Ascaris lumbricoides: Coproporphyrinogen oxidase activity in eggs and muscle

The possible presence of coproporphyrinogen oxidase (EC 1.3.3.3), an oxygen-requiring enzyme in the porphyrin biosynthetic pathway, was investigated in supernatant fractions of homogenized Ascaris lumbricoides muscle and developing eggs, and in mitochondrial preparations of muscle. Compared with rat liver controls, low levels of enzyme activity were found in A. lumbricoides gut, 6-day eggs, and muscle mitochondria. Enzyme activity in muscle, 8-day, and 25-day eggs was not measurable under the conditions employed.     

The occurrence of the second moult of Ascaris lumbricoides and Ascaris suum

Maung M. 1978. The occurrence of the second moult of Ascaris lumbricoides and Ascaris suum. International Journal for Parasitology 8: 371–378. Eggs of Ascaris lumbricoides and A. suum were cultured at 28°C and observed daily. Larvae were released by pressure, by artificial hatching with CO₂, and by natural hatching after infection of laboratory mice. The early stages of development in the egg were observed to comprise two moults, one occurring immediately after the other. Both moults were initiated within the egg, but the time of completion of the second moult varied considerably, and in some instances was not completed until the larvae reached the liver of experimentally infected animals.     

Identification and quantification of some elements in the hog roundworm, Ascaris lumbricoides suum, and certain tissues of its host

Atomic absorption spectrophotometric and fluorometric analyses were utilized for the determination of several elements in the whole bodies of both male and female Ascaris lumbricoides suum and from the muscle and kidney of the swine host. Concentrations of cadmium, calcium, copper, lead, magnesium, manganese, iron, selenium, potassium, and zinc in these tissues are reported. Statistical analysis (Tukey's procedure) of the data indicated no differences in metal concentrations between male and female ascarids. There were three instances in which the metal concentrations were statistically different in worm tissues and both hog tissues.     

Biosynthesis of Biopterin by Rat Brain

Abstract: A method for the determination of [¹⁴C]biopterin biosynthesis from [¹⁴C]guanosine-5'-triphosphate by a desalted preparation from rat striatum, based on sequential reverse-phase and cation-exchange high performance liquid chromatography, is described. Synthesis of reduced forms of biopterin by this striatal extract was found to be dependent on enzymatic activity, guanosine-5'-triphosphate, magnesium ions, and a reduced pyridine nucleotide. As demonstrated by the technique of isotope dilution, isotope trapping, 6-lactyl-7,8-dihydropterin (sepiapterin) was found to be an intermediate in biopterin biosynthesis that is catalyzed by the striatal extract. Rat brain was also shown to synthesize biopterin in vivo from intraventricularly administered [¹⁴C]guanosine or sepiapterin. Intraventricular injection of sepiapterin increased dihydro- and 5,6,7,8-tetrahydrobiopterin levels in rat brain by more than eightfold. The temporal relationship between the appearance of dihydro- and 5,6,7,8-tetrahydrobiopterin following intraventricular injection of sepiapterin suggests that dihydrobiopterin is the immediate product of sepiapterin reduction which is then reduced further to the functional cofactor 5,6,7,8-tetra-hydrobiopterin. Therefore, in contrast to previous reports, the biosynthesis of biopterin by rat brain does not appear to differ from that occurring in other, nonneural tissues.     

Fine binding specificities to Ascaris suum and Ascaris lumbricoides antigens of the sera from patients of probable visceral larva migrans due to Ascaris suum

Fine binding specificities to Ascaris suum and A. lumbricoides antigens of the sera from patients with probable visceral larva migrans (VLM) due to A. suum infection were examined. Although multiple-dot enzyme-linked immunosorbent assay (ELISA) was found to be useful for the primary screening of patients, identification of the responsible species was sometimes difficult due to extensive cross reactions with other ascarid parasite antigens. Fine resolution to determine the causative pathogen was obtained by a rather classical Ouchterlony's double immunodiffusion test. The difference in the binding of the patients' sera to A. suum and A. lumbricoides antigens was also demonstrated by an inhibition ELISA. The patients' antibodies bound with higher avidity to the A. suum antigen than to the A. lumbricoides and Toxocara canis antigens. Combination of at least two different immunological assay methods is recommended for the diagnosis of VLM due to ascarid parasites.     

Ascaris suum and Toxocara canis: Egg extract antigens in guinea pigs and the macrophage migration inhibition test

Guinea pigs immunized with 500 μg of either Ascaris suum or Toxocara canis egg extracts, emulsified with Freund's complete adjuvant, were skin tested with both the homologous and heterologous antigens. Cross-reactions were observed in both groups. Migration of macrophages from sensitized animals was more inhibited by homologous than by heterologous antigens. Lymph-node lymphocytes from sensitized animals were stimulated to incorporate [⁸H]thymidine similarly by both antigens.     

Expression of biopterin transporter (BT1) protein in Leishmania

The present work focuses on the growth phase regulated expression of biopterin transporter gene (BT1) from the LD1 locus on chromosome 35 of Leishmania donovani. Antiserum against recombinant BT1 detected a polypeptide of 45 kDa of equal intensity at lag, log and stationary phases of promastigote growth, both in L. donovani strain LSB-7.1 (MHOM/BL/67/ITMAP263), and strain LSB-146.1 (HOM/IR/95/X81), a natural isolate from Isfehan, Iran that caused cutaneous leishmaniasis. However, in both these strains an additional polypeptide of higher molecular mass (50 kDa) was also observed during lag phase only. In addition, polypeptides of 40, 20, 18 and 16 kDa were seen only during the lag and log phases of both strains. Analysis of L. donovani single, double and triple (null) BT1 knockout mutants confirmed that the 45-kDa polypeptide was the BT1 gene product, as it was absent in the null mutant. These results indicate that 45-kDa BT1 protein in Leishmania is consistently and constitutively expressed in all the growth stages of the parasite.     

Ascaris lumbricoides and Ascaridia galli: Biogenic amines in adults and developmental stages

Ascaris lumbricoides var. hominis and Ascaridia galli contain 5-hydroxytryptamine, histamine, dopamine, and norepinephrine. The chick parasite showed lower levels of monoamines compared to human ascaris. Amine concentrations in females were higher than in males. In all specimens, 5-hydroxytryptamine was the highest while norepinephrine was found to be uniformly low. The female reproductive organ contained the maximum amount of dopamine while intestine was rich in histamine. A progressive increase in the concentrations of biogenic amines was noticed during development.     

Development of larvae of Ascaris suum from the third to the fourth stage in a chemically defined medium

Third stage larvae of Ascaris suum, recovered from the lungs of rabbits 7 days after oral infection with eggs, were cultured to the fourth larval stage in chemically defined, low molecular weight medium. The medium consisted of tissue culture medium 199, supplemented with glucose and glycyl-hystidyl-lysine and gassed with a mixture of N₂-CO₂-O₂ (90:5:5). Growth and development in this medium were similar to that in media supplemented with whole serum or with a serum dialysate.     

An in vitro Cr release assay for Ascaris suum infective larvae

This report describes the use of ⁵¹Cr release as a measure of the viability of Ascaris suum inefective larvae after treatment with sodium hypochlorite or kalafungin. Inefective larvae of A. suum were treated with varying dilutions of these compounds and percent ⁵¹Cr release and percent visual larval damage were recorded. An 8% solution of sodium hypochlorite released >90% of the ⁵¹Cr and produced >90% visual larval damage. Kalafungin released >32% ⁵¹Cr and produced >60% visual larval damage at a concentration of 1 μg·ml⁻¹. The results demonstrate that both assays are equal indicators of the viability of A. suum infective larvae when the cytotoxic agent is sodium hypochlorite. Kalafungin, an inhibitor of mitochondrial function, is detected more efficiently using percent visual larval damage when compared to ⁵¹Cr release.     

Synthesis of biopterin from dihydroneopterin triphosphate by rat tissues

High performance liquid chromatography procedure for the analysis of pterins of biopterin synthesis from dihydroneopterin triphosphate via sepiapterin in rat tissues has been described. Sepiapterin-synthesizing enzyme 1, which catalyzes in the presence of Mg²⁺ the conversion of dihydroneopterin triphosphate to an intermediate designated compound X was assayed by determining pterin which is formed from compound X under acidic conditions. Sepiapterin- and biopterin-synthesizing activity were also assayed by determining sepiapterin and biopterin, respectively. Analytical results revealed the presence of these activities in most rat tissues examined and high levels were found in kidney, pineal gland and liver. Activities were also detectable in peripheral erythrocytes.     

Biosynthesis of an acidic galactan and sulfated glycosaminoglycans during embryonic development of the mollusc Pomacea sp.

The synthesis of acidic polysaccharides by eggs of Pomacea sp. at different stages of development was studied. By day 3 after oviposition an acidic galactan became apparent. This compound reaches its maximum concentration by day 6 and then slowly decreases in concentration, no longer being detectable by day 12. Among the sulfated glycosaminoglycans, chondroitin sulfate is the first to appear (day 10), followed by heparan sulfate and other sulfate glycosaminoglycans, which were synthesized in large amounts until hatching (around day 15). Each one of the compounds was purified and characterized by chemical analysis and enzymatic degradation. The synthesis and characterization of the sulfated glycosaminoglycans was confirmed by the use of radioactive sulfate applied to intact eggs at different stages of development. These studies suggest that the main difference between vertebrate and mollusc development regarding the acidic polysaccharides is that hyaluronic acid seems to be absent during early mollusc development. It is proposed that the acidic galactan may be synthesized from the neutral galactan, already present in the eggs in high amounts, and may replace this glycosaminoglycan in the mollusc embryogenesis.     

Succinate dehydrogenase and fumarate reductase activity in Aspiculuris tetraptera and Ascaris suum and the effect of the anthelmintics cambendazole,thiabendazole, and levamisole

Comley John C. W. and Wright Spdenis J. 1981. Succinate dehydrogenase and fumarate reductase activity in Aspiculuris tetraptera and Ascaris suum and the effect of the anthelmintics cambendazole, thiabendazole, and levamisole. International Journal for Parasitology11: 79–84. Succinate dehydrogenase and fumarate reductase activities from a particulate fraction of A. tetraptera and a soluble extract of A. suum have been determined using spectrophotometric methods. Fumarate reductase activity in A. suum could only be detected anaerobically. Succinate dehydrogenase activity from A. suum was partially characterized and shown to exist in several multimolecular forms (isoenzymes). The in vitro effect of the anthelmintics cambendazole, thiabendazole and levamisole on succinate dehydrogenase and fumarate reductase activity from the above nematodes are described. Significant inhibition of fumarate reductase activity of both nematodes was only achieved using 5 mM levamisole and 1 mM thiabendazole. After in vivo anthelmintic treatment of A. tetraptera only thiabendazole significantly inhibited fumarate reductase. It is suggested that the succinate dehydro-ogenase-fumarate reductase complex in these nematodes is unlikely to be the primary site chemotherapeutic attack for any of the anthelmintics tested.     

Development of nested multiplex polymerase chain reaction (PCR) assay for the detection of Toxocara canis, Toxocara cati and Ascaris suum contamination in meat and organ meats

Ascarid Larva Migrans Syndrome (ascarid LMS) is a clinical syndrome in humans, caused by the migration of animal roundworm larvae such as Toxocara canis, Toxocara cati and Ascaris suum. Humans may acquire infection by ingesting embryonated eggs, or infective larvae of these parasites in contaminated meat and organ meats. To detect these pathogenic contaminations, a novel nested multiplex PCR system was developed. Our novel nested multiplex PCR assay showed specific amplification of T. canis, T. cati and Ascaris spp. Detection limit of the nested multiplex PCR was tested with serial dilution of T. canis, T. cati or A. suum genomic DNA (gDNA) from 100?pg to 100 ag and found to be 10?fg, 1?fg and 100?fg, respectively. When larvae were spiked into chicken liver tissue, DNA of T. canis and A. suum was detected from the liver spiked with a single larva, while the assay required at least 2 larvae of T. cati. Moreover, the ascarid DNA was detected from the liver of mice infected with 100 and 300 eggs of T. canis, T. cati or A. suum. This nested multiplex PCR assay could be useful for the detection of contamination with ascarid larvae in meat and organ meats.     

Immunological Evidence for the Requirement of Sepiapterin Reductase for Tetrahydrobiopterin Biosynthesis in Brain

Specific antibodies to sepiapterin reductase were used to investigate its involvement in de novo (6R)-5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis in rat brain. Antisepiapterin reductase (anti-SR) serum totally inhibited NADPH-dependent sepiapterin reductase activity in supernatants from discrete rat brain areas and liver. The anti-SR serum also inhibited the conversion of 7,8-dihydroneopterin triphosphate to BH4 in rat brain extracts. The inhibition was accompanied by a concentration-dependent increase in the formation of 6-lactoyltetrahydropterin (6LPH4), a proposed intermediate in BH4 biosynthesis. In addition, anti-SR serum was used to characterize the distribution and molecular properties of sepiapterin reductase in rat tissues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting indicated that there was a single polypeptide with the same molecular weight (28,000) as that of the subunit of pure sepiapterin reductase present in all tissues examined except for liver, where an immunoreactive protein of higher molecular weight (30,500) also was detected. Two-dimensional gel electrophoresis of rat striatum and liver demonstrated that the isoelectric point of sepiapterin reductase from both tissues was 6.16 and that the higher molecular weight immunoreactive material in liver had an isoelectric point of 7.06. Our studies with specific anti-SR serum confirmed the results of previous studies using chemical inhibitors of sepiapterin reductase, which suggested that sepiapterin reductase activity was essential for BH4 biosynthesis in the CNS and that 6LPH4 could be a precursor of BH4.     

Identification of tubulin isoforms in different tissues of Ascaris suum using anti-tubulin monoclonal antibodies

Three monoclonal antibodies specific to α- and β-tubulin were used to examine the expression of tubulin isofoms in the intestine, reproductive tract and body wall muscle of A. suum. The tubulins were found to be different in their isoelectric points, number of isoforms and peptide maps with Western blot analysis of one-dimensional polyacrylamide gel confirming the presence of α-, β₁- and β₂- tubulin. Commercial cross-reactive anti-α and anti-β MAbs 356 and 357 recognized tubulin from A. suum tissues as well as from pig brain, whereas anti-A. suum β-tubulin specific MAb P3D6 recognized tubulin from the A. suum tissues only. Two-dimensional gel analysis showed different isoform patterns in different A. suum tissues with anti-A. suum β-tubulin MAb P3D6 and cross-reactive β-tubulin MAb 357 recognizing 2–4 β- tubulin isoforms and anti-α-tubulin MAb 356 recognizing 1–6 α-tubulin isoforms. Different peptide maps of tubulin were observed in the three tissues, when subjected to limited proteolysis followed by SDS-PAGE. The data indicate that different tubulins are found in different tissues of adult A. suum.     

Stimulation of dehydrogenase synthesis by cadmium in a cadmium-resistant strain of Saccharomyces cerevisiae

The activity of dehydrogenase in Saccharomyces cerevisiae was estimated by reduction of 2,3,5-triphenyltetrazolium chloride. By the adaptation of yeast to cadmium, the high activity of dehydrogenase was observed. Furthermore, the activity of dehydrogenase in Cd-resistant cells was increased by growing in medium containing CdSO₄. However, the activity of dehydrogenase was inhibited by the addition of CdSO₄ to the reaction mixture. The activity of dehydrogenase in Cd-sensitive cells was increased slightly by incubation with low concentrations of CdSO₄.High activity of dehydrogenase in Cd-resistant cells was completely negated by the addition of cycloheximide to the incubation medium. The increase of dehydrogenase activity is due partly to de novo synthesis of protein.     

Biosynthesis of ticloidine in Physalis peruviana

The aerial parts and roots of Physalis peruviana (Cape Gooseberry) have been shown to contain tigloidine (3β-tigloyloxytropane) and 3α-tigloyloxytropane. The tiglic acid moiety of these alkaloids is derived from l-iSoleucine.     

13C-NMR study of the glucose synthesis pathways in the bacterium Chlorobium thiosulfatophilum

Photoassimilation of ¹³CO₂ and acetate by the photosynthetic bacterium Chlorobium thiosulfatophilum was investigated using ¹³C-NMR as the method for determination of the labelling pattern of the glucose synthesized by the bacterium. Metabolic pathways functioning in the bacterium were identified by analysis of the multiplet structure of the spectra of tightly coupled systems. The labelling pattern showed that glucose was synthesized in C. thiosulfatophilum mainly by the gluconeogenesis pathway. In agreement with previous investigations, the reserve polysaccharide of C. thiosulfatophilum was shown to be polyglucose, with the glucose units linked predominantly by (1→4)α-glucosidic linkages.