Correlation between lipid partition coefficients and surface permeation inSchistosoma japonicum

Summary Comparison of transintegumental membrane permeability and partition coefficients of selected nonelectrolytes was attempted to correlate the parameters of lipid solubility, hydrophilicity, and membrane permeation in male and female schistosomes (parasites of the portal venous tributaries of man). Surface permation (measured by the triple isotope technique) and octanol/water partition coefficients were determined for 17 compounds (acetamide, aminopyrine, antipyrine, benzyl alcohol, butanol, caffeine, ethanol, ethylene glycol, glycerol, inosine, mannitol, methanol, polyethylene glycol, propylene glycol, sucrose, thiourea, and urea).Linear regression analyses comparing the logarithm of the partition coefficient to transintegumental uptakes indicate a positive correlation in both sexes:R=0.76 (P<0.001) for males, andR=0.77 (P<0.001) for females. Similarly, linear regression analyses comparing hydrogen bond number with the logarithm of tissue uptake index demonstrate a high (negative) correlation in both males (R=–0.85,P<0.001) and females (R=–0.90,P<0.001). The male and female schistosomes showed no statistically significant differences in correlation of these parameters. Surface permeation was the same in male and female schistosomes, suggesting that male-female variations in integumental uptake rates previously observed may be restricted to metabolites which enter by way of a selective carrier system.     

Schistosoma mansoni, S. japonicum, S. haematobium: glycogen content and glucose uptake in parasites from fasted and control hosts

The glycogen content of male and female Schistosoma mansoni has been measured in flukes from normally fed hosts and those from fasted hosts. In infections from both the mouse and the hamster, a significant reduction in schistosomal glycogen of males is seen hours after food is withdrawn from the host. Reductions in protein content of the schistosomes were only observed in hamster infections fasted at least 72 hr. The livers of infected mice not only decrease in size during fasting, but there is a concomitant reduction in glycogen per unit wet weight. Comparisons of glycogen:protein ratios of mansonian males, females, and host livers indicate that the fasting-induced loss of liver glycogen is also observed in the male schistosome, but not the female. Studies of both S. mansoni and S. haematobium pairs from fed hosts suggest that the ratio of glycogen:protein contents in the male schistosome correlates with the glycogen:protein ratio of the female partner. Measurements of glucose uptake in vitro suggest that greater uptake rates may be observed in flukes perfused from fasted hosts. In S. japonicum from infected mice, a reduction in male glycogen was also detected as early as after a 6-hr fasting period, but changes in the females were not significant. Unmated male S. japonicum also exhibit a reduction in glycogen levels after fasting, but the quantity of worm glycogen present in these males remains higher than comparable mated males. In mice entrained to a regulated pattern of available food, fluctuations in glycogen content of the male schistosomes were observed, but in the female partners fluctuations were of a smaller magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tegumental glucose permeability in male and female Schistosoma mansoni

Tegumental hexose transporters have been kinetically characterized in mated and separated male and female Schistosoma mansoni 8-12 wk postinfection. Significant gender-specific differences in Km and Vmax were observed. In mated males, the estimated constants (mean +/- SE) were: Km = 0.63 +/- 0.31 mM, Vmax = 0.93 +/- 0.44 nmol/mg worm water/min, and the Kd = 0.25 +/- 0.09 microliter/mg worm water/min. In mated females the kinetics were: Km = 0.99 +/- 0.40 mM, Vmax = 1.22 +/- 0.42 nmol/mg worm water/min, and Kd = 0.60 +/- 0.14 microliter/mg worm water/min. The influx of 2-deoxy-D-glucose and 3-O-methylglucose has been similarly characterized; these analogs share the same glucose transporter in male and female schistosomes. 2-Deoxy-D-glucose has a higher affinity, and 3-O-methylglucose a lower affinity, than does glucose. Because mated male schistosomes supply glucose to female partners, similarities between the free glucose concentration of the male and the affinity of the transporter determined for mated female schistosomes suggest that male-to-female transfer may be a potentially rate-limiting step in glucose utilization by the female. Permeability x surface are (PS) products and Vmax/Km ratios were significantly elevated in mated schistosomes, suggesting that the transporter is primarily localized to the dorsal surface of the male. Gender- and mating-specific analyses of PS products indicate that tegumental permeability to glucose is significantly increased in mated schistosomes, and compares very favorably to that of the host liver.     

Schistosoma mansoni: Correlation between lipid partition coefficients and the transintegumental uptake of nonelectrolytes

Comparison of transintegumental membrane permeability and partition coefficients of selected nonelectrolytes attempted to correlate the parameters of lipid solubility and membrane permeation in male and female Schistosoma mansoni. Transintegumental uptakes and octanol:water partition coefficients were determined for 10 nonelectrolytes (acetamide, antipyrine, benzyl alcohol, caffeine, ethanol, ethylene glycol, propylene glycol, sucrose, thiourea, and urea). Linear regression analyses comparing the logarithm of the partition coefficient to the transintegumental uptakes yielded values of R = 0.80 (P < 0.001) for males, R = 0.84 (P < 0.001) for females, and R = 0.82 (P < 0.001) for a combined analysis of males and females. The male and female schistosomes showed no statistically significant differences in correlation of these parameters. The evidence, then, suggests that the multilaminate membrane functions in a way similar to the function of a lipid bilayer with regard to the parameters studied.     

Schistosoma mansoni: Uptake in vitro and incorporation of adenine by adults

The rates of adenine uptake and incorporation into nucleic acids by adult male and female Schistosoma mansoni were determined during periods of up to 10 days in vitro, and comparisons were made between paired and separated worms. Adenine uptake by separated males and females exceeded that exhibited by equivalent paired worms. The rate of incorporation of adenine into nucleic acids was higher in separated females than in paired females. In contrast, the state of pairing had little effect on adenine incorporation by male S. mansoni. There was no correlation between rates of adenine uptake and incorporation and the reproductive activity of S. mansoni adults in vitro. Uptake and incorporation rates appeared to reflect the changing somatic requirements of both male and female worms.     

Comparative glucose utilization rates in separated and mated schistosomes

The rate of phosphorylation of 2-deoxyglucose (2DG) was determined by sequential pulsing of schistosomes (Schistosoma mansoni, S. japonicum, and S. haematobium) with 3H- and 14C-labeled 2-deoxy-D-glucose. Subsequent column chromatographic separation of the neutral [3H]2DG and [14C]2DG from the 3H- and 14C-labeled 2-deoxy-D-glucose 6-phosphate permitted estimation of the quantity of [3H]2DG phosphorylated in 2 min, and the proportion of [14C]2DG phosphorylated in 1 min; thus a phosphorylation rate was determined from a single tissue sample. The relative phosphorylation rate of 2-[3H]2DG to D-1-[14C]glucose (i.e., the phosphorylation coefficient) was also measured in male and female schistosomes. It was demonstrated that even though 2DG is taken up more rapidly than glucose, it is phosphorylated at a much slower rate in both S. mansoni and S. japonicum. In both of these species, mated males phosphorylate 2DG and glucose at a greater rate than do unmated males. Similarly, mated females phosphorylate and consume more glucose than do separated females. In contrast, the phosphorylation coefficient is greater in separated than in mated schistosomes. Intraspecific comparisons suggest that, at reduced substrate concentrations, glucose utilization rates are higher in S. japonicum, intermediate in S. mansoni, and lower in S. haematobium.     

Schistosoma mansoni: influence of the female parasite on glutathione biosynthesis in the male

The glutathione (GSH) content of male Schistosoma mansoni increases in the absence of the female. This phenomenon, originally observed in vitro, also occurs within the host. At the time of recovery from mice, the GSH content of males from single-sex infections was 1.7-fold higher than that of paired males from mixed sex infections (P less than 0.01). The effect of mating status on male GSH biosynthetic and turnover rates was examined to determine the basis for increased GSH content in unpaired males. GSH turnover rates, measured when GSH biosynthesis was inhibited by greater than 95% with 5.0 mM DL-buthionine-SR-sulfoximine, were indistinguishable between unpaired and paired males with a first-order rate constant of 0.018 hr-1. In contrast, incorporation of L-[35S]cysteine into GSH revealed that GSH biosynthesis was 5-fold higher in unpaired than in paired males. Transport of L-cystine into male schistosomes, the presumed rate-limiting step in GSH biosynthesis, was unaffected by mating status. The GSH content increased when males were incubated in medium that had previously contained females or when separated from females by a microporous membrane. Males paired to 50% ethanol-fixed females had unchanged GSH content in vitro. It appears that male GSH biosynthesis may be regulated by a response stimulated by the female's physical presence in the gynechophoral canal and not by a soluble factor released from the female.     

Sugar transport and metabolism in Schistosoma mansoni.

The absorption kinetics of some 14-C-labeled simple sugards in adults of Schistosoma mansoni are described. The influx of fructose and 3-0-methylglucose was by diffusion alone, while glucose, 2-deoxyglucose (2DOG), galactose, glucosamine, and mannose were absorbed by mediated transport as well as by diffusion. Although absorbed glucose was rapidly metabolized, uptake rates of radio-glucose in 2-min incubations corresponded with the amount of glucose (determined chemically) removed from the incubation medium. In 30-min incubations 2DOG was slowly metabolized and accumulated against an apparent concentration difference. The mediated transport of glucose and 2DOG was inhibited in Na+-free media, and by the presence of ouabain, phlorizin, phloretin, and other sugars. Accordingly, influxes of glucose of 2DOG and 22-Na+ were coupled. On a per mg protein basis, female worms transported more 2DOG and glucose, but less glycine, than did males. However, the rate of glucose metabolism by male and female worms incubated together was greater than that of either males or females incubated separately. The nature of sugar transport in schistosomes and other flatworms is similar to that in vertebrates.     

Glucose transport in Acholeplasma laidlawii B: dependence on the fluidity and physical state of membrane lipids.

The uptake of D-glucose by Acholeplasma laidlawii B occurs via a mediated transport process, as shown by the following observations: (i) glucose permeates A. laidlawii B cells at a rate at least 100 times greater than would be expected if its entry occurred only by simple passive diffusion; (ii) the apparent activation energy for glucose uptake in A. laidlawii is significantly lower than that expected and observed for the passive permeation of this sugar; (iii) glucose uptake appears to be a saturable process; (iv) glucose uptake can be completely inhibited by low concentrations of phloretin and phlorizin; and (v) glucose uptake is markedly inhibited at temperatures above 45 C, whereas the passive entry of erythritol continues to increase logarithmically until at least 60 C. The metabolism of D-glucose by this organism is rapid and, at low glucose concentrations, the intracellular radioactivity derived from D-[14-C]glucose is at any given time a reflection of the net effect of glucose transport, glucose metabolism, and loss from the cell of radioactive metabolic products. Care must thus be taken when attempting to determine the rate of glucose transport by measuring the accumulation by the cells of the total radioactivity derived from D-[14-C]glucose. The rate of uptake of D-glucose by A. laidlawii B cells is markedly dependent on the fatty acid composition and cholesterol content of the plasma membrane and exhibits a direct dependence on the fluidity of the membrane lipids as measured by their reversible, thermotropic gel to liquie-crystalline phase transition temperatures. In contrast to the transport rates, the apparent activation energy for glucose uptake above the phase transition temperature is not dependent on membrane lipid composition. At the temperature range within the membrane lipid phase transition region, the apparent activation energy of glucose uptake is different from the activation energy observed at temperatures above the phase transition. This may reflect the superimposed operation within the phase transition region of more than one temperature-dependent process.     

The effect of maintenance in vitro on glucose uptake and the incorporation of glucose into glycogen by adult Schistosoma mansoni

The effect of maintenance in vitro on glucose uptake and the incorporation of glucose into glycogen by adult Schistosoma mansoni. International Journal for Parasitology16: 253–261. Adult male Schistosoma mansoni rapidly depleted their glycogen reserves in vitro. Both sexes also exhibited a gradual reduction in glycogen content during prolonged maintenance. Paired and separated worms were incubated in [³H] glucose and rates of glucose uptake and incorporation into glycogen were determined following periods of maintenance in vitro. The glucose uptake rate declined during long-term maintenance and was higher for separated males and females than for equivalent paired worms. Increasing the medium glucose concentration also increased the rate of uptake. Glucose continued to be incorporated into glycogen throughout 10 days in vitro, with evidence from paired schistosomes suggesting that the rapid depletion of male glycogen could be due to a decrease in incorporation rate in vitro. The incubation of separated worms and the use of higher glucose concentrations in media both effected an increase in incorporation rate. These results are discussed in the light of observations of the depletion of schistosome glycogen in vitro.     

Polarity of transport of 2-deoxy-D-glucose and D-glucose by cultured renal epithelia (LLC-PK1).

At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.6 mM) and 1 mM 2dglc (Km 1.5 mM) were greater than apical uptake rates and the (apical-to-basolateral)/(basolateral-to-apical) flux ratio was high for glucose (9.4) and low for 2dglc (0.8), thus, confirming the lack of interaction of 2dglc with the apical SGLT. Specific glucose transport inhibitor studies using phlorizin, phloretin and cytochalasin B confirmed the polarised distribution of SGLT and GLUT in LLC-PK1 cells. Basolateral sugar uptake could be altered by addition of insulin (1 mU/ml) which increased 2dglc uptake by 72% and glucose uptake by 50% and by addition of 20 mM glucose to the medium during cell culture which decreased 2dglc uptake capacity at confluence by 30%. During growth to confluence, 2dglc uptake increased to a maximum, then decreased at the time of confluence, coincident with a rise in uptake capacity for alpha-methyl-D-glucoside, a hexose that interacts only with the apical SGLT. It was concluded that the non-metabolisable sugar 2dglc was a useful, specific probe for GLUT in LLC-PK1 cells and that GLUT was localised at the basolateral membrane after confluence.     

Schistosoma mansoni: nucleic acid synthesis in immature females from single-sex infections, paired in vitro with intact males and male segments

1. The object of this study was to see whether stimulation of nucleic acid synthesis in immature females by male Schistosoma mansoni is mediated locally by contact, or is propagated systemically in the female. 2. Immature females perfused from single-sex animal infections were paired for one week in vitro with segments of males cut transversely into thirds; others were paired with intact males, or maintained without males; all were then incubated with [3H]-thymidine or tyrosine. 3. Washed females were bisected transversely and isotope uptake counted separately in the anterior and posterior halves. 4. The halves in contact with cut male segments showed significantly higher uptake of [3H]-thymidine than the non-contact halves, indicating increased DNA synthesis and cell division, but non-contact halves had greater uptake of [3H]-tyrosine. 5. Dot-blot hybridization with a female specific single stranded cDNA failed to detect production of the corresponding mRNA in females paired with male segments.     

A genetically diabetic model "KK-CAy mice" for a pharmacological assay.

A genetically diabetic model, KK-CAy mice which were bred by mating female KK mice (aa, BB, cc) with male KK-CAy mice (Aya, BB, CC) was studied on the usefulness as a tool for a pharmacological assay. Body weights of KK-CAy mice increased more rapidly than those of control mice, KK-C. When the body weights of male KK-CAy mice reached about 30 g 10 weeks after birth, their blood glucose levels increased. Severe hyperglycemia (over 300 mg/100 ml) was often observed in the males, but not in the females. Glucose tolerance in the KK-CAy mice was more markedly impaired than that in the control mice. The increase in blood FFA level correlated with the increase in body weight on both KK-CAy mice and the controls. On hyperinsulinemia observed, the ratio of plasma immunoreactive insulin (IRI) level to blood glucose level in the male mice was lower than that seen in the female mice. On hyperglucagonemia observed, elevation of plasma immunoreactive glucagon (IRG) was more remarkable in the males than in the females. Morphological study showed insular degranulation only in the males. Since the dose-dependent insulin-induced falling was observed on blood glucose level in nonfasted KK-CAy mice, they could be used as a feasible tool for an assay of antidiabetic drugs.     

Diet-induced obesity increases NF-κB signaling in reporter mice

The nuclear factor (NF)-κB is a primary regulator of inflammatory responses and may be linked to pathology associated with obesity. We investigated the progression of NF-κB activity during a 12-week feeding period on a high-fat diet (HFD) or a low-fat diet (LFD) using NF-κB luciferase reporter mice. In vivo imaging of luciferase activity showed that NF-κB activity was higher in the HFD mice compared with LFD-fed mice. Thorax region of HFD females displayed fourfold higher activity compared with LFD females, while no such increase was evident in males. In male HFD mice, abdominal NF-κB activity was increased twofold compared with the LFD males, while females had unchanged NF-κB activity in the abdomen by HFD. HFD males, but not females, exhibited evident glucose intolerance during the study. In conclusion, HFD increased NF-κB activity in both female and male mice. However, HFD differentially increased activity in males and females. The moderate increase in abdomen of male mice may be linked to glucose intolerance.     

Sex-dependent antioxidant enzyme activities and lipid peroxidation in ageing mouse brain

We investigated whether oxidant status and antioxidant enzyme activities during ageing of mouse brain are regulated in sex-dependent manner. In the homogenate from the brain of 1, 4, 10 and 18 months old male and female CBA mice, lipid peroxidation (LPO), total superoxide dismutase (tSOD), catalase (CAT) and glutathione peroxidase (Gpx) were determined. LPO was age- and sex-related, favoring males over females throughout the lifespan with the peak in both sexes at 10 months of age. Throughout ageing, no difference in tSOD activity between male and female brains was observed, except in immature 1 month old mice. Gender-related difference in Gpx activity was observed, with higher level in females comparing to males, reaching statistical significance in senescent (18 months old) animals. CAT activity was drastically changed with ageing in both the male and female brain. We found different age associated trends in CAT activity in males and females: decreased with age in males and increased with age in females. Taken together, the present findings indicate that brains of female mice have lower oxidant and higher antioxidant capacity mostly related to CAT and to a lesser extent to Gpx activity.     

Kinetics of Cerebral Uptake Processes In Vitro of L-Glutamine, Branched-Chain L-Amino Acids, and L-Phenylalanine: Effects of Ouabain

Abstract: Estimates have been made of the amounts and rates of uptake of radioactive branched-chain i-amino acids, L-phenylalanine, and L-glutamine into incubated rat brain cortex slices. Estimates have also been made of the binding of these amino acids to brain cell fragments. It is shown that such binding, as well as the process of passive diffusion, is not affected by the presence of ouabain (0.2 mM), which suppresses the energy-dependent concentrative uptakes of the amino acids investigated. The maximum specific binding of L-glutamine is about three times that of the other amino acids and amounts to about 11% of the total uptake of the amino acid by rat brain cortex slices in 12 min from a medium containing 0.25 mM-glutamine. The sodium-ion concentration of the medium appears not to play a significant role in determining the rate of L-glutamine uptake in brain slices except at relatively low concentrations (<20 mequiv./l). The presence of Na⁺, however, is essential for the attainment of a tissue-to-medium concentration ratio greater than 2.0 for L-glutamine. At relatively low concentrations (0.25 mM) the rapidity of uptake of L-glutamine into a suspension of nerve terminals exceeds that into brain cortex slices. The uptakes of L-glutamine (K_m's = 0.66 mM and 2.25 mM) and of the branched chain L-amino acids (K_m's approx. 0.3 mM and 2 mM) by rat brain cortex slices are characterized by a double affinity system, but that of L-phenylalanine has only one affinity system (K_m= 0.23 mM). The K_m's have been calculated after subtracting the ouabain-insensitive passive uptakes of the amino acids from the total observed uptakes.     

Schistosoma mansoni: sex-specific modulation of parasite growth by host immune signals

Development of female schistosomes from infectious cercariae to mature egg-producing adults requires both male schistosomes and an intact adaptive immune system. By examining single sex infections in immunodeficient mice, we provide evidence that female schistosome development is not directly influenced by the adaptive immune system, whereas male development is. Our data are consistent with a sequential model of schistosome development, where the adaptive immune system signals development of mature males, which subsequently stimulate development of mature females. The male schistosome therefore appears to play a central role both in transducing signals from the adaptive immune system and in facilitating female development.     

Effect of insulin on glucose transport and metabolism in isolated fat-cells of gonadal adipose tissue from mature age-matched male and female rats.

Insulin action on glucose transport and metabolism was studied in paraovarian adipocytes from 3-month-old female rats and compared with insulin action in epididymal adipocytes from closely age-matched males. At maximal insulin concentrations the stimulations of 2-deoxyglucose uptake (4-fold the basal value) and of [U-14C]glucose incorporation into CO2 and total lipids (3- and 2-fold the basal values respectively) were similar in adipocytes from rats of both sexes. At submaximal insulin concentrations (less than 0.2 nM) the ability of paraovarian adipocytes to transport and to metabolize glucose was higher than that of epididymal adipocytes; accordingly an increase in insulin binding was observed in paraovarian adipocytes as compared with epididymal adipocytes. These results show that paraovarian adipocytes from mature female rats were highly responsive to insulin, and exhibited a higher sensitivity to the hormone than did epididymal adipocytes from male rats of the same age.     

Metabolism of alpha-D-[1,2-13C]glucose pentaacetate and alpha-D-glucose penta[2-13C]acetate in rat hepatocytes

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.