Nucleotide hydrolysis by solubilized membrane-bound enzymes of the brush border plasma membrane of Hymenolepis diminuta.

Pyrophosphate, p-nitrophenyl phosphate and a variety of pyrimidine and purine nucleotides are hydrolyzed by the solubilized membrane-bound enzymes of the brush border plasma membrane of Hymenolepis diminuta. The pH optima (or ranges) for hydrolysis of substrates are 8.0 (pyrophosphate), 8.8 (p-nitrophenyl phosphate), 8.4-8.9 (nucleoside monophosphates), and 7.1-8.1 (nucleoside triphosphates); all substrates, with the exception of nucleoside triphosphates, have a higher affinity for the solubilized enzyme at pH 7.4 than at their optimal pH for hydrolysis. ATP is degraded completely by the enzyme preparation to adenosine and inorganic phosphate, but since neither ADP nor ATP accumulate in the incubation medium it is not known whether ATP hydrolysis involves the sequential hydrolysis of terminal phosphate groups. Isoelectric focusing and various chromatographic procedures (gel permeation, ion-exchange and hydrophobic interaction chromatography) fail to separate the alkaline phosphatase, phosphodiesterase, 5'-nucleotidase, adenosine triphosphatase and ribonuclease activities associated with the solubilized membrane preparation. Additionally, inhibitor studies indicate that only a single enzyme with low substrate specificity is involved in the hydrolysis of nucleotides, p-nitrophenyl phosphate, pyrophosphate and hexose phosphate esters. Purines and pyrimidines and their nucleosides interact with the active site, and in some instances activity of the enzyme is stimulated by an unknown mechanism.

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.

Partial characterization of the protein and carbohydrate constituents of the egg shell of Hymenolepis diminuta (Cestoda).

The egg shell of Hymenolepis diminuta is composed of 31.7% protein and 2.9% carbohydrate (by weight), as determined using the Lowry and phenol-sulfuric acid assays and bovine serum albumin and glucose, respectively, as standards. Amino acid analyses of egg shells demonstrated the presence of 16 naturally occurring amino acids and six unidentified residues. Histidine accounted for > 22% of the amino acid residues. Hypochlorite-solubilized egg shells were fractionated using a combination of gel, hydroxyapatite and anion-exchange chromatography, and the fractions were analyzed by isoelectric focusing and gel chromatography. The results demonstrate that the shell consists of a complex mixture of proteins (almost all of which have acidic isoelectric points), glycoproteins and possibly free complex carbohydrates.

Mechanism of 3-0-methylglucose uptake by Hymenolepis diminuta.

The mechanism by which Hymenolepis diminuta (Cestoda) absorbs 3-0-methylglucose (30MG) in vitro was analyzed. Influxes of 0.1 and 0.01 mM-3H-30MG during incubations ranging from 5 s to 60 min were not affected significantly when 10 mM-unlabeled 30MG was present as an inhibitor. After 60 min in 0.1 mM-3H-30MG, the concentration of labeled substrate within tapeworms (0.04 mumol ml-1 worm water = 0.04 mM) was less than that of the bathing medium. Tapeworms incubated for 1 h with either 5 mM-glucose or 5 mM-beta-methylglucose (beta MG) gained 15-20% more water than did tapeworms in saline alone, but addition of 5 mM-30MG to the saline had no significant effect on weight change. When the 3H-30MG concentration was varied from 0.01 to 10 mM, influxes were a linear function of substrate concentrations. These analyses show that H. diminuta absorbs 30MG by simple diffusion alone. Thus, use of this monosaccharide to estimate the internal concentration of actively transported sugars (e.g. glucose or beta MG) in H. diminuta is invalid.

Purification, quantification and mechanical hatching of eggs of the tapeworm, Hymenolepis diminuta.

Eggs of Hymenolepis diminuta were separated from host's feces using a combination of NaCl flotation, filtration through nylon monofilament screen cloths, and centrifugation. The resulting purified egg preparations (PEPs) were verified microscopically to be virtually free of contaminating debris. The relationship of absorbance (500 nm) of PEPs and 'number of eggs per unit volume' was linear, and such measurements provided a rapid, reproducible method for quantifying PEPs. The average wet and dry weights of individual eggs were 2.86 x 10(-7) and 0.458 x 10(-7) g, respectively. Eggs were mechanically hatched using a Dounce homogenizer with pestle 'A'. This technique caused no detectable damage to larvae and resulted in a high percentage of 'activated' (i.e. motile) larvae.

Increased coprophagic activity of the beetle, Tenebrio molitor, on feces containing eggs of the tapeworm, Hymenolepis diminuta.

When provided with fecal pellets from uninfected (control) rats and rats infected with the tapeworm Hymenolepis diminuta, more fed and starved (72 h) female and starved male Tenebrio molitor fed on fecal pellets from infected- than from control rats; compared to fecal pellets from controls rats, fed males avoided the infective fecal pellets. Uninfective and infective fecal pellets had similar moisture contents, so increased coprophagic activity was not due to differences in moisture content. Fed and starved males and females were fed on fecal pellets containing tapeworm eggs and examined for cysticercoids. Significantly greater numbers of starved beetles than fed beetles were infected with cysticercoids, but the numbers of infected males and females within each treatment were not significantly different. On the other hand, males contained significantly greater numbers of cysticercoids than did females, and there was no significant difference between the numbers of cysticercoids recovered from fed and starved beetles. The data support the hypothesis that the feeding behavior of T. molitor on rat feces is altered by the presence of tapeworm eggs. The data demonstrated further that transmission dynamics are affected by a complex interaction of the beetle's sex and nutritional status.

Partial biochemical characterization of venom from the ant, Pseudomyrmex triplarinus.

Venom from the ant, Pseudomyrmex triplarinus, contains 12 proteins with mol. wts of > 100,000-4200, and they constitute 41.5% of the dry weight. In comparison with published data on ant, wasp, and bee venoms, whole venom has intense phospholipase activity and intermediate hemolytic activity. Four major proteins were isolated and purified by low pressure chromatography. The most abundant protein had a mol. wt of 4200 and weak hemolytic activity. The second most common protein was 20,400 and had phospholipase A2 activity. The other two major proteins had mol. wts of 24,500 and 14,100 and both exhibited phospholipase and direct hemolytic activities. There are eight minor proteins (> 100,000-40,000), each present at about 1% or less of the total protein. Assayed as a mixture, they had hyaluronidase activity. Seventeen free amino acids were detected with aspartic acid, glutamic acid, and proline together making up 72% of the total mass of amino acids. Glycerol was present at a concentration of 3.1% of the dry weight and the venom was devoid of lipids.

Solubilization of the membrane-bound enzymes of the brush-border plasma membrane of Hymenolepis diminuta (Cestoda) using nonionic detergents.

The ability of nonionic detergents to solubilize the membrane-bound enzymes of the brush-border plasma membrane of Hymenolepis diminuta was investigated. Of the detergents tested (Triton X-100, Tween 80, Brij 35, Lubrol PX and WX, W-1, and beta-octyl-D-glucoside), only Triton was an effective solubilizing agent. Optimal solubilization was achieved by incubating an isolated fraction of the brush-border membrane in the presence of 1% Triton X-100 for 60 min at 37 C, followed by centrifugation at 100,000 g for 60 min at 25 C. This treatment resulted in solubilization of 94% of the alkaline phosphohydrolase, 91% of the phosphodiesterase and ribonuclease, and 88% of the 5'-nucleotidase activities. The pH optima for enzymes solubilized in nonionic and ionic detergents (Triton and sodium dodecyl sulfate, respectively) did not differ. Isoelectric focusing of the Triton-solubilized material demonstrated the presence of at least 14 polypeptides, a majority of which had isoelectric points below pH 7.

Phosphohydrolase activity of the isolated, brush-border membrane of Hymenolepis diminuta (Cestoda) following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.

Following electrophoresis of isolated, brush-border membranes of Hymenolepis diminuta on SDS-polyacrylamide gels, three distinct areas of alpha-naphthyl phosphate (NP) hydrolysis were detected; these corresponded to proteins with molecular weights of 106,800, 172,700, and greater than 340,000 Daltons. Hydrolysis of NP was inhibited by adenosine triphosphate, adenosine;5'-monophosphate, p-nitrophenyl-phosphate, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, molybdate, ethylenediaminetetraacetate (EDTA), and ethyleneglycol-bis-(beta-amino-ethyl)-N,N'-tetraacetate (EGTA), but not by fluoride. Inhibition of NP hydrolysis by EDTA was relieved in the presence of Mg++ or Ca++. Heating the isolated, brush-border membrane in the presence of SDS for 5 min at 95 C destroyed all enzymatic activity. These characteristics indicated that the enzyme(s) responsible for NP hydrolysis (following separation of membrane proteins by SDS-polyacrylamide gel electrophoresis) were the same enzymes responsible for the phosphohydrolase activity associated with intact and solubilized, brush-border membrane preparations of H. diminuta.

Glycosyl transferase activity in the brush border membrane of Hymenolepis diminuta.

The isolated brush border membrane of Hymenolepis diminuta incorporates radiolabeled glucose when incubated in the presence of uridine diphospho(UDP)-D-(6-3H)glucose. The pH optimum for incorporation was 7.0 to 7.2 regardless of the buffer used. Transferase activity was maximal in 200 mM Tris buffer; 100 mM phosphate buffer inhibited significantly the incorporation of radiolabeled glucose, whereas 50 mM Tris-maleate and 100 mM PIPES resulted in moderate inhibition of activity. Incorporation of labeled glucose was not inhibited by low concentrations (0.01%) of Triton X-100, but activity was inhibited 50% by 0.25% Triton X-100. Addition of divalent cations to the brush border membrane preparation did not activate transferase activity, but addition of chelating agents (i.e., EDTA or EGTA) inhibited transferase activity nearly 90%. Incorporation of labeled glucose was inhibited by UDP, guanosine diphosphate (GDP), UDP- and GDP-activated monosaccharides, and monosaccharides, indicating that the transferase activity lacked substrate specificity.

Variation in the sizes of eggs and oncospheres and the numbers and distributions of testes in the tapeworm, Hymenolepis diminuta.

Four "strains" of Hymenolepis diminuta were examined for morphological variation. These included the ARME "strain" (currently maintained at the University of Keele, U.K.), the OSU "strain" (currently maintained at The Ohio State University) and the TOR (or UT) "strain" (currently maintained at the University of Toronto), all of which were derived from the parental RICE "strain," and the ANU "strain" (currently maintained at the Australian National University). Additionally, 2 separate "clonal" populations (populations derived from single cysticercoids) from both the OSU and ANU "strains" were examined. All "strains" and "clones" were maintained under identical conditions using Tenebrio molitor and male Sprague-Dawley rats as the intermediate and definitive hosts, respectively. The lengths and widths of eggs and larvae (oncospheres) passed in the hosts' feces, and the numbers and distributions of testes in proglottids were quantified and the data analyzed. Although analyses of the lengths and widths of eggs and larvae demonstrated significant differences among some "strains" and "clones," a discriminate analysis of the data indicated these parameters to be of questionable taxonomic significance. The eggs of all "strains" and "clones" consisted of 2 distinct populations differing in density and size but not infectivity; the relative proportions of eggs in the 2 populations were not determined. Considering all possible numbers and distributions of testes, 17 variations were seen in the strobilae of tapeworms. Analyses of the data demonstrated that the "strains" and "clones" could be differentiated clearly using only the frequencies of the 1p2a (1 poral and 2 aporal testes) or 1p3a distribution, or the frequencies of proglottids containing 3 or 4 testes; all other variations failed to clearly differentiate or group the various "strains" and "clones."(ABSTRACT TRUNCATED AT 250 WORDS)

Hymenolepis diminuta metacestodes (Cysticeroids) are unable to utilize exogenous trehalose.

Cysticercoids (metacestodes) of Hymenolepis diminuta were incubated in Tyrode's salt solution (pH 7.2) containing 1.25 mM or 40 mM trehalose for 5 or 18 hr, and the amounts of trehalose remaining in the media were determined using high-pressure liquid chromatography. No differences were detected in the amounts of trehalose remaining in control (no cysticercoids) or experimental incubations under any of the experimental conditions. Thus, cysticercoids are apparently unable to utilize trehalose present in the external medium.

Trypsin adsorption by Hymenolepis diminuta (Cestoda).

Significant amounts of radioactivity were associated with Hymenolepis diminuta following incubation in 3H-trypsin. Autoradiography of worms incubated in 3H-trypsin for 30 min demonstrated that all radioactivity was associated with the worm's surface (tegument). The amount of 3H-trypsin adsorbed by the worms was not sufficient to account for the inactivation of this enzyme in the presence of intact worms. Unlabeled trypsin and poly-L-glutamate (but not poly-L-lysine) inhibited adsorption of 3H-trypsin, but were without effect on trypsin inactivation by H. diminuta. Therefore, trypsin was adsorbed by intact H. diminuta, but the process of adsorption apparently did not play any role in inactivation of the enzyme.

Solubilization of membrane-bound ribonuclease (RNAse) and alkaline phosphatase from the isolated brush border of Hymenolepis diminuta (Cestoda).

Plasma membrane from the brush border isolated from the tegument of Hymenolepis diminuta contains membrane-bound ribonuclease (RNase) and alkaline phosphatase activities. RNase (yeast RNA substrate), alkaline phosphatase (p-nitrophenyl phosphate substrate), and additional membrane proteins were solubilized by sonication or treatment with the detergents dodecyl trimethylammonium bromide, beta-octyl-D-glucopyranoside, sodium dodecyl sulfate (SDS), or ZwittergentTM 3-12 (N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). At optimal conditions, greater than 90% of both enzymes and total protein were solubilized by the latter two detergents, whereas beta-octyl-D-glucopyranoside, dodecyl trimethylammonium bromide, and sonication were only partially effective. Nonionic detergents did not solubilize the membrane effectively.

Beetle-to-beetle transmission and dispersal of Hymenolepis diminuta (Cestoda) eggs via the feces of Tenebrio molitor.

When grain beetles (Tenebrio molitor) were fed eggs of Hymenolepis diminuta, many of the eggs passed intact through the beetles' intestines, and eggs were present in the beetles' feces for at least 48 hr after feeding. When uninfected T. molitor were fed beetle feces containing H. diminuta eggs, they became infected. Tenebrio molitor were fed on H. diminuta eggs and then placed in fresh bran for 48 hr. When uninfected T. molitor were placed in this bran, they became infected. Thus, feces from beetles that have ingested H. diminuta eggs serve as a source of eggs for other beetles, as well as a mechanism of egg dispersal.

Type I phosphodiesterase in the isolated, brush-border membrane of Hymenolepis diminuta.

The isolated, brush-border membrane of Hymenolepis diminuta contained an enzyme which hydrolyzed phosphodiester bonds. This enzyme appeared to be a Type I phosphodiesterase (E. C. 3.1.4.1) (produces nucleoside 5'-phosphates) and had no activity against synthetic, Type II phosphodiesterase substrates (mononucleotides substituted at the 3' position). The effects of various potential inhibitors of enzymatic activity, and cation requirements of this enzyme, demonstrated a distinct difference between the phosphodiesterase and alkaline phosphatase activities of the isolated, brush-border membrane. SDS-polyacrylamide gel electrophoresis of the isolated membrane preparation, followed by localization of phosphodiesterase activity in the gels, indicated the enzyme had a molecular weight of approximately 87,000. Thus, the phosphodiesterase activity represents a previously undescribed, membrane-bound enzyme of the brush-border of Hymenolepis diminuta.

Partial characterization of ribonuclease (RNase) activity from the isolated and solubilized brush border of Hymenolepis diminuta.

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.

Chloride-sensitive glucose transport in Hymenolepis diminuta.

The maximal rate of glucose influx in Hymenolepis diminuta decreased when CL- in the incubation medium was replaced with acetate, benzoate, bicarbonate, formate, hippurate, iodide, lactate, mandelate, or nitrate. The effect of Cl- deletion on glucose influx using any of these anions was reversed when worms were incubated in Krebs-Ringer saline for 15 min. Glucose in the incubation medium increased 36Cl- influx in worms, while in Na+-free media the presence of glucose had no effect on 36Cl- influx. Influx of 10 mM 36Cl- in H. diminuta was inhibited by unlabeled Cl- in the incubation medium. Hymenolepis diminuta accumulated glucose aginst an apparent concentration differnce when Cl- in the incubation media was replaced with acetate, bicarbonate, or nitrate. The influx of Cl- appears couples with the influxes of Na+ and glucose, but the data do not show whether the influxex of these molecules are mediated through a common "carrier."

Purine base and nucleoside uptake in Plasmodium berghei and host erythrocytes.

The absorption of 3H-labeled adenine, adenosine, hypoxanthine, and 14C-labeled inosine by normal rat erythrocytes, Plasmodium bergheri-infected erythrocytes and saponin released "free parasites" was measured. The uptake of these labeled substrates by normal rat erythrocytes occurs both by diffusion and mediated transport systems. Similar absorptive mechanisms for these substrates also were observed for both Plasmodium berghei-infected erythrocytes and "free parasites." Data from inhibition studies using purine base and nucleoside analogues indicate the presence of three distinct transport loci in the normal erythrocyte for adenosine-inosine, hypoxanthine, and adenine and two loci in the infected erythrocyte and "free parasite" for adenosine-inosine-hypoxanthine and adenine. The initial metabolism of 3H-adenosine by the "free parasite" also was examined. A double isotope technique was used to follow the separate metabolic fates of the purine base and ribose moieties of adenosine. The data suggest a possible conversion of adenosine to the purine base and ribose moiety and subsequent uptake of the purine base by the parasite. In addition, a powerful adenosine deaminase inhibitor (2-deoxcoformycin) significantly reduced the uptake of 3H-adenosine by the "free parasites." Chromatographs of aliquots from postincubation media show the tritium label to be associated predominately with adenosine in the presence of 2-deoxycoformycin and with isoine and hypoxanthine in the absence of the inhibitor.

Effect of tunicamycin on the uptake and incorporation of galactose in Hymenolepis diminuta.

The effects of tunicamycin (TM) on the uptake and incorporation of tritiated galactose into the tegumental membrane and carcass from adult Hymenolepis diminuta were examined to assess the potential usefulness of this inhibitor for studying the function of the tapeworm surface glycocalyx. Hymenolepis diminuta adults (11 days old) were preincubated for 1 hr, pulsed for 30 min with [3H]galactose and [14C]leucine, and chased for 2 hr; replicate experiments were conducted in which all media contained no TM or TM at 10 micrograms/ml. Tunicamycin significantly inhibited the incorporation of tritiated galactose into the tapeworm's carcass and 30,000-g tegumental membrane fraction. Incorporation of tritiated galactose into the tapeworm's tegumental surface membrane also was inhibited significantly when expressed relative to the incorporation of [14C]leucine. Tunicamycin did not affect the amounts of free, i.e., soluble, [3H]galactose or [14C]leucine recovered from the tapeworms not did it affect the short-term (2 min) uptake of [3H]galactose by tapeworms. Thus, the inhibitory effect of TM appears to be at the level of protein glycosylation rather than carbohydrate (galactose) transport. The data indicate that TM might be useful for producing tapeworm surface membranes with diminished carbohydrate moieties.