the percentage of L4. The presence or absence of hyaluronic acid plays a key role in basic developmental processes of vertebrates and is regulated, in part, by hyaluronidases. Developmental processes occurring during the transition of A. suum L3 to L4 may likewise depend on hyaluronidase. In addition, the infection process of a number of organisms, including some nematodes, depends on hyaluronidase. A. suum may likewise utilize hyaluronidase to facilitate larval migration within the host.

Animals↗

PubMed2001

Cuticular collagen synthesis by Ascaris suum during development from the third to fourth larval stage: identification of a potential chemotherapeutic agent with a novel mechanism of action.

The dominant proteins released by Ascaris suum during development in vitro from the L3 to L4 stage were identified as collagenous cuticular proteins by sequence analysis and susceptibility to digestion by collagenase. Under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the collagen proteins separated into 3 groups with molecular weights estimated at 32 kDa, 54-60 kDa, and 71-91 kDa. The 32-kDa protein represents monomeric collagen; the 54-60- and 71-91-kDa components represent dimeric and trimeric forms, respectively, polymerized by nonreducible cross-links. Furthermore, the release of these forms of collagen was developmentally regulated, as exemplified by a sequential temporal progression from monomeric to dimeric to trimeric forms in association with the in vitro transition from L3 to L4. The data suggest that collagen released in vitro during development of A. suum L3 to L4 reflects the increased translation of collagen gene products and their initial assembly into higher molecular weight molecules associated with the synthesis of the L4 cuticle. A biotinylated dipeptidyl fluoromethylketone cysteine protease inhibitor (Bio-phe-ala-FMK) bound specifically to the 32-kDa collagen and, to a lesser extent, to a 30-kDa protein; binding was dependent on the presence of dithiothreitol (DTT) and was prevented by iodoacetamide. Because cysteine residues play an essential role in the initial assembly of the collagen monomers into the higher molecular weight oligomers present in the mature nematode cuticle, inhibition of molting of A. suum L3 to L4 by the cysteine protease inhibitor Z-phe-ala-FMK might be due to its binding to thiol groups of collagen monomers during a critical phase of collagen assembly. Prevention of cystine cross-links during this critical period of cuticle assembly by peptide-FMK inhibitors may represent a potential control mechanism having a novel mechanism of action.

Amino Acid Sequence↗

PubMed2000

Trichuris suis: A secretory serine protease inhibitor.

A trypsin inhibitor was identified in extracts of adult Trichuris suis and culture fluids from 24-h in vitro cultivation of adult parasites. The inhibitor was isolated by acid precipitation, affinity chromatography (trypsin-agarose), and reverse phase HPLC as a single polypeptide with a molecular weight estimated at 6.6 kDa by laser desorption mass spectrometry. The purified inhibitor associated strongly with trypsin (equilibrium dissociation inhibitory constant (K(j)) of 3.07 nM) and chymotrypsin (K(j) = 24.5 nM) and was termed TsTCI. Elastase, thrombin, and factor Xa were not inhibited. The cDNA-derived amino acid sequence of the mature TsTCI consisted of 61 residues including 8 cysteine residues with a molecular mass of 6.687 kDa. The N-terminal region of TsTCI (46 residues) showed limited homology (36%) to a protease inhibitor from the hemolymph of the honeybee Apis mellifera, which is considered to be a member of the Ascaris inhibitor family. However, TsTCI did not display sequence homology with other members of this family or the distinctive cysteine residue pattern which distinguishes this family. However, similarity of a region of TsTCI (11 residues) with the reactive site regions of inhibitors from the nematodes Ascaris suum, Anisakis simplex, and Ancylostoma caninum was apparent.

Amino Acid Sequence↗

PubMed2000

Trichuris suis: a secretory chymotrypsin/elastase inhibitor with potential as an immunomodulator.

A serine protease inhibitor, termed TsCEI, was purified from adult-stage Trichuris suis by acid precipitation, affinity chromatography (elastase-agarose), and reverse-phase HPLC. The molecular weight of TsCEI was estimated at 6.437 kDa by laser desorption mass spectrometry. TsCEI potently inhibited both chymotrypsin (K(i) = 33.4 pM) and pancreatic elastase (K(i) = 8.32 nM). Neutrophil elastase, chymase (mouse mast cell protease-1, mMCP-1), and cathepsin G were also inhibited by TsCEI, whereas trypsin, thrombin, and factor Xa were not. The cDNA-derived amino acid sequence of the mature TsCEI consisted of 58 residues including 9 cysteine residues with a molecular mass of 6.196 kDa. TsCEI displayed 48% sequence identity to a previously characterized trypsin/chymotrypsin inhibitor of T. suis, TsTCI. TsCEI showed 36% sequence identity to a protease inhibitor from the hemolymph of the honeybee Apis mellifera. Sequence similarity was also detected with the trypsin/thrombin inhibitor of the European frog Bombina bombina, the elastase isoinhibitors of the nematode Anisakis simplex, and the chymotrypsin/elastase and trypsin inhibitors of the nematode Ascaris suum. The inhibitors of T. suis, an intestinal parasite of swine, may function as components of a parasite defense mechanism by modulating intestinal mucosal mast cell-associated, protease-mediated, host immune responses.

Amino Acid Sequence↗

PubMed2000

Efficacy of sperm mobility assessment in commercial flocks and the relationships of sperm mobility and insemination dose with fertility in turkeys.

Our objectives were to evaluate: 1) the efficacy of the Sperm Mobility Test on commercial turkey farms, and 2) the influence of sperm mobility phenotype on fertility when insemination parameters are varied. In research flocks, differences in sperm mobility among toms are predictive of fertility. We wanted to test the efficacy of this sire selection test in practical, real-world situations, evaluating its usefulness in terms of assessing large numbers of toms, different strains of turkeys, and variable management practices. Utilizing field study results, controlled studies were then conducted to improve test parameters. For the field trials, semen from each of 405 breeder toms (11 strains or lines) was evaluated either in duplicate (n = 285) or in triplicate (n = 120). Sperm mobility was normally distributed among all toms tested, except for one strain. Because the sperm mobility indices for toms evaluated in these field trials were higher than those observed in research flocks, the Sperm Mobility Test was modified to increase the separation between high and low sperm mobility phenotypes by increasing the concentration of Accudenz. To determine the effects of sperm mobility and insemination dose on sustained fertility through time, hens from a research flock were inseminated twice before the onset of lay with sperm from toms classified as high-, average-, or low-mobility in concentrations of 25 to 400 million sperm per artificial insemination dose, and egg fertility was evaluated over a 5-wk period. Toms with the high-mobility sperm phenotype maintained higher fertility (P < 0.05) over the 5-wk period at all insemination doses compared with toms with low-mobility sperm. Toms with high-mobility sperm sired equal numbers of poults in a sperm competition study in which numbers favored low-mobility toms by 3:1. These results demonstrate that the Sperm Mobility Test can be used for on-farm evaluation of semen quality of toms in commercial flocks and that sperm mobility influences fertility and sire fitness.

Animals↗

PubMed2000

Characterization of acid phosphatase and phosphorylcholine hydrolase in adult Haemonchus contortus.

An acid phosphatase (AP) and a phosphorylcholine hydrolase (PCH) were detected in excretory-secretory (ESP) products from adult Haemonchus contortus. The AP had a pH optimum of 4.5 and was inhibited by tartaric acid and sodium fluoride, but not by o-phenanthroline. The AP hydrolyzed paranitrophenol (pnp)-phosphate and to a lesser extent pnp-phenyl-phosphonate but did not hydrolyze diester substrates. Purified AP consisted of heterodimers with relative molecular weight (Mr) of 41.9 and 48.7 kDa and had a native molecular weight of 98 kDa by size-exclusion chromatography (SEC). The PCH had a pH optimum of about 9.5 and was inhibited by EDTA and o-phenanthroline but not by the specific phospholipase inhibitor D609. The specific activity of PCH in the ESP was approximately 25-fold less than that of AP. PCH also hydrolyzed 5'-thymidine monophosphate-pnp at a rate about 40% lower than pnp-phosphorylcholine but did not hydrolyze 3'-thymidine monophosphate-pnp. Partial purification of PCH suggests an Mr of 50.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Mr of 102 kDa by SEC. Both AP and PHC were secreted in vitro in a time-dependent manner and had their highest concentrations in the intestine. The results indicate that H. contortus adults secrete significant amounts of AP that might be a digestive enzyme. PCH is also an intestinal enzyme and is secreted in lesser amounts than AP. The PCH is probably not a phospholipase C but has some characteristics of a type I phosphodiesterase.

Acid Phosphatase↗

PubMed2000

A developmentally regulated hyaluronidase of Haemonchus contortus.

The trichostrongylid nematode Haemonchus contortus released a hyaluronic acid-degrading enzyme during in vitro development from the third (L3) to fourth (L4) larval stage. The enzyme did not degrade chondroitin sulfate A. Enzyme activity was optimal between pH 4.0 and 6.0, and the enzyme was inhibited by high concentrations of NaCl; the divalent cations Cu2+, Zn2+, Ca2+, and Mn2+ were not inhibitory. The hyaluronidase had a molecular mass estimated at 57 kDa by sucrose density gradient centrifugation and at 111 kDa by substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (reducing and nonreducing conditions), suggesting the formation of a dimer during the electrophoretic separation conditions. The level of hyaluronidase released during in vitro development peaked between 24 and 48 hr in culture and then gradually decreased, with little or no activity present in the 168-hr culture fluid. The enzyme was not detected in culture fluid from 24-hr incubations of either the mid-L4 stage (obtained from sheep 7 days postinfection) or the adult stage (obtained from sheep 30-35 days postinfection). The temporal expression of the hyaluronidase suggested a role for this enzyme in the early stages of the L3-L4 developmental process.

Animals↗

PubMed1999

Neandertal knees: power lifters in the Pleistocene?

It has been proposed (Trinkaus, 1983 a; Miller & Gross, 1998) that the marked thickness of Neandertal patellae and/or the posterior displacement of their tibial condyles increased their relative M. quadriceps femoris moment arms, thereby making their legs powerful in extension. However, it is necessary to compare these reflections of muscle moment arm length to appropriate measures of the body weight moment arm and body mass estimates, both of which are influenced by ecogeographically determined body proportions. Reassessment of tibial condylar displacement and patellar thickness, as well as patellar height, relative to an appropriate measure of the moment arm for the baseline load on the knee (body weight), to that moment arm times estimated body mass, and to that moment arm times a skeletal reflection of body mass (femoral head diameter) rejects the hypothesis that the Neandertals had exceptionally powerful knee extension. Relative tibial condylar displacement remains above that of a modern industrial society sample, but similar to that of the Broken Hill tibia, Late Pleistocene early modern humans and a recent human nonindustrial sample. Relative patellar thickness is similar to that of early modern humans, who have relatively thick patellae compared to the late Holocene human samples. Consequently, once body proportions are taken into account, there is little difference between the Neandertals and other later Pleistocene humans in knee extensor mechanical advantage, and all of these fossil hominids are similar in the more important proximal tibial proportions to those of nonindustrial recent humans.

Animals↗

PubMed1999

Effects of transient treatment with 6-N-propyl-2-thiouracil on testis development and function in breeder turkeys.

Experiments were conducted to address recent reports of precocial testis development or testicular hypertrophy induced by the anti-thyroid agent 6-N-propyl-2-thiouracil (PTU) in domestic fowl and rodents. In three experiments, PTU was administered to male turkeys during different stages of development. The PTU was given in the feed at a concentration of either 0.1 or 0.5% and effects on thyroid hormones were measured. Periods of PTU treatment were 8 to 16 or 10 to 18 wk of age (Experiment 1); 0 to 8, 0 to 16, and 8 to 16 wk of age (Experiment 2); and 8 to 16 and 20 to 35 wk of age (Experiment 3). Data were collected to determine the effect of PTU treatment on testicular growth (weight), sexual maturation (semen onset and plasma testosterone concentrations), and early adult testicular function in terms of semen quantity (semen volume and sperm concentration) and quality (sperm viability and fertilizing ability). The 0.1% PTU treatment at 8 to 16 wk of age in Experiment 1 resulted in greater testis weights and sperm production than those of untreated controls at 24 wk of age. The difference was absent by 36 wk of age. Likewise, in Experiment 2, onset of semen production was advanced by about 2 wk by the 8 to 16 wk treatment as compared to untreated controls (23.3 vs 25.9 wk), although, at 32 and 36 wk of age neither the quantity nor quality of semen were significantly affected. Also in Experiment 2, 0.1% PTU treatment from 0 to 8 or 0 to 16 wk of age delayed the onset of semen production and depressed semen volumes at 32 and 36 wk of age without affecting semen quality. In Experiment 3, the mean age at the onset of semen was not significantly affected by 0.5% PTU treatment from 8 to 16 or 20 to 35 wk of age. In addition, semen volume and quality at 32 and 36 wk of age was similar to controls. It was concluded that antithyroid treatment with PTU was dependent on dose and time of treatment for effects on both sexual development and early reproductive performance. Precocial development and associated hypertrophy of the testes occurred but was transient. Thereafter, during initial reproductive function, semen quantity and quality were normal.

Aging↗

PubMed1999

Sperm mobility: A primary determinant of fertility in the domestic fowl (Gallus domesticus).

Previous research demonstrated that sperm mobility is a quantitative trait of the domestic fowl. The trait is quantified by measuring the absorbance of an Accudenz solution after overlay with a sperm suspension and brief incubation at body temperature. In the present work, average and high sperm mobility phenotypes (n = 30 males per phenotype) were selected from a base population. Differences were found between sperm oxygen consumption (p < 0.0001), acylcarnitine content (p < 0.05), linear velocity (p < 0.001), and straightness (p < 0.001), a trajectory variable measured with the Hobson SpermTracker. Oxygen consumption and stearoylcarnitine content of sperm from the high-mobility phenotype were twice those observed with sperm from average males, implying a pivotal role for mitochondria. On the basis of these results, a graded relationship was predicted between fertility and sperm mobility. Males (n = 48) were chosen at random from another base population, sperm mobility was measured per male, and each ejaculate was used to inseminate 8-12 hens (8 x 10(7) viable sperm per hen). When fertility was plotted as a function of sperm mobility, data points approximated a skewed logistic function. The hypothesis that vaginal immunoglobulins constitute an immunological barrier to sperm transport was tested and rejected. Therefore, we concluded that sperm mobility is a primary determinant of fertility in the fowl.

Animals↗

PubMed1999

Characterization of a hemoglobin-like protein from adult Haemonchus contortus.

A hemoglobin-like protein was purified from supernatants of adult Haemonchus contortus extracts by high-pressure liquid chromatography. The purified protein had an M(r) of 33 kDa as determined by size-exclusion chromatography under non-denaturing conditions and an M(r) of 19 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the hemoglobin may exist as a dimer. The sequences of 3 peptides resulting from proteolytic digest of the purified protein were determined and demonstrated greater than 50% identity to the globin from Trichostrongylus colubriformis. Adult H. contortus incubated overnight in [3H]leucine, incorporated radioactivity into a peak that coeluted with parasite hemoglobin, indicating the adults synthesize hemoglobin in vitro. The L3-stage lacked hemoglobin, but the L4-stage contained a hemoglobin with an M(r) of 19.6 kDa.

Amino Acid Sequence↗

PubMed1998

A hemolytic factor from Haemonchus contortus alters erythrocyte morphology.

A hemolytic factor from adult Haemonchus contortus caused distinct morphological changes in the surface of sheep red blood cells (RBCs). After a 15 min exposure to the hemolytic factor, hemolysis was not detected in incubation media, but RBCs were spherical in shape with numerous surface projections compared to control cells that were smooth-surfaced biconcave disks. After 30 min, a time at which significant hemolysis occurred, echinocytes were formed, and after 90 min, cells were severely disrupted with many visible holes in membranes. No RBC ghosts were observed. RBCs from four other mammalian species were lysed by the H. contortus hemolytic factor. However, the rate of hemolysis varied with a relative order of sheep approximately rabbit>goat>pig>calf. The morphology of RBCs from all four species was significantly altered after 30 min incubation with the degree of morphological changes related to the degree of hemolysis. These results support the hypothesis that the hemolytic factor acts as a pore-forming agent, although a phospholipase or other enzyme might play a role in solubilization of cell membranes.

Animals↗

PubMed1998

Purification and characterisation of a secreted aminopeptidase from adult Ascaris suum.

A metalloaminopeptidase was identified in culture fluids collected during in vitro cultivation of adult Ascaris suum. The enzyme was purified by anion-exchange and size-exclusion HPLC. The M(r) of the enzyme was estimated at 293 kDa and consisted of subunits with M(r)s of 153 and 142kDa. The isoelectric point of the aminopeptidase was 4.7. The aminopeptidase displayed a substrate preference for terminal arginyl residues. Aminopeptidase activity was also present in muscle, female reproductive tissue, pharynx, pseudocoelomic fluid and intestine. Among the various tissues, aminopeptidase activity was highest in the intestines; the highest activity was found in culture fluids (three-fold higher than intestinal tissue). The aminopeptidase released by adult A. suum was enzymatically and biochemically identical to an aminopeptidase released during in vitro development of A. suum third- to fourth-stage larvae.

Aminopeptidases↗

PubMed1998

Effect of protease class-specific inhibitors on in vitro development of the third- to fourth-stage larvae of Ascaris suum.

Third-stage larvae (L3) of Ascaris suum develop and molt to fourth-stage larvae (L4) during in vitro cultivation; consistently greater than 80% of the larvae develop to L4 during 7 days in culture (DIC). To assess the role of proteases in this process, the effect of protease class-specific inhibitors was studied. The presence of either a serine protease inhibitor (AEBSF, 100 microM) or an aspartic protease inhibitor (pepstatin A, 100 microM) had no effect on the percentage of L4 after 7 DIC. However, the presence of either a cysteine protease inhibitor (Z-Phe-Ala-FMK, 100 microM) or an aminopeptidase inhibitor (amastatin, 100 microM) resulted in 77% and 34% reductions, respectively, in the percentage of L4 compared to untreated cultures; viability of the larvae was not affected. The effect of Z-Phe-Ala-FMK on molting was time and dose dependent. In contrast to Z-Phe-Ala-FMK, E-64, another specific inhibitor of cysteine proteases, had no effect on molting. The data support a role for an aminopeptidase and suggest a role for a cysteine protease in the development of the L3 to L4 stage of A. suum.

Animals↗

PubMed1997

The in vitro uptake of albumin by adult Haemonchus contortus is altered by extracorporeal digestion.

FITC (fluorescein isothiocyanate)-labeled albumin was used as a substrate to further characterize the digestion of host blood proteins by adult Haemonchus contortus. Isolated H. contortus intestine degraded FITC-albumin; degradation was completely inhibited by E-64, a specific inhibitor of cysteine proteases. The in vitro uptake and degradation of FITC-albumin by parasites was also evaluated. Uptake of fluorescence was demonstrated; greater than 50% of the fluorescence was associated with degraded FITC-albumin. However, both the uptake and degradation of FITC-albumin were reduced by about 85%. Additionally, when parasites were incubated in the presence of specific inhibitors of cysteine proteases, degradation of FITC-albumin was shown to occur rapidly in the media during in vitro incubation. This degradation was blocked completely by the presence of cysteine protease inhibitors. These results indicate that the rapid extracorporeal digestion of FITC-albumin (mediated by secreted cysteine proteases) produce relatively high concentrations of low molecular weight FITC-labeled fragments and alters the kinetics of uptake of fluorescence by the parasite. These fragments rather than FITC-albumin are rapidly taken up by the parasites. Thus in vitro demonstration of a definitive role for the cysteine proteases as intestinal digestive enzymes in H. contortus is compromised by the enzyme's extracorporeal activity. This extracorporeal cysteine protease activity may represent the enzyme's predominant activity.

Abomasum↗

PubMed1997

Characterization of haemolytic activity from adult Haemonchus contortus.

Adult Haemonchus contortus contain a detergent-soluble factor that haemolyses sheep red blood cells in a time- and concentration-dependent manner. This factor had comparable haemolytic activity at pH 5.0 and 8.0; activity was lower at pH 6.0 and 7.0. The activity was heat-stable, unaffected by proteolytic inhibitors, and inhibited by 20 mM polyethyleneglycol. Haemolytic activity was associated with the particulate fraction of the isolated intestine, suggesting an essential role for this activity in the acquisition of nutrients by disrupting host red blood cells. The data are consistent with the hypothesis that the mechanism of action of the haemolytic factor is as a pore-forming agent.

Abomasum↗