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Biomedical subjects

E L Jarroll

Publications and source records attributed to E L Jarroll.

At least 19 recordsLinked to original sources
PubMed1993

The effects of oxygen on fermentation in Giardia lamblia.

Detailed study of the effects of oxygen on the carbohydrate metabolism of Giardia lamblia revealed that low concentrations of oxygen (< 0.25 microM) produced profound alterations in the carbon balance of this organism. Although this concentration of oxygen could not be detected by mass spectrometry, a marked stimulation of ethanol production was observed. Associated with this was an inhibition of alanine production and oxidation of the intracellular NAD(P)H pool. Higher concentrations of oxygen inhibited ethanol production and further reduced levels of alanine. These results suggest that this stimulation is due to changes in carbon flux. Analysis of cell and medium hydrolysates after the growth of trophozoites in [U-14C]glucose suggests that G. lamblia does not synthesise detectable levels of labelled amino acids, except alanine and to a lesser extent valine, from this sugar. Trophozoites of G. lamblia have both glutamate dehydrogenase and alanine aminotransferase activity. As glutamate is taken up from the medium, it is suggested that glutamate dehydrogenase and alanine aminotransferase cooperate to convert pyruvate to alanine, with the concomitant oxidation of NAD(P)H.

Alanine↗
PubMed1992

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.

The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.

Animals↗
PubMed1992

Galactosamine-synthesizing enzymes are induced when Giardia encyst.

Galactosamine, a Giardia filamentous cyst wall specific-sugar, is below the limits of detection in non-encysting trophozoites. Radiolabeling studies suggest that Giardia synthesize galactosamine primarily from endogenous glucose rather than salvage it from the environment. Enzymes responsible for galactosamine synthesis from glucose are induced during encystment and have been characterized in crude homogenates and in supernatant (soluble) fractions. These enzymes (specific activity; time after encystment is induced for maximal activity; x-fold increase) include glucosamine 6-phosphate isomerase (in the deaminating direction, 167 mU mg protein-1; 20 h; x 182-fold; in the aminating direction, 258 mU mg protein-1; 20 h; x 13-fold), glucosamine 6-phosphate N-acetylase (11 mU mg protein-1; 20 h; x 20-fold), phosphoacetylglucosamine mutase (160 mU mg protein-1; 20 h; x 12-fold), UDP-N-acetylglucosamine pyrophosphorylase (22 mU mg protein-1; 48 h; x 8-fold), and UDP-N-acetylglucosamine 4'-epimerase (13 mU mg protein-1; 48 h; x 4000-fold). This represents the first report of these enzymes and of an inducible carbohydrate-synthesizing pathway in any protozoan.

Animals↗
PubMed1991

Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris.

In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.

Amino Acid Sequence↗
PubMed1990

Giardia sp.: comparison of electrophoretic karyotypes.

Species in the genus Giardia have been named on the basis of host specificity, cell dimensions, and median body morphology. Despite these criteria, the species taxonomy of Giardia is still in question. To investigate Giardia taxonomy on a molecular level, Giardia chromosomal DNA was analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE) and transverse alternating field electrophoresis (TAFE). Chromosomal DNA of G. duodenalis isolates (human, muskrat, sheep, dog, beaver), G. muris (mouse), and G. ardeae (great blue heron) were subjected to OFAGE and TAFE analyses. Comparable DNA patterns were obtained by both electrophoretic methods, but OFAGE required 8 days while TAFE required only 3 days. DNA patterns among all G. duodenalis isolates, although quite similar to each other, were distinctly different from those of G. muris and G. ardeae; G. muris and G. ardeae DNA patterns were distinctly different from each other. A G. duodenalis (Portland 1) total DNA probe hybridized to the DNA of all G. duodenalis isolates on Southern blots, but not detectably to G. muris and G. ardeae DNA. Similarly, G. muris and G. ardeae total DNA probes only hybridized detectably to their respective DNA. One probe that appears to hybridize to the DNA of all G. duodenalis and to G. ardeae DNA rather than G. muris DNA has been developed. Another probe that hybridizes only to G. muris and G. ardeae DNA has been developed. These data suggest that the differentiation of Giardia isolated from host and environmental samples may eventually be accomplished by DNA probes. Additionally, these techniques perhaps combined with other criteria may lead to the establishment of a sound taxonomic scheme for this genus.

Animals↗
PubMed1990

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging.

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.

Animals↗
PubMed1990

Axenic culture and characterization of Giardia ardeae from the great blue heron (Ardea herodias).

Trophozoites of Giardia ardeae were obtained from the great blue heron (Ardea herodias) and established in axenic culture using the TYI-S-33 medium. The generation time in culture for G. ardeae was 22-25 hr, which was 3-fold longer than for Giardia duodenalis (WB strain). A morphological comparison of trophozoites in the original intestinal isolate to those grown in culture revealed that they were identical for the following characteristics: a pyriform-shaped body, a ventral adhesive disc with a deep notch in the posterior border, teardrop-shaped nuclei, pleomorphism in median body structure ranging from a round-oval appearance (Giardia muris type) to that of a clawhammer (G. duodenalis type), and a single caudal flagellum on the right side (as viewed dorsally) with the left one being rudimentary. Analysis of the chromosomal migration patterns was performed by orthogonal-field-alternation gel electrophoresis and demonstrated that the pattern for G. ardeae was distinctly different from that for G. duodenalis (Portland 1-CCW strain). Bacterial symbionts were seen attached to trophozoites in the original isolate but could not be detected in cultured trophozoites using scanning electron microscopy, fluorescence light microscopy using the Hoechst 33258 dye for DNA localization, or by standard microbiological techniques using nonselective media for growing aerobic or anaerobic bacteria. This study demonstrated that avian-derived Giardia could be grown in axenic culture; based on morphological criteria and chromosomal migration patterns, that G. ardeae should be considered a distinct species; and that rationale for determining Giardia spp., based on median body structure alone, should no longer be considered adequate for classification at the species level.

Animals↗
PubMed1989

Giardia cyst wall-specific carbohydrate: evidence for the presence of galactosamine.

Gas chromatographic (GC), mass spectrometric (MS), lectin binding and enzymatic analyses of the carbohydrates from Giardia cyst walls, intact cysts and trophozoites were performed to investigate the carbohydrate composition of Giardia cyst walls and to test the hypothesis that the Giardia cyst wall is composed largely of chitin. Galactosamine, verified by MS, was present in Giardia cyst walls and intact cysts (ca. 47 nmol 10(-6) cysts). Since not even trace amounts of it were detected in trophozoites by either GC or lectin binding, galactosamine is hypothesized to be a cyst wall-specific amino hexose. Based on the putative binding affinity of Phaseolus limensis lectin, galactosamine may be present in cyst walls as N-acetylgalactosamine. Neither glucosamine nor sialic acid were detected in as much as 11 mg dry weight of cysts, cyst walls, or trophozoites. Glucose, the most abundant carbohydrate, and ribose were detected in Giardia cysts and trophozoites. Galactose (ca. 10 nmol 10(-6) cysts) was detected in cysts but not in trophozoites. The lack of detectable levels of (1) glucosamine in cyst wall hydrolysates, (2) cyst staining by Calcofluor M2R, (3) endogenous chitinase activity and (4) N-acetylglucosamine when cysts served as a substrate for exogenous chitinase suggests that the Giardia cyst wall is not composed largely of chitin as previously reported. beta-N-Acetylgalactosaminidase, EC 3.2.1.32, activity was detected in cysts and trophozoites and represents the first carbohydrate splitting hydrolase detected in Giardia.

Animals↗
PubMed1989

Giardia lamblia: characterization of proteinase activity in trophozoites.

The proteinase activity of Giardia lamblia trophozoites, Portland 1 strain, was characterized with respect to substrate specificities and inhibitor sensitivities. Proteinase activity with urea-denatured hemoglobin (UDH), alpha-N-benzoyl-DL-arginine-2-naphthylamide (BANA), and alpha-N-benzoyl-argininamide (BAA) as substrates exhibited pH optima of 5.8, 3.8, and 5.0, respectively. For BANA, the apparent Km was 0.20 mM and the Vmax was 2.56 microM. For BAA, the apparent Km was 4.0 mM and the Vmax was 8.69 microM. Dithiothreitol (DTT, 5 mM) enhanced proteinase activity threefold for UDH, fourfold for BAA, and fivefold for BANA. Iodoacetamide, L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), each at 1 mM, inhibited proteinase activity by greater than 90% with BANA and BAA. Iodoacetamide inhibited proteinase activity by 35% with UDH; TPCK and TLCK inhibited activity greater than 70% with UDH. Activity on BAA was inhibited by 91% with Zn2+ and activity on UDH was inhibited by 30% with Cu2+. Virtually complete inhibition of proteinase activity on BANA and BAA was obtained with leupeptin and chymostatin at 1 microgram/ml. Pepstatin A, chelators, and other heavy metals had no apparent effect on proteinase activity. Two polypeptide bands (ca. 105 and 40 kDa) indicative of proteinase activity were visualized by sodium dodecyl sulfate-gelatin polyacrylamide gel electrophoresis. The 105 kDa band was visible over the pH range of 4 to 7, but with greater intensity from pH 5 to 7. The 40 kDa band, while present at pH 5, was most intense at pH 6 and 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Respiration in the cysts and trophozoites of Giardia muris.

Cysts and trophozoites of the parasitic protozoon Giardia muris both showed respiratory activity but respiration in cysts was only 10 to 20% that of trophozoites. The O2 dependence of respiration in cysts and trophozoites showed O2 maxima above which respiration decreased. The O2 concentration at which the respiration rate was greatest was higher for cysts than trophozoites. The effects of various inhibitors on cyst and trophozoite respiration suggested that flavoproteins and quinones play some role in respiration. The substrate specificities and the effects of inhibitors on G. muris trophozoites were similar to those observed for Giardia lamblia. Metronidazole, the drug most commonly used in the treatment of giardiasis completely inhibited respiration and motility in trophozoites; however, it had no effect on either respiration or viability in cysts. Menadione, a redox cycling naphthoquinone, stimulated then completely inhibited respiration in cysts and trophozoites; a complete loss of cyst viability or trophozoite motility was also observed. The effects of menadione on G. muris may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.

Animals↗
PubMed1989

Inactivation of gerbil-cultured Giardia lamblia cysts by free chlorine.

Giardia lamblia cysts were harvested from Mongolian gerbils and exposed to free chlorine in buffered water at pH 5, 7, and 9 at 15 degrees C. The contact times required to obtain a 2-log reduction in cyst survival (i.e., a 99% kill) were interpolated from survival curves generated at fixed concentrations of chlorine in the range of 0.25 to about 16 mg/liter. Concentration-time (C.t') products for 99% inactivation ranged from about 120 to nearly 1,500 mg.min/liter. These values are higher than those reported previously for free chlorine using G. lamblia cysts from infected humans. The cysts isolated from gerbils, as with other Giardia cysts, were unusually sensitive to chlorine in alkaline solutions.

Animals↗
PubMed1987

Giardia lamblia: uptake of pyrimidine nucleosides.

The aerotolerant, anaerobic parasite Giardia lamblia, which depends solely upon salvage pathways for its pyrimidine requirements, was found to transport uridine, cytidine, and thymidine by a carrier mediated mechanism. Support for this conclusion comes from the facts that uptake of radiolabeled uridine, cytidine, and thymidine exhibited saturation kinetics, and uptake of these same radiolabeled nucleosides was inhibited by unlabeled homologs, certain pyrimidine analogs, iodoacetate, and N-ethylmaleimide. Uridine and cytidine (perhaps uracil and cytosine also) are postulated to be transported at a common site which is distinct from the site for thymidine transport. Thymidine does appear to bind nonproductively to the uridine/cytidine transport site, but the reverse of this does not appear to occur.

Animals↗
PubMed1984

Giardia lamblia: autoradiographic analysis of nuclear replication.

Giardia lamblia trophozoites, grown in axenic culture, were labeled for various periods of time with [3H]thymidine. After autoradiography, grains were counted over each of the two nuclei in each trophozoite. Analysis of the fraction of trophozoites labeled for each time period resulted in an estimate of a generation time of 15 hr. The DNA synthetic or S phase for a trophozoite in culture was calculated to be 1.8 hr. G1 and G2 periods were determined to be 8.5 and 3 hr, respectively. A comparison of the labeling density between the two nuclei indicated that replication takes place simultaneously in both nuclei for at least 70% of S period. The fraction of asymmetrically labeled trophozoites is consistent with a model in which the nuclei replicate out of phase by 15-30 min, but, due to the small diameter of the nuclei relative to the grain size, the possibility that replication takes place simultaneously in both nuclei of a trophozoite throughout the S phase cannot be ruled out.

Animals↗
PubMed1983

Pyrimidine metabolism in Tritrichomonas foetus.

The pyrimidine metabolism of Tritrichomonas foetus (KV 1) was studied using whole cells and cell homogenates. Pyrimidines and pyrimidine nucleosides were readily incorporated into nucleic acids. Orotate and aspartate were not incorporated into pyrimidine bases. Enzymes of the pyrimidine salvage pathway (i.e., thymidine and uridine phosphorylases and uridine kinase) were detected in trophozoite homogenates, but the activities of de novo pyrimidine synthesis enzymes (i.e., carbamoylphosphate synthase, aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase) were below the level of detection in these same homogenates. The evidence presented supports the proposal that T. foetus is incapable of synthesizing pyrimidines de novo but is capable of salvaging preformed pyrimidines and pyrimidine nucleosides from the growth medium and that enzymes of this parasite's pyrimidine salvage pathway are not organelle-associated.

Animals↗
PubMed1982

Pyrimidine metabolism in Giardia lamblia trophozoites.

The pyrimidine metabolism of Giardia lamblia trophozoites (Portland I strain) was studied using whole trophozoites and trophozoite homogenates. Pyrimidines and pyrimidine nucleosides were readily incorporated into nucleic acids. Orotic and aspartic acid incorporations were below the level of detection. Enzymes of the pyrimidine salvage pathway (i.e., thymidine and uridine phosphorylases and thymidine and uridine kinases) were detected in trophozoite homogenates, but the activities of de novo pyrimidine synthesis enzymes (i.e., carbamoyl-phosphate synthase, aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase) were below the level of detection in these same homogenates. The evidence presented supports the conclusion that G. lamblia trophozoites appear incapable of synthesizing pyrimidines de novo but are capable of salvaging preformed pyrimidines and pyrimidine nucleosides from the growth medium and the enzymes of this pyrimidine salvage pathway are not organelle associated.

Animals↗
PubMed1982

Energy metabolism of the contagious equine metritis bacterium.

The energy metabolism of the English E-CMO strain of contagious equine metritis bacterium was studied in whole cells and cell extracts. This bacterium appears to have an active Krebs cycle and probably obtains energy by oxidative phosphorylation since glycolysis and the hexose monophosphate pathways appear to be absent. These conclusions are based on the findings that [U-14C]glucose incorporation by this bacterium is below the level of detection, and that respiration is stimulated by Krebs cycle intermediates (i.e., malate, citrate, and succinate), but not by glucose, fructose, maltose, or sucrose. Furthermore, support comes from the fact that enzymes generally associated with the Krebs cycle and electron transport (i.e., malate dehydrogenase, succinate dehydrogenase, isocitrate dehydrogenase, fumarate hydratase, malate dehydrogenase [decarboxylating], cytochrome oxidase, superoxide dismutase, NADH dehydrogenase, and catalase) were detected. Those enzymes normally associated with glycolysis and the hexose monophosphate pathways (i.e., hexokinase, glucose 6-phosphate dehydrogenase, fructose biphosphate aldolase, glycerol 3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, pyruvate kinase, phosphate acetyl transferase, acetate kinase, alcohol dehydrogenase, and lactate dehydrogenase) were below the level of detection.

Animals↗
PubMed1981

Lipid and carbohydrate metabolism of Giardia lamblia.

The lipid and carbohydrate metabolism of Giardia lamblia was studied using trophozoites isolated from a human and axenically grown in vitro in medium containing fetal bovine serum. The phospholipid, fatty acid and neutral lipid composition of the G. lamblia trophozoites was similar to that of the medium. Phosphatidylethanolamine, phosphatidylcholine and sphingomyelin were the major phospholipids detected; monoacyl-, diacyl-, triacylglycerides, sterols, and sterol esters were the major neutral lipids found. Several unidentified glycolipids were also detected. Glucose and threonine were readily incorporated by the trophozoites, but not into cellular phospholipids or sterols. However, approximately 86% of the glucose incorporated into the trophozoites was found in the nucleic acids, and 38% of the threonine incorporated was detected in the cellular proteins. Small amounts of the glucose and threonine were incorporated into glycolipid-containing fractions. Glycerol and acetate were not appreciably incorporated into trophozoites while glycerol 3-phosphate incorporation was not detected. Cholesterol was readily assimilated by the trophozoites; 98% of the incorporated was found in the sterol fraction. Radiorespirometric data suggest that the major routes of glucose metabolism in G. lamblia are via Embden- Meyerhof-Parnas and pentose phosphate pathways. However, endogenous acetate (as acetyl-CoA) formed during the metabolism of glucose is not used for lipid biosynthesis. These findings suggest that G. lamblia trophozoites are incapable of synthesizing cellular phospholipids or sterols de novo, but rather, utilize lipids already present in the medium.

Animals↗