Search

Purpose: To implement and test a Web-based tracking and feedback (T&F) tool to close referral loops and reduce adjuvant breast cancer treatment underuse in safety-net hospitals (SNHs). Patient and Methods: We randomly assigned 10 SNHs, identified patients with new stage 1 to stage 3 breast cancer, assessed their connection with the oncologist, and relayed this information to surgeons for follow-up. We interviewed key informants about the tool’s usefulness. We conducted intention-to-treat and pre- and poststudy analyses to assess the T&F tool and implementation effectiveness, respectively. Results: Between the study start and intervention implementation, several hospitals reorganized care delivery and 49% of patients scheduled to undergo breast cancer surgery were ineligible because they already were in contact with an oncologist. One high-volume hospital closed. Despite randomization of hospitals, intervention (INT) hospitals had fewer white patients (5% v 16%; P = .0005), and more underuse (28% v 15%; P = .002) compared with usual care (UC) hospitals. Over time, INT hospitals with poorer follow-up significantly reduced underuse compared with UC hospitals (INT hospitals, from 33% to 9%, P = .001 v UC hospitals, from 15% to 11%, P = .5). There was no difference in underuse (9% at INT hospitals, 11% at UC hospitals; P = .8). Hospitals with better follow-up (odds ratio, 0.85; 95% CI, 0.73 to 0.98) had less underuse. In settings with poor follow-up and tracking approaches, key informants found the tool useful. The rapidly changing delivery landscape posed significant challenges to this implementation research. Conclusion: A T&F tool did not significantly reduce adjuvant underuse but may help reduce underuse in SNHs with poor follow-up capabilities. Inability to discern T&F effectiveness is likely due to encountered challenges that inform lessons for future implementation research.

Hymenolepis diminuta adults were killed and fixed in fully extended positions, and the total lengths of the strobilae, numbers of proglottids, and distances of proglottids along the strobilae were determined. The relationship of proglottid number to distance along the strobila was exponential. Beginning at proglottid 100 (P100), the lengths and widths of proglottids at 100 proglottid intervals were determined, and the surfaces areas were calculated. The relationships of proglottid length and width to proglottid number were linear, but the relationship of proglottid number to surface area (SA) was exponential. The volumes of proglottids were calculated, and the relationship of volume (V) to proglottid number was exponential. The relationship of surface area to volume ratio (SAVR) to proglottid number was exponential; at the anterior end of the worm (P100), the SAVR was 14.6, while at the posterior end of the worm (P1300) the ratio was 4.2. A single exponential equation describing the relationships among proglottid number, SA, and V was derived.

Sexual characters may reveal the quality of a potential mate, including the mate's level of infection with parasites. Females that prefer males with low levels of infection or no infection may benefit in several ways. Direct benefits may include avoidance of infection, acquistition of larger nuptial gifts or enhancement in fecundity due to differences in male fertility. Females may also benefit indirectly by producing offspring that are more resistant to infections. We measured female preference for odours produced by male grain beetles, Tenebrio molitor, that were either infected by a tapeworm, Hymenolepis diminuta, or uninfected. This parasite is not transmitted directly between conspecifics. Females were attracted to odours of all males, but they were less attracted to those from parasitized males. To the contrary, females were preferentially attracted to infected females. Males did not show any biased attraction to odours from infected and uninfected male beetles. Females that mated with highly infected males produced fewer offspring than females mated to uninfected males, indicating parasitic infection inflicts multiple costs to males. These results are consistent with models of parasite-mediated sexual selection. Copyright 2000 The Association for the Study of Animal Behaviour.

Pappas, P. W., Barley, A. J., and Wardrop, S. M. 1999. Hymenolepis diminuta: Glucose and glycogen gradients in the adult tapeworm. Experimental Parasitology 91 , 315–326. Adult (20-day-old) Hymenolepis diminuta were cut into 12 pieces of equal length, and the individual pieces of the tapeworm's strobila were analyzed. There was a continuous gradient of decreasing concentrations of glucose (mM) and decreasing levels of glycogen (μg/mg wt) in the strobila. The ethanol extracts of the individual pieces of strobila contained four compounds tentatively identified as disaccharides; the distributions of these compounds were different from those of glucose and glycogen. The distributions of glucose, glycogen, and the “disaccharides” changed when tapeworms were incubated for 1 h in saline or glucose. When compared, on a per-weight basis, to the most posterior sections of the strobila, the anterior sections absorbed more glucose and incorporated more glucose into glycogen. There was a continuous gradient of decreasing values along the tapeworm's strobila of the V max for glucose uptake, while the K t values for glucose uptake changed only slightly. The data indicate that glucose and glycogen metabolism are most active in the anterior part of the tapeworm's strobila where new proglottids are produced and the initial stages of organogenesis occur.

SUMMARYWhen incubatedin vitrofor 24 h, oncospheres ofHymenolepis diminutaabsorb and metabolize radioactive glucose. Between 0 and 12 h post-activation, oncospheres absorb glucose, but glucose is neither metabolized into other carbohydrates nor incorporated into the ethanol-precipitable fraction (which would contain glycogen). Between 12 and 24 h post-activation glucose is incorporated into a number of higher molecular weight carbohydrates that are demonstrable in ethanol extracts of the larvae, as well as the incubation media. Furthermore, measurable amounts of radioactivity are incorporated into the ethanol-precipitable carbohydrate fraction of oncospheres. To determine if these temporal changes in carbohydrate metabolism occurred spontaneously following activation, oncospheres were pre-incubated for 12 h (0–12 h post-activation) in the absence or presence of glucose, and then transferred to media containing radioactive glucose for an additional 12 h (12–24 h post-activation). In these latter experiments, glucose absorption and metabolism between 12 and 24 h post-activation were virtually identical to glucose metabolism in oncospheres that were incubated in radioactive glucose for 0–12 h immediately following activation. Thus, these data do not support the hypothesis that the temporal shift in carbohydrate metabolism occurs spontaneously.

SUMMARYWhen incubatedin vitrofor 24 h, intact eggs, chemically shelled eggs (obtained by treating intact eggs with NaOCl), activated larvae (eggs in which the outer shell and inner envelope were removed), and oncospheres (activated larvae treated with papain to remove the embryophore) absorb and metabolize radioactive glucose. Intact eggs, which are covered by the impermeable shell, absorb only small amounts of exogenous radioactive glucose, while chemically shelled eggs, activated larvae, and oncospheres absorb much larger amounts. Only very small amounts of the exogenous glucose are incorporated into the ethanol-precipitable carbohydrate fraction (which would include glycogen) by any of the preparations of eggs/larvae. However, the glucose is incorporated into higher molecular weight end-products that are liberated into the incubation medium. There is a temporal shift in the ability of activated larvae and oncospheres to metabolize exogenous glucose. Activated larvae and oncospheres absorb but do not metabolize glucose during the first 8 h post-activation. Between 8 and 16 h post-activation, however, virtually all of the absorbed glucose is metabolized into higher molecular weight end-products that are liberated into the incubation media. This temporal shift suggests that activation of oncospheres and cysticercoid morphogenesis are accompanied by distinct changes in carbohydrate metabolism.

The acid phosphate activity (APA) associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta , hydrolyzed p -nitrophenyl phosphate (PNPP), pyrophosphate ( PP i ), and β-glycerophosphate (βGP). Inhibition of PNPP hydrolysis at pH 4.0 was inhibited in a competitive manner by the following compounds (listed in order of decreasing affinity with their apparent inhibitor constants ( K ′ i )): molybdate (0.031 m M ); PP i (0.147 m M ); NaF (0.150 m M ); o -carboxyphenyl phosphate (0.261 m M ); inorganic phosphate (0.770); arsenate (3.45 m M ); tartrate (22.1 m M ); and βGP (29.8 m M ). Cu 2+ , formaldehyde, and arsenite at 10:1, 80:1, and 200:1 inhibitor to substrate ratios did not inhibit APA. The maximal rate of hydrolysis ( V max ) of each substrate was greater at pH 4.0 than 5.0. The apparent Michaelis constant ( K ′ m ) for PNPP increased from 0.233 to 0.351 m M when the pH was raised from 4.0 to 5.0. The K ′ m for PP i decreased from 0.101 to 0.046 m M , while the K ′ m for βGP changed from 2.04 to 2.22 m M under similar circumstances. APA and alkaline phosphatase activity increased as a function of temperature up to 45 °C.

The strobila of an adult tapeworm represents a continual gradient of developmental stages from immature to gravid proglottids. The purpose of this study was to determine if organogenesis (as measured by the develop mental gradient) in tapeworms within a single host and among different hosts occurred at the same rates. Rats were infected with Hymenolepis diminuta and the tapeworms were recovered 20 days post-infection. The total number of proglottids in each worm was determined, and five “benchmarks” of organogenesis were quantified. The data demonstrated that organogenesis in worms from a single host occurred at a relatively constant rate, but that rates in tapeworms from different hosts were different.

The defensive glands of beetles, Tenebrio molitor, infected with metacestodes (cysticercoids) of Hymenolepis diminuta are everted less frequently upon stimulation, and contain less toluquinone (methylbenzoquinone) and m-cresol, than glands of uninfected controls. These differences, as shown in predation trials with wild rats, increase the likelihood that both cysticercoids and beetles will be ingested by the tapeworm's definitive host. This is the first documented case of a parasite inhibiting the chemical defence of an intermediate host, and one of only a few reports of parasite-induced manipulation of host biology supported by empirical evidence implicating facilitated parasite transmission between host species.

Faecal pellets were collected from uninfected rats and rats infected with Hymenolepis diminuta, and analysed for caloric value, lipids, carbohydrates, glucose, amino acids, and proteins. Faeces from infected rats contained significantly more lipid and less carbohydrate and glucose than faeces from control rats. The total free amino acid content of faeces from infected and control rats did not differ, but there were significant differences in the amounts of individual free amino acids. The data support the hypothesis that the nutrient composition of faeces from infected and control hosts differ. Beetles that serve as the intermediate host for H. diminuta may be able to detect these differences, and this may explain why beetles preferentially ingest faeces containing the tapeworm's eggs.

Benzoquinones were extracted from flour beetles by rinsing them for 8 min in methanol followed by homogenization in fresh methanol. This procedure recovered > 96% of the total benzoquinones from both species, with the methanolic rinse and supernatant representing the "surface" and "total" benzoquinones. As determined by UV/VIS spectrometry and reversed-phase HPLC, the methanolic rinse contained methyl- and ethyl-1,4-benzoquinone (MBQ and EBQ), but no substituted hydroquinones, The methanolic supernatant contained MBQ and EBQ, and methyl- and ethyl-hydroquinone. When stored at -20 degrees C for 72 hr, the chemical composition of the methanolic rinse did not change, but the composition of the methanolic homogenate did change. Notably, the amounts of benzoquinones in the methanolic supernatant decreased during storage. The method described represents an improvement over those described previously in that it uses less hazardous solvents for extraction (methanol vs more non-polar solvents such as hexane) and allows for the differentiation and quantification of the surface and total benzoquinones of these beetles.

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.

SUMMARYThe carbohydrate constituents of whole eggs and fractions derived from the eggs of Hymenolepis diminuta were partly characterized. Whole eggs contained 9·6 ng of carbohydrate (phenol-sulphuric acid-positive material) per egg, and 3·6 ng of glucose. Analysis of hydrolysed eggs by HPLC demonstrated the presence of glucose, galactose, glycerol, N-acetylgalactosamine, and 2-deoxyribose. Isolated egg shells and the KOH-stable, ethanol-precipitable fraction (putative glycogen) of eggs contained 26 and 76%, respectively, of the total carbohydrate associated with eggs. Glucose and galactose were present in both of these samples, but only glucose was present in the KOH-stable, ethanol-precipitable fraction isolated from chemically deshelled eggs; thus, the source of the galactose in the KOH-stable, ethanol-precipitable fraction isolated from whole eggs was the egg-shells. These data confirm earlier reports that the shells contain carbohydrate; the data demonstrate further that only two monosaccharides (glucose and galactose) are present in the shells, and that they are present as high molecular weight polymers. The ethanol-soluble fraction of eggs contained 2·5% of the total carbohydrate in eggs, and glucose accounted for < 1% of the ethanol-soluble carbohydrate. Analysis of the ethanol-soluble fraction by HPLC demonstrated that the predominant monosaccharides were glycerol and mannose, with smaller quantities of 2-deoxyribose and 2-deoxyglucose. The absence of a ‘free pool’ of glucose in eggs, and the presence of large amounts of glycerol and mannose, suggest that the pathways of intermediary carbohydrate metabolism in eggs might be very different from those in adult tapeworms.

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.

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.

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.

An acid phosphatase activity (APA; EC 3.1.3.2) was demonstrated in homogenates of adultHymenolepis diminuta. The APA was soluble based on the observation that it did not sediment at 130 000 g. APA was partially purified using a combination of differential centrifugation, ammonium sulphate precipitation, chloroform extraction, and gel and fast-protein-liquid-chromatography. This combination of techniques resulted in a preparation with a specific activity approximately 500 times greater than the crude enzyme preparation. The temperature and pH optima of the partially purified APA were 44°C and pH 5·0. The enzyme appeared to be a monomer with a molecular weight of approximately 62 000. APA had a higher affinity for a greater activity towards aromatic than aliphatic phosphoesters and phosphoryl transferase activity was demonstrable using 1-butanol and ethylene glycol as acceptors. APA was inhibited significantly by sodium dodecyl sulphate, fluoride, molybdate and tartrate, but CuSO4and Fast Garnet GBC were poor inhibitors. The precise cellular localization and function of this enzyme remains unknown since it possesses characteristics of both cytoplasmic and lysosomal APA's of other organisms.

SUMMARYIn the absence of exogenous divalent cations, the isolated brush-border plasma membrane of Hymenolepis diminuta possesses alkaline phosphatase activity (APA). APA is stimulated in the presence of exogenous Mg2+ and inhibited by low concentrations of Zn2+ or high concentrations of Ca2+, and inhibition of APA by Zn2+ is reversed by both Mg2+ and Ca2+. APA is inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol-bis-(β-aminoethyl ether) N, N′-tetraacetic acid, and 1, 10-phenanthroline in time- and concentration-dependent fashions, with EDTA being the most effective inhibitor. Following treatment with EDTA, APA is restored by Mg2+ and, to a lesser extent, by Ca2+, but not by Zn2+. Thus, APA represents a Mg2+-dependent enzyme that can be partly activated by Ca2+ but only in the absence of Mg2+.

Eggs of the tapeworm, Hymenolepis diminuta, and adults of the beetle, Tenebrio molitor, were incubated at high and low temperatures. The tapeworm's eggs survived at higher and lower temperatures and for longer periods of time than did adult beetles, indicating that the thermal tolerance of the eggs does not limit the parasite's distribution.

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.

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.

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.

SUMMARYPreparations of isolated brush border plasma membrane of Hymenolepis diminuta and H. microstoma possess the following enzymatic activities: alkaline phosphohydrolase (E.C. 3.1.3.1); Type I phosphodiesterase (E.C. 3.1.4.1); ribonuclease (E.C. 3.1.4.22); adenosine triphosphatase (E.C. 3.6.1.3); and 5′-nucleotidase (E.C. 3.1.3.5). The following enzymatic activities could not be demonstrated in either membrane preparation: Type II phosphodiesterase (E.C. 3.1.4.18); cyclic adenosine-3′, 5′-monophosphate phosphodiesterase (E.C. 3.1.4.17); leucine aminopeptidase (E.C. 3.4.11.1); maltase (α-glucosidase; E.C. 3.2.1.20); and lactase (β-galactosidase; E.C. 3.2.1.23). These data generally agree with those of previous studies in which similar membrane-bound enzymes were demonstrated in intact (living) worms.

Hymenolepis diminuta possesses a tegumental ribonuclease (RNase) which hydrolyzes rat liver and degraded yeast RNA. Polyacrylamide gel electrophoresis and gel chromatography of rat liver RNA after incubation with intact worms demonstrated significant hydrolysis of the high molecular weight RNA fractions (28 S and 18 S), with the appearance of fractions of intermediate molecular weight (i.e., between 18 S and 4 S), as well as ethanol-soluble fractions. Hydrolysis of degraded yeast RNA (with a molecular weight of approximately 25,000) yielded a single ethanol-precipitable hydrolysis product, as well as ethanol-soluble hydrolysis products.

Proteins of the isolated brush border membrane of Hymenolepis diminuta were hydrolyzed in vitro by chymotrypsin, papain, pepsin, subtilopeptidase A (= subtilisin Carlsberg), and trypsin. Neither proteolytic nor amidase activity was demonstrable in the isolated membrane using proteinaceous (casein and hemoglobin) or chromogenic (benzoyl-arginine- p -nitroanilide and succinyl-alanyl-alanyl-propyl-phenylalanine p -nitroanilide) substrates, and the membrane preparation did not inhibit the proteolytic and amidase activities of these enzymes. Thus, the isolated tegumental membrane of H. diminuta is not inherently resistant to the action of proteolytic enzymes, and it does not inhibit proteolytic activity. In control incubations containing only buffer, the alkah'ne phosphatase activity of the brush border membrane decreased in a time dependent manner, but in the presence of chymotrypsin, subtilopeptidase A, and trypsin, the membrane retained greater alkaline phosphatase activity (pepsin and papain could not be tested for this effect on alkaline phosphatase activity). A similar time dependent decrease in activity was also noted for each of the proteolytic enzymes in control assays, but subtilopeptidase A and papain retained greater activity in the presence of the isolated membrane preparation when these assays were compared to controls.

The metabolic rate of uninfected Mus musculus (CF-1 strain) at 30 C was 1.668 ± 0.032 ccO2/g/hr (mean ± SE, n = 35). At 2 days postinfection (PI)the metabolic rate of infected mice was 2.64 ± 0.15 ccO2/g/hr (n = 6), or 58% higher than that of uninfected control mice. Between 2 and 8 days PI there was a steady decrease in the metabolic rate of infected mice, and by Day 15 PI the metabolic rates of infected and uninfected mice were the same. Since gross histopathological changes (e.g., fibrosis of the bile duct and liver) in infected mice are not evident until Day 4 or 5 PI, the increased metabolic rate during the early stage of infection may be a direct response of the mouse to excretory (secretory) products of the developing parasite.

The uptake, localization, and fate of tritiated arginine in Haematoloechus medioplexus was studied using autoradiography of paraffin and frozen tissue sections and liquid scintillation counting techniques. Autoradiographic and liquid scintillation studies on ligated and unligated parasites demonstrated that, during in vitro incubation, only the gut epithelium was active in arginine absorption. Autoradiographic studies also showed that arginine was not preferentially assimilated into any organ or tissue studied. Preincubation of parasites in cycloheximide inhibited the initial assimilation of arginine; autoradiography of frozen sections showed that cycloheximide did not inhibit absorption through the gut lining. Iodoacetate and dinitrophenol (DNP) were both effective inhibitors of arginine absorption. Liquid scintillation studies demonstrated that parasites must first be preincubated in iodoacetate for 5 min for this inhibitor to be totally effective. Dinitrophenol was instantaneous in inhibiting arginine absorption; i.e., preincubation was not a prerequisite for inhibition. Apparently the iodoacetate must first have been absorbed to inhibit absorption, while the DNP affected the permeability of cell membranes bordering the gut lumen. Of the arginine assimilated, 94% was incorporated into the protein fraction of these parasites; the remainder was found in the carbohydrate and lipid fractions. Using pulse labeled parasites, it was found that these worms could excrete, or secrete, radioactive compounds. The tegument and/or excretory pore was responsible for the excretion of a radioactive, ninhydrin positive compound, which was probably the original arginine, or a small peptide containing the original arginine, or an amine. These worms also regurgitated a radioactive compound from the gut which was not reactive with ninhydrin. No correlation between total uptake of labeled arginine and length of incubation could be found. This was likely due to the ability of these worms to absorb, as well as excrete, arginine and the end products of arginine metabolism during short-term incubations, or to the exhaustion of the amino acid pool in these worms resulting in cessation of protein synthesis.

1. 1. Anodal polyacrylamide-gel electrophoresis of Drosophila melanogaster larvae , pupae and adults has shown lactate dehydrogenase (LDH) to consist of six, three and six isoenzymes, respectively. 2. 2. In terms of electrophoretic mobility, LDH-6 of adults corresponds to LDH-3 of pupae, and LDH-1 and 4 of adults correspond to LDH-1 and 5 of larvae, respectively. 3. 3. These results suggest the possibility of a dimeric structure for LDH in larvae and pupae, and that different loci are responsible for synthesis of LDH in these two life-cycle stages. 4. 4. A combination of the loci that are active in larvae and pupae may be responsible for LDH synthesis in adults. 5. 5. Dramatic differences were found when the protein and lipoprotein banding patterns of larvae, pupae and adults were compared. 6. 6. These differences were manifest in changes in the number, density and electrophoretic mobility of protein and lipoprotein bands.

When specimens of intact Hymenolepis diminuta were incubated in the presence of α- or β-chymotrypsin, assays for proteolytic activity following removal of the worms showed an inactivation of both enzymes. The amount of inactivation in either case was dependent upon the enzyme concentration, total number of worms (total worm weight) present, period of time worms were incubated with the enzymes, and pH of the assay medium. Inactivation of α- and β-chymotrypsin was independent of available surface area and the presence of polyions, was irreversible, and ceased upon removal of the worms from the medium. Intact worms also inactivited the zymogen, α-chymotrypsinogen A. The data suggest that the inactivation of α- and β-chymotrypsin resembles that previously reported for inactivation of trypsin by intact H. diminuta.

Surface aminopeptidase (APase) in the acanthocephalan, Moniliformis dubius, was studied using leucylleucine, leucylglycylglycine, glycylglycine, alanylalanine, tri-alanine and tetra-alanine as substrates. In the presence of intact worms all peptides, except glycylglycine, were hydrolysed liberating amino acids and/or peptide subunits into the incubation medium. In 5 min incubations with 5mM leucylleucine, 93% of the liberated leucine was absorbed by the worms indicating a kinetic advantage for absorption of the leucine. In media with leucylleucine plus 10 mM methionine as an inhibitor of leucine uptake, the liberated leucine was not absorbed by the worms, but accumulated in the incubation medium. The inhibitory effects of leucylleucine and alanylalanine (or products of hydrolysis) on the uptake of 0·1 mM [14C]leucine in 2 min incubations were examined. Inhibition of [14C]leucine uptake by these peptides was non-linear up to 5 mM inhibitor concentration. Concentrations of either peptide greater than 5 mM did not produce further inhibition. A significant portion of [14C]leucine transport was not inhibited by these peptides. Inclusion of 0·5 mM Pb2+, an APase inhibitor, blocked the inhibition of [14C]leucine uptake by leucylleucine indicating that the inhibition is caused by the liberated leucine alone, and that interaction of the intact dipeptide and the leucine transport system is negligible. It is concluded that the surface membrane of adult worms has APase which can hydrolyse peptides in the ambient medium. The spatial arrangement of the APase and the leucine transport system is such that the hydrolysis of a peptide confers a kinetic advantage for absorption of the liberated amino acids. Encysted or mechanically excysted cystacanth larvae from the haemocoele of the cockroach showed no APase activity. Activation of the APase required a 30 min treatment in lipase, [Na+]taurocholate or other surface active agents. Since the APase in adult worms was unaffected by washing, the data strongly suggest that this enzyme is of parasitic origin.

The uptake of 14C-nicotinamide was linear over the concentration range of 0.025 to 20 mM (Y = 58.1[x] + 0.8; r > 0.99), and the uptake of 0.05 m M 14 C-nicotinamide was not inhibited by unlabeled nicotinamide at concentrations as high as 10 mM (200:1 inhibitor/substrate ratio). Absorption appeared to occur by diffusion only. The uptake of 0.1 m M 14 C-nicotinamide was not linear with respect to time, again indicating a nonmediated mechanism of absorption. Analyses of worm extracts and incubation media after 30 min incubations demonstrated that all the radioactivity remained as labeled nicotinamide. Worms removed from rats which had received intraperitoneal injections of 14C-nicotinamide were found to contain radioactivity. On the basis of comparison of chromatograms of worm extracts with chromatograms of standard nicotinamide derivatives, this radioactivity was present as nicotinamide, nicotinic acid, and two unidentified compounds which did not correspond to nicotinuric acid, 1-methyl nicotinamide, or the reduced forms of nicotinamide adenine dinucleotide or trinucleotide.

The free pool amino acids ofTaenia crassicepsmetacestodes (advanced larvae) were analyzed quantitatively. In addition, the uptake of L-glutamic acid, L-proline, L-phenylalanine, L-lysine, and L-methionine was studied. Proline and glutamic acid absorption followed straight-line kinetics with respect to substrate concentration, and were not inhibited by their own molecular species. Lysine, phenylalanine and methionine were found to enter larvae by a combination of diffusion and mediated processes. Lysine absorption was inhibited only by lysine, arginine and ornithine. Phenylalanine and methionine uptake was not inhibited by lysine or arginine, but was inhibited by several other amino acids. The data suggested the presence of a basic amino acid transport locus and two distinct transport loci for methionine absorption. In addition, both immature and advanced larvae were found to be impermeable to [14C]inulin (M.W.ca.5000). These results are discussed and compared with the results of earlier investigations of protein and amino acid absorption byT. crassicepslarvae.

1. In Na+-free media, 2 min glucose influx(JGi) was sharply reduced. Li+ activated JGi to a small extent in the absence of Na+, while K+, choline and tris did not. When Na+ was varied with tris, choline, or Li+ substituted for deleted Na+, JGi was a hyperbolic function of Na+ concentration. When K+ was substituted, JGi was a linear function of Na+ concentration (Fig. 1). When K+ was varied at a low, constant Na+ concentration, K+ behaved as a fully competitive inhibitor of Na+ activation of JGi (Fig. 2). 2. At various fixed concentrations of Na+, JGi obeyed Michaelis-Menten kinetics. With decreasing Na+, there was a decrease in maximal glucose influx (JGimax) and no significant change in the apparent Michaelis constant (Kt) (Fig. 3, Table 2). Similar results were obtained with fixed glucose concentrations and varying Na+ (Fig. 4, Table 2). 3. Simultaneous determinations of22Na+ and14C-glucose influxes at 15 sec intervals showed that zero order kinetics pertained over a 2 min incubation period (Fig. 5). Influx coupling coefficients (Jnai/JGi) were determined in double labeling experiments with constant Na+ and varying glucose concentrations or with constant glucose and varying Na+. The experimentally determined coefficient was higher than 1.8 indicating a true coefficient of 2 or more. This coefficient was independent of Na+ and glucose concentrations (Fig. 6, Table 3). 4. After 30 min preloading with glucose, worms were incubated for 30 min in media with modified cation compositions containing 10.5 mM glucose. In low Na+, low K+ media, accumulation of glucose continued. In low Na+, high K+ media, there was no change in tissue glucose. In Na+-free, high K+ media, there was a net glucose efflux against a concentration difference (Table 4). Efflux of22Na+ from worms previously equilibrated in 250 mM22Na+ was not affected by the presence of glucose in the external medium. 5. The data are consistent with Crane's Na+-gradient hypothesis for the transport and accumulation of sugars.Calliobothrium is compared with some other Na+-glucose coupled animal systems and the data are discussed in terms of a model showing first order dependence on concentration and a coupling coefficient of 2 or more.

1. Labeled glycerol influx (J G i ) in the cestode,Hymenolepis diminuta, occurs by diffusion and two mediated systems which were distinguished by their sensitivity to Na+ and inhibitors. The Na+-activated system had a maximal glycerol influx (J G i max) of 91.5 μmoles/g ethanol extracted dry wt/hr and an apparent transport constant (K t ) of 0.24 mM; fluxes of glycerol and Na+ did not appear to be coupled. The second transport system, which operated in the absence of external Na+, had aJ G i max of 83.8 μmoles/g ethanol extracted dry wt/hr and aK t of 0.69 mM (Figs. 1 and 2). 2. DL-β-glycerophosphate, glyceraldehyde, monoacetin, and phloretin inhibited both transport systems, while 1, 2-propanediol inhibited only the Na+-dependent system (Fig. 3). 3. The inhibition ofJ G i by DL-β-glycerophosphate was relieved by ammonium molybdate, adenosine triphosphate, adenosine monophosphate and glucose-6-phosphate (Fig. 4, Table 1). These compounds had little or no effect onJ G i indicating that inhibition was produced by glycerol liberated from the hydrolysis of the phosphorylated compound by surface phosphohydrolases of the worm. 4. Li+, K+, Tris and choline did not replace Na+ as an activator ofJ G i (Fig. 7). Glycerol diffusion rates in media where Li+ or K+ were used to replace Na+ were identical; however, replacement of Na+ with Tris lowered the diffusion rate (Table 2). Diffusion-corrected rates forJ G i were the same in Li+-, K+-, and Tris-substituted media, but were significantly lower in choline-substituted media; choline appeared to act as a competitive inhibitor of mediatedJ G i (Fig. 8, Table 2). 5. Phloretin competitively inhibitedJ G i while phlorizin had no effect. The effects of pH on the inhibition by phloretin indicated that the ketonic form, and not the enolic form, of phloretin was responsible for inhibition (Fig. 5).

Adenosine triphosphatase (ATPase; EC 3.6.1.3) and 5′-nucleotidase (5′-NTase; EC 3.1.3.5) activities of the isolated brush border membrane of Hymenolepis diminuta have been studied. The pH optimum for ATPase activity is 7.4, and divalent cations are necessary for maximum activity; no Na+-K+ activated ATPase is present in the isolated brush border membrane. ATPase activity is inhibited by molybdate and phosphorylated monosaccharides, but not by N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB), or fluoride. The pH optimum for 5′-NTase activity is 9.6–10.2, and divalent cations are necessary for maximum activity. 5′-NTase activity is inhibited by molybdate at pH 9.6 and 7.4, and activated by NEM and pCMB and pH 9.6 and 7.4, respectively; fluoride has no effect on 5′-NTase activity. Solubilization of the brush border membrane fraction in 1% sodium dodecyl sulfate has no inhibitory action on either enzyme activity.

SUMMARYSeveral major groups of parasitic helminths (Monogenea, Digenea and Nematoda) possess two surfaces that are potentially absorptive in nature. These are an external surface, a tegument in the platyhelminths and cuticle in the nematodes, and the intestine. This paper discusses the relative contributions of these absorptive surfaces in the nutrition of these parasitic helminths. There are many factors that determine the availability of, and a parasite's ability to absorb nutrients via either of these surfaces, and this review discusses individually some of the more important morphological, physiological and environmental factors affecting the potential nutritional roles of these surfaces. It is clear from such a summary of previous studies that the intestines and teguments (cuticles) of helminth parasites can each serve an important nutritional role. However, insufficient data make it impossible at this time to determine the relative nutritional roles of these surfaces in any single parasitic helminth.

Several compounds were tested as inhibitors of the alkaline phosphatase (AlkPase) activity associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta. Molybdate, arsenate, arsenite and beta-glycerophosphate (BGP) were competitive inhibitors of the hydrolysis of p-nitrophenyl phosphate, while levamisole and clorsulon were uncompetitive and mixed inhibitors, respectively. Molybdate was also a competitive inhibitor of the hydrolysis of BGP and 5'-adenosine monophosphate, and levamisole was an uncompetitive inhibitor of BGP hydrolysis. The apparent inhibitor constants (Ki') for molybdate and levamisole were virtually identical regardless of the substrate, and these data support the hypothesis that the AlkPase activity is represented by a single membrane-bound enzyme with low substrate specificity. Quinacrine, Hg2+, and ethylenediaminetetraacetate were also potent inhibitors of the AlkPase activity, but the mechanisms by which these latter three inhibitors function were not clear.

When Hymenolepis diminuta was preincubated in Na+-free KRT (tris-maleate buffered Krebs-Ringer saline) for varying time intervals, followed by incubation in 14C-glucose in Na+-fee KRT, the influx of glucose in worms was lowered significantly. This effect of Na+ deletion on glucose influx was totally reversible by incubating worms in KRT ([ Na+] = 154 meq/l).When Na+ in the medium was replaced with K+, tris, or choline, a similar decrease in glucose influx in worms was noted; replacement of Na+ with Li+ resulted in a glucose influx rate significantly higher than that obtained with K+, tris, or choline as the replacement cation. In media with a suboptimal Na+ concentration (25 meq/l), influx of 0.5 mM glucose in worms was unaffected by varying concentrations of K+ (0-100 meq/l).Apparent Michaelis-Menten kinetics were observed when glucose influx as a function of glucose concentration was determined in media with Na+ concentrations of 154, 50, 25, and 10 meq/l. There was a slight decrease in the apparent tra...

The membrane-bound and solubilized (using Triton ×-100 or sodium dodecyl sulfate (SDS)) alkaline phosphohydrolase (APase) activities of the isolated brush border membrane of Hymenolepis diminuta require a divalent cation for maximum activity. Highest rates of substrate (p-nitrophenyl phosphate) hydrolysis are obtained with low concentrations of Mg2+ (1 mM), although low concentrations of Mn2+, Ca2+, or Zn2+ will also partially satisfy this requirement; higher concentrations of Mg2+ and Mn2+, and other divalent cations (Cu2+, Fe2+, and Pb2+), inhibit the membrane-bound APase activity. Solubilization of the membrane-bound enzyme in either Triton or SDS results in an increase in specific activity and Km, but has little effect on thermal stability of the APase activity. Phosphate, pyrophosphate, adenosine 5′-triphosphate, adenosine 5′-monophosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate inhibit substrate hydrolysis, and the relative affinities of these inhibitors for the APase enzyme are altered only slightly upon solubilization. Graphic analyses of data from inhibitor studies indicate that all eight inhibitors will inhibit membrane-bound and solubilized APase activities 100% at high inhibitonsubstrate ratios. Molybdate, F−, 2-mercaptoethanol, cysteine, and p-chloromercuribenzoate inhibit membrane-bound APase activity. Inhibitor data indicate that if more than one enzyme is responsible for the APase activity of the brush border membrane of H. diminuta, the enzymes cannot be differentiated on the basis of substrate specificity.

The specific activities of the alkaline phosphatase (APase), type I phosphodiesterase and 5'-nucleotidase activities associated with the brush-border plasma membrane of the tapeworm, Hymenolepis diminuta, decrease significantly as the tapeworm grows and matures. Kinetic analyses of the APase activity associated with membrane preparations from whole 6-, 12-, and 18-d-old H diminuta, and individual pieces of 18-d-old H diminuta cut into ten pieces of equal length, failed to demonstrate qualitative changes in the APase activity. Therefore, the decreased specific activities are apparently due to changes in the ratios of enzymatically active to enzymatically inactive membrane proteins (ie, quantitative changes in the membrane proteins) which occur as the tapeworm grows.

The histological host response of female CF-1 mice to Hymenolepis microstoma involves dramatic changes in the host's bile duct and liver. These changes are similar to those described by previous investigators using different strains of mice. The histological changes which occur in bile duct and liver tissues of female CF-1 mice are accompanied by a significant hypertrophy of these organs; the wet weights of the bile duct and liver increase almost 2000 and 50%, respectively, in infected mice. No significant histopathological changes are found in the spleen or small intestine tissues of infected mice, yet the wet weight of both of these organs increases almost 100%. The magnitude of the histopathological changes which occur in the bile duct could possibly account for the increase in wet weight, i.e., there is significant fibrosis, but the histological changes in the liver, spleen, and small intestine do not appear to be of sufficient magnitude to account for the increases in wet weight.

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.

The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hy-drolyzes p-nitrophenyl phosphate over a broad pH range. Acid phosphatasc activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2-mercaptoethanol, and ethylenediaminetetra-acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X-100, while such treatment causes a significant loss of acid phosphalase activity. Both activities are nonspecific and hydrolyze a variety of phos-phorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.

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.

Under the described experimental conditions, lactate dehydrogenase (LDH) of Helisoma antrosum is characterized as follows: 1. 1. At low substrate (lithium lactate) concentrations (0·01–0·03 M), LDH of whole snail homogenates appears to display hyperbolic kinetics. At higher substrate concentrations (0·04–0·16 M), LDH displays sigmoid kinetics. 2. 2. LDH exists as a multiple molecular form displaying five isoenzymes. 3. 3. A Hill plot of the data for whole snail homogenates indicates that at least one of the LDH isoenzymes possesses at least two interacting binding sites. 4. 4. Isoelectric focusing demonstrated four molecular forms of LDH, the isoelectric points (pI) of these forms being 3·8, 5·2, 6·2 and 8·5. 5. 5. The Michaelis constant (apparent Km) of the LDH from whole snail homogenates is 5·5 × 10−2 M. 6. 6. LDH-4, and possibly LDH-5, correspond to a thick band which had been previously demonstrated with amino black (protein stain) and Ponceau red (lipoprotein stain). 7. 7. LDH-4 is the predominant band in acrylamide gels.

The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l -amino acids than d -amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l -methionine uptake by larvae is inhibited more by d -alanine and d -valine than by their respective l -isomers, while d - and l -methionine inhibit l -methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na + -insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na + . Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.

1. 1. After incubation in tritiated arginine for one and two hour periods, autoradiography was used to demonstrate the presence of a radioactive, water insoluble excretory product in the excretory system of Cephalogonimusamericanus. 2. 2. The probable composition of this compound is discussed.