Characterization of polysaccharides of the eggs and adults of Hymenolepis diminuta.

Polysaccharides and other complex carbohydrates were released by proteolysis of the chloroform-methanol insoluble residue of 10 day-old worms and eggs of Hymenolepis diminuta. Gas-liquid chromatographic analysis of alditol acetate derivatives of monosaccharides released from the polysaccharides by hydrolysis revealed that in the 10 day-old worm, glucose was the most abundant sugar, followed by galactose, glucosamine, galactosamine, fucose and possibly rhamnose. Mannose was least abundant and xylose was absent. In the egg, glucose and galactose were equally abundant, followed by the same sugars found in 10 day-old worms, and xylose was present. Uronic acid was detected in both fractions by specific chemical tests. None of the saccharide material from eggs and worms was susceptible to degradation by Streptomyces hyaluronidase, chondroitinase AC, and slightly susceptible to chondroitinase ABC, as shown by electrophoretic analysis on composite 2.2% acrylamide-agarose slab gels and 4.5/12.5% polyacrylamide gels before and after enzymatic treatment. One of the gel-separable bands, however, was degradable by both nitrous acid and Flavobacterium heparinase. Both bands from eggs were degradable by nitrous acid. These results suggest that eggs contain heparin and/or heparan sulfate and perhaps dermatan sulfate and that 10 day-old worms also have these polyglycans but possibly not chondroitin sulfate or hyaluronic acid.

Glycosaminoglycans of tegumental fractions of Hymenolepis diminuta.

The teguments of 6 and 10 day-old Hymenolepis diminuta were removed with Triton X-100 and separated into brush border and vesicular fractions by differential centrifugation. Glycosaminoglycans (GAG) isolated from these tissues and from the denuded carcass were treated with specific GAG-degrading enzymes and other chemical agents and analyzed by sodium dodecyl sulfate-polyacrylamide, agarose gel and cellulose acetate electrophoresis. Both 6 and 10 day-old worm carcasses contained chondroitin sulfate, heparin/heparan sulfate and hyaluronic acid. The 10 day-old worm brush border and vesicle fractions contained chondroitin sulfate but no heparin-like material. Colorimetric analysis showed that the carcasses of both 6 and 10 day-old worms contained uronic acid. About 98% of the detectable uronic acid of 10 day-old worms was found in the carcass, and only 2% in the brush border fraction. No uronic acid was detected in the other tegumental fractions.

Analysis of ecdysteroids in different developmental stages of Hymenolepis diminuta.

Prepatent and patent adult Hymenolepis diminuta from the intestines of rats, H. diminuta eggs recovered from the faeces of rats harbouring patent infections, and infective cysticercoids from the beetle intermediate host were analysed for free and conjugated ecdysteroids. Adult worms and eggs contained both free ecdysteroids and hydrolysable polar conjugated ecdysteroids, with comparatively large amounts of immunoreactive material also being detected following hydrolysis of the possible apolar conjugated ecdysteroid fraction. Free ecdysteroids were not detected in the cysticercoid sample. The concentration of free ecdysteroids in H. diminuta eggs was higher than that detected in the tissues of the adult worms. Ecdysone and 20-hydroxyecdysone were the major identified compounds of the free ecdysteroid fraction, whereas in the hydrolysed polar conjugated ecdysteroid fraction these two compounds were accompanied by 20,26-dihydroxyecdysone. The free ecdysteroid fraction also contained comparatively large amounts of unidentified immunoreactive material.

Alterations in brush border membrane proteins and membrane-bound enzymes of the tapeworm, Hymenolepis diminuta, during development in the definitive host.

During growth and maturation of the tapeworm, Hymenolepis diminuta, significant decreases occur in the brush border membrane-bound alkaline phosphatase, phosphodiesterase, 5'-nucleotidase, adenosine triphosphatase and ribonuclease activities. These decreases are accompanied by qualitative and quantitative changes in the polypeptide profiles of the brush border membrane fraction. Gradients of enzymatic activities and polypeptide profiles are also demonstrable when mature tapeworms are cut into pieces and the brush border membrane of each piece analyzed individually. In fully developed tapeworms the enzymatic activities and polypeptide profiles of membrane preparations reflect mainly the contributions of the more mature proglottids; these proglottids constitute most of the tapeworm biomass. The most anterior sections of these fully developed worms are biochemically similar to young, developing worms.

Evidence for a Ca2+-dependent activator protein in the rat tapeworm Hymenolepis diminuta.

A low molecular weight, acidic, heat stable protein has been characterised from the rat tapeworm Hymenolepis diminuta. This protein was found to activate cyclic 3', 5'-nucleotide phosphodiesterase in a Ca2+-dependent manner. The activation process was inhibited by the phenothiazine drug trifluoperazine. The biochemical properties of this protein clearly resemble those of ovine brain calmodulin. Our investigation thus concludes that there is a calmodulin-like activator protein in this cestode.

Rhodoquinone requirement of the Hymenolepis diminuta mitochondrial electron transport system.

The occurrence of rhodoquinone as a mitochondrial membrane component was demonstrated in adult Hymenolepis diminuta. Chromatographic separation of pentane extracts, from lyophilized mitochondrial membranes, coupled with spectral analyses of separated material demonstrated the presence of rhodoquinone. The presence of ubiquinone was not apparent. Rhodoquinone content of membranes was about 1.2 micrograms (mg protein)-1. The rhodoquinone requirement of the H. diminuta electron transport system was demonstrated both in terms of the less active NADH oxidase and the physiologically required, NADH-dependent fumarate reductase employing lyophilized mitochondrial membranes as the source of activities. Pentane extraction of membranes virtually abolished the oxidase and fumarate reductase systems. Supplementation of pentane-treated membranes with H. diminuta rhodoquinone restored oxidase and fumarate reductase activities to levels simulating those of lyophilized membranes. Ubiquinone did not substitute for rhodoquinone. The rhodoquinone-reconstituted membranes displayed rotenone sensitivity. These findings represent the first direct demonstration of the rhodoquinone requirement of helminth electron transport-coupled oxidase and fumarate reductase.

Influence of 0.02 M EDTA and 3 M KCl on surface of Hymenolepis diminuta and composition of isolated proteins.

The glycocalyx of Hymenolepis diminuta (Cestoda, Cyclophyllidea) was isolated using 0.02 M EDTA or 3 M KCl. It was shown in the electron micrographs that 0.02 M EDTA did not damage the tapeworm plasma membrane, eliminating glycocylax only, in contrast to 3 M KCl which disrupted tegument up to the basal membrane. The protein analysis of extracts and the supernatant of homogenate of the whole tapeworm strobila by polyacrylamide gel electrophoresis (PAGE) and dodecyl sulphate-polyacrylamide gel electrophoresis (SDS) electrophoresis revealed that the substance extracted with 3 M KCl was more abundant in protein fractions than the two remaining ones. The substance extracted with 0.02 EDTA, collecting the tapeworm glycocalyx possessed the smallest amount of protein fractions, however, some of them were more abundant.

Quantitative determination of inositol in Hymenolepis diminuta (Cestoda).

Myoinositol and scylloinositol have been identified qualitatively and quantitatively by gas-liquid chromatography in Hymenolepis diminuta. No myoinosose-2 was detected. Myoinositol was unevenly distributed throughout the worm. The scolex and neck regions contained more free- and phosphatidyl-bound inositol. This region also contained more lipid-bound phosphorus, but less total lipid and water.

The occurrence and distribution of 5-hydroxytryptamine in Hymenolepis diminuta and H. nana.

The presence of 5-HT in Hymenolepis diminuta and Hymenolepis nana was detected by 2 biochemical methods and as yellow fluorescence in a histochemical method. In H. diminuta, 5-HT was found in a concentration of about 1.2 micron/g; this amount did not vary significantly in worms aged 6 to 18 days or more or in various regions of the worm. In H. nana, 5-HT was found in a concentration of about 1.8 micron/g. It was histochemically localized in H. diminuta and H. nana in a pattern similar to that of acetylcholinesterase previously described in these 2 cestodes, and it may be the opposing neuro-transmitter to acetylcholine. The lack of 5-HT in the vestigial rostellum of H. diminuta may be correlated with loss of function of this organ.

Developmental physiology of cestodes. XIV. Roughage and carbohydrate content of host diet for optimal growth and development of Hymenolepis diminuta.

Diets of rats infected with Hymenolepis diminuta (CESTODA: Cyclophyllidea) were altered with respect to carbohydrate content and to roughage, and the effects on worm growth and development were studied. Compared to worms from rats fed a 56% glucose diet, those on a 56% starch diet were heavier at 10 and 15 days and had more immature proglottids at 5 days, mature prglottids at 10 days, and mature and gravid proglottids at 15 days postinfection. In addition, worms from rats fed the starch diet contained a higher carbohydrate concentration and a lower lipid concentration from those fed the glucose diet. Worms from rats fed diets with combinations of carbohydrates such as 51% starch-5% sucrose and 51% starch-5% lactose were not different from those fed the 56% starch diet. If rats were fed a pellet diet (Purina Laboratory Chow), the worms grew substantially larger than those from rats fed the 56% starch or combination diets. The differences could be overcome if a 6% roughage component were included in the 56% starch diet. Therefore, the starch-roughage diet here presented is recommended as the optimal defined diet for studies of the development of H. diminuta in the definitive host.

An explanation of the apparent reversal of the circadian migration by Hymenolepis diminuta (Cestoda) in the rat.

Hymenolepis diminuta exhibits 2 concurrent migrations: an age-dependent, forward migration and a circadian migration. In experiments where the age of worms was assumed to be uniform throughout the test-day, 2 patterns (7-day-old and 16-day-old) of circadian migration were seen and an apparent reversal in circadian pattern suggested. In experiments where the age of worms became progressively older during the test-day, only the 16-day-old pattern was seen and no reversal was observed. The 7-day-old pattern and hence the apparent reversal in circadian migration is postulated to be an artifact of the method whereby both the age and size of worms were presumed to be uniform throughout the test-day. The 7-day-old pattern results from comparing worms of unequal size and thus at different positions in their forward migration. Data on the daily variation of stomach contents of rats and the results from protein determinations of 6-day-old worms support this hypothesis. Therefore H. diminuta is believed to exhibit only the 16-day-old pattern of circadian migration: an anterior migration between 12 midnight and 6 AM, and a posterior migration between 12 noon and 6 PM.

Protein, glycogen, and water content in schistosomes.

The volume of distribution (= VD) of water was measured in Schistosoma mansoni, S. haematobium, S. japonicum, and Hymenolepis diminuta. In the rat tapeworm, H. diminuta, the volume of distribution of 3H-water was positively correlated with wet weight (r = 0.87, P less than 0.001) and this same phenomenon also was demonstrated in S. mansoni (r = 0.90, P less than 0.001). The 5-sec VDwater was constant over a range of glucose (0.01-50 mM) and phenylalanine (0.01-20 mM) concentrations in both male and female schistosomes. The VD of antipyrine, which permeates by virtue of its lipophilic properties, also was shown to correlate with protein content in S. mansoni. Protein content determined in single, isolated schistosomes was correlated with the volume of distribution of water in S. haematobium, S. japonicum, and S. mansoni males and females. Age-related variations in the protein content (of S. mansoni) and volume of distribution of water (in both S. japonicum and S. mansoni) were also defined, and the use of tritiated water content as an indicator of mass in small tissue samples was thus established. Female blood flukes recovered from mice infected for more than 90 days appeared to be characterized by a slight reduction in size with age. Schistosoma mansoni reared in the golden hamster may be slightly smaller than schistosomes of the same strain raised in outbred mice. These results provide a baseline that should be useful for future physiological and immunological studies.

Developmental physiology of cestodes: characterization of putative crowding factors in Hymenolepis diminuta.

It was shown previously that worm-conditioned saline (WCS) prepared from crowded 10-day-old H. diminuta inhibited the incorporation of 3H-thymidine into DNA in the anterior regions of uncrowded worms and that the inhibition was partially accounted for by succinate and acetate excreted by the worms. The present study describes further characterization of the active components of WCS. An ultrafiltrate was fully as potent as untreated WCS, indicating that all detectable inhibitory components were less than about 500 daltons in molecular mass. Inhibitory factors in WCS were stable to heat (80 C for 30 min), cold (4 C for 48 hr), drying and reconstitution, alkaline pH (11 to 12 for 3 hr), and ethanolic extraction. Active compounds were probably not lipoidal in nature. Although the acidic ethanol extract of WCS was inhibitory, no activity was observed in fractions of WCS that contained basic, acidic and neutral amino acids. Amino compounds in the WCS were further investigated. Twenty-four amino acids were identified, 3 of which (phosphoserine, 1-methylhistidine, and 3-methylhistidine) have not been reported previously for H. diminuta. On a molar basis, alanine accounted for 40-50% of the amino acids released. The amino sugar, D-glucosaminic acid, was found in the WCS and also has not been heretofore reported from H. diminuta or any other cestode. In concentrations comparable to those in the WCS, D-glucosaminic acid inhibited incorporation of 3H-thymidine into the DNA of the tapeworms by 25-35%, suggesting that D-glucosaminic acid may be one of the crowding factors.

Biochemical effects of thiabendazole and cambendazole on Hymenolepis diminuta (Cestoda) in vivo.

An investigation of the chemotherapeutic and biochemical effects of two benzimidazole anthelmintics, thiabendazole (TBZ) and cambendazole (CBZ), on Hymenolepis diminuta in experimentally infected rats is reported. Thiabendazole was active against H. diminuta at a relatively high dosage. A single oral dose of TBZ at 250 mg/kg body weight on day 15 of infection eliminated 100% of the tapeworms as determined at necropsy 5 days after treatment. The chemotherapeutic actions of TBZ on H. diminuta were accompanied by marked changes in worm weight and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of TBZ 24 hr earlier were significantly smaller and contained much less glycogen (as a percent of the wet weight) than worms from unmedicated controls. Protein concentrations increased in TBZ-treated worms and at a rate sufficient to offset the decline in glycogen concentration. Glycogen/protein ratios in TBZ-treated worms were significantly lower than the corresponding control values. Cambendazole proved to be five times more potent than TBZ against H. diminuta and produced the same basic changes in worm weight and chemical composition within 18 hr of treatment of the host. Administration of a single oral dose of TBZ or CBZ to the host produced in H. diminuta another change, the onset of which coincided with, or preceded, the gross alterations in worm weight and chemical composition. That change, observed in in vitro studies carried out 14 hr after treatment, revealed that tapeworms from drug-treated rats absorbed and metabolized much smaller quantities of exogenous glucose than did the controls, and the ability of the worm to accumulate glucose against a concentration difference was significantly depressed.

Effect of host feeding and available glucose on glycogen synthase and phosphorylase activities in Hymenolepis diminuta and Vampirolepis microstoma.

The influences of host feeding and the availability of glucose in vitro on the activities of glycogen synthase and glycogen phosphorylase in Hymenolepis diminuta and in Vampirolepis microstoma were studied. The worms were recovered from hosts that had been fed ad libitum, starved for 24 hr, or starved 24 hr and then refed for 1 hr immediately prior to worm recovery. The ratios of active to inactive glycogen synthase and phosphorylase were correlated with the host feeding regimen prior to recovery. Glycogen synthase in H. diminuta was predominately in the inactive D form in worms from both fed and fasted hosts. One hour after refeeding, up to 80% of the synthase was in the active I form. Phosphorylase in H. diminuta was predominantly in the active a form in worms from fed and fasted hosts, but activity of this enzyme was suppressed in worms from refed hosts. When H. diminuta from fasted hosts was incubated in a balanced salt solution containing 40 mM glucose, glycogen synthase I increased, and phosphorylase a decreased. Glycogen synthase in V. microstoma was predominantly in the inactive D form in worms from both the fed and fasted hosts, but the proportion in the active I form increased to over half the total synthase by 1 hr of host refeeding. The proportion of glycogen phosphorylase a was high in worms from fed hosts and decreased, but not dramatically, in worms from fasted hosts. The results suggested that the worms had access to another source of glucose, probably from the host bile, and we measured a low but significant concentration of carbohydrate in the gall bladder bile of mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acids of Hymenolepis palmarum (Johri, 1956) and chemotaxonomic studies on hymenolepidid cestodes.

Chemotaxonomic patterns in the distribution of amino acids of Hymenolepis palmarum (Johri, 1956) and other hymenolepidids revealed the common presence of beta-aminoisobutyric acid, lysine, phenylalanine and tyrosine but 3,4 dihydroxyphenylalanine and norleucine were exclusive to H. palmarum. Both qualitative and quantitative differences in amino acids have been recorded.