Schistosoma mansoni: female-dependent lipid secretion in males and corresponding changes in lipase activity

Eight to 10-week old Schistosoma mansoni males from unisexual infections were examined histochemically for neutral lipids and lipase activity. In addition, in situ fixed pairs were examined for lipase activity. Neutral lipid content of males from unisexual infections was variable and lipase activity was minimal. Following 1 h incubation at 37 degrees C in Earle's balanced salt solution (EBSS) in which females had previously been maintained for 1 h, males showed moderate increase in lipid content and diminished lipase activity. In contrast, unisexually developed males incubated for 1 h with females from bisexual infections showed increased lipid accumulation and lipase activity. Unisexually developed males incubated for 1 h in EBSS showed both lipid accumulation and release from the dorsal surface. Worm-pairs fixed in situ showed greater lipase activity in females than in males. These observations suggest that a factor(s) released by females affects the physiology of males.     

Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro

Haseeb M. A., Eveland L. K. and Fried B. 1984. Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro. International Journal for Parasitology14: 83–88. Schistosoma mansoni male and female adults were incubated at 37°C for 0.5 and 1.0 h in Earle's balanced salt solution containing 0.1% glucose and 0.5% lactalbumin hydrolysate, then examined by histochemistry and scanning electron microscopy. Histochemical analysis of cryostat sections stained with Oil Red O showed that males contain neutral lipid mainly in the parenchyma and tubercles, while females contain neutral lipid in the vitellaria. Neutral lipids are released from the tubercles of both paired and unpaired males maintained in vitro. There is evidence of in situ lipid transfer from males to blood vessel walls. Neutral lipid was not seen in females from unisexual infections. Sudan Black B staining fo total lipids is positive in tubercles, parenchyma, and vitellaria. Nile Blue Sulphate stains acidic lipids in male caecal walls. Scanning electron microscopy reveals no tegumental damage.     

The uptake, localization and transfer of [4-14C]cholesterol in Schistosoma mansoni males and females maintained in vitro

Schistosoma mansoni male and female adults incorporated [4-14C]cholesterol in vitro. Males incorporated about three times more cholesterol than females. The major sites of cholesterol deposition in males were the tegument and parenchyma. The tegument and vitelline glands were the primary sites of cholesterol accumulation in females. In males, dorsal tegument showed greater cholesterol uptake than ventral tegument. Radiolabelled males and females transferred cholesterol to unlabelled members of the opposite sex.     

Transmission electron microscopic observations on lipid release in Schistosoma mansoni maintained in vitro

Transmission electron microscopy was done on Schistosoma mansoni adult males and females unpaired immediately following recovery from mice and on worms maintained in Earle's balanced salt solution (EBSS) containing 0.1% glucose and 0.5% lactalbumin hydrolysate for 1.0 h at 37°C. Males incubated for 0.5 and 1.0 h released lipids from non-tuberculate and tuberculate areas of the dorsal tegument. Lipid release was not seen in females. Transmission electron microscopy revealed no apparent damage in worms maintained in BBSS for 1.0 h. Our observations suggest that structures described as “spheres” (Weisberg, Carlisle &; Bentley, 1983) are lipid droplets.     

Changes in ornithine decarboxylase activity in response to temperature stress and stimulation of juvenile hormone in Anastrepha fraterculus (Diptera, Tephritidae)

Ornithine decarboxylase (ODC) activity was analyzed in Anastrepha fraterculus (Diptera) females (4 days old) submitted to temperature stress (6 degrees C and 20/6 degrees C) and the topical application of juvenile hormone (JH). ODC activity and ejaculatory apodeme measurements (length and width) were made in males (15 days old) after 6 degrees C stress. JH dose of 500 ng and incubation of 3, 7, and 18 h increased ODC activity. Females reared at 6 degrees C and 20/6 degrees C had higher ODC activity than those reared at 25 degrees C. The treatment of 6 degrees C and JH incubation for 1 h increased ODC activity when compared to 6 degrees C treatments only. However, the treatment of 20/6 degrees C only after 3 or 18 h of JH incubation resulted in higher ODC activity than controls (20/6 degrees C) or 20/6 degrees C plus 1 h of JH incubation. Males did not undergo differences in ODC activity when reared at 6 degrees C or 25 degrees C but the ejaculatory apodeme measurements was higher in those reared at 25 degrees C than in those reared at 6 degrees C. The results can be considered an adaptive process to environmental changes.     

Properties of intracellular ribonuclease utilized for RNA reduction in disintegrated cells of Saccharomyces cerevisiae.

The properties of intracellular RNase in disintegrated cell suspensions of Saccharomyces cerevisiae have been studied. The influence of salt addition and/or incubation of the suspension on the activity of RNase and on the degradation of endogenous RNA was determined. No significant change in the RNase activity in the disintegrated suspensions was obtained by addition of 3% NaCl or by incubation at 50 degrees C with 3% NaCl. During the incubation with NaCl the active RNase was able to degrade endogenous RNA. By incubation without salt the RNase was inactivated. Inactivation also occurred after extraction at alkaline pH. The RNase had an optima at pH 5-6 and temperatures between 50-60 degrees C. The main part of the RNase in the unincubated suspension was soluble also at pH 4.0. No serious protein degradation occurred during the short time incubation needed for RNA reduction. 70% of the protein in the suspensions was recovered in the precipitate at pH 4.0 after 20 min of incubation. The corresponding protein recovery from unincubated suspensions was 77%.     

Lipoprotein lipase in rat lung. The effect of fasting.

We measured lipoprotein lipase activity in dried defatted preparations of rat lung using doubly labeled chylomicron triglyceride as substrate. The enzyme activity was linear for the first hour of incubation at 37 degrees C, had a pH optimum of 8.1 and was completely inhibited by 0.5 M NaC1. Lungs from fed rats hydrolyzed chylomicron triglyceride at a rate of 13.00 mumoles/g per h; the activity rate was unchanged by fasting 8-72 h. Heparin infusion into isolated lungs caused immediate release of lipoprotein lipase to the venous effluent. The activity released was equivalent to about 10% of total lung lipoprotein lipase activity in both fed and fasted rats. Since the ability to remove blood triglyceride is directly related to the level of lipoprotein lipase activity, these findings indicate that the lung is one of the few tissues able to remove efficiently blood triglyceride during fasting.     

Lipoprotein lipase activity of rat cardiac muscle. Changes in the enzyme activity during incubations of isolated cardiac-muscle cells in vitro.

1. Isolated cardiac-muscle cells from the hearts of adult rats were shown to retain a high amount of viability during 4 h of incubation when viability was assessed by Trypan Bue stain exclusion and intracellular enzyme leakage. 2. The cells also retained their ability to take up O2 and utilize added substrates over the period of incubation at both 25 and 30 degrees C. 3. When cells from the hearts of fed rats were incubated in a buffered-salts solution at pH 7.4 in the presence of amino acids and heparin, lipoprotein lipase activity in the medium increased progressively. 4. During these incubations the intracellular activity of the enzyme remained constant and the total activity of lipoprotein lipase in the system (cells plus medium) increased by 80% over the 4 h of incubation at 25 degrees C. 5. In the absence of heparin only low amounts of enzyme activity were detectable in the medium and the total lipoprotein lipase activity in the system remained constant. 6. The measurement of lipoprotein lipase activity in either fresh homogenates of the cells or in homogenates of acetone/diethyl ether-dried powders of the cells had no effect on the overall pattern of activity change during the incubations, although as reported previously the total activity detected with acetone/diethyl either-dried preparations was approx. 3-fold higher than with fresh cell homogenates. 7. The observations were compared with published data on lipoprotein lipase activity changes in neonatal heart cell cultures maintained in vitro.     

Laundry detergent compatibility of the alkaline protease from Bacillus cereus

The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C.     

Esterification of cholesterol in high density lipoprotein decreases its ability to support ACTH-stimulated steroidogenesis by rat adrenocortical cells

Addition of high density lipoprotein 3 (HDL3) isolated from human plasma of d greater than 1.125 g/ml which had been preincubated for 24 h at 37 degrees C enhanced steroidogenesis by cultured rat adrenal cells only 38% as well as HDL3 isolated from unincubated plasma. Loss of steroidogenic activity due to preincubation was associated with a decrease in the percent HDL3 cholesterol remaining unesterified. Inhibition of lecithin-cholesterol acyltransferase activity by heating (60 degrees C, 1 h) or addition of dithionitrobenzoic acid (1.4 mM) prevented esterification of cholesterol in HDL and also prevented loss of steroidogenic activity. Although incubation of plasma of d greater than 1.125 g/ml prior to isolation caused cholesterol esterification, there was no change in the ratio of total cholesterol to protein in HDL, size and shape of the HDL particle as assayed by measurement of sedimentation velocity, nor affinity for the putative HDL receptor. Addition of unesterified cholesterol to preincubated HDL restored steroidogenic activity. These results indicate that unesterified cholesterol in HDL is preferentially used as substrate for rat adrenal steroidogenesis. The effects of nonlipoprotein serum proteins on HDL action in the adrenal were also examined. The ability of HDL3 to enhance rat adrenal steroidogenesis was not significantly less in serum-free media than in media supplemented with lipoprotein-poor fetal calf serum or human plasma of d greater than 1.21 g/ml, suggesting that rat adrenal uptake of HDL cholesterol does not depend on participation of plasma enzymes or transport proteins.     

Chylomicron-remnant uptake by freshly isolated hepatocytes. Effect of heparin and of hepatic triacylglycerol lipase.

Chylomicron remnants labelled biologically with [3H]cholesterol were efficiently taken up by freshly isolated hepatocytes during a 3 h incubation in Krebs bicarbonate medium. Their [3H]cholesteryl ester was hydrolysed (74% net hydrolysis), and 0.1 mM-chloroquine could partially inhibit this hydrolysis, provided that hepatocytes were first preincubated for 2 h 30 min at 37 degrees C. This hydrolysis was also measured in preincubated cells with remnants double-labelled (3H and 14C) on their free cholesterol moiety; [3H]cholesterol arising from [3H]cholesteryl ester hydrolysis was recovered in the free [3H]cholesterol pool. A dose-response study showed saturation of remnant uptake at 180 micrograms of remnant protein/10(7) cells. Heparin (10 units/ml) increased remnant uptake by 63% (P less than 0.01), [3H]cholesteryl ester accumulation in the cell pellet by 110% (P less than 0.025) and hepatic lipase activity secreted in the medium by 2.4-fold (P less than 0.01) and by 3.3-fold (P less than 0.01) at the end of the preincubation and incubation periods respectively. Addition of 100 munits of semi-purified hepatic lipase preparation/flask stimulated remnant uptake by 44-69%, and [3H]cholesteryl ester accumulation in the presence of chloroquine by 2.1-fold (P less than 0.025). When hepatic lipase was incubated solely with the remnants, it decreased their triacylglycerol and phospholipid contents by 24% and 26% respectively. Thus freshly isolated hepatocytes may be used to study chylomicron-remnant uptake. Hepatic lipase, which seems to underly the stimulating effect of heparin, facilitates remnant uptake in vitro, and this could be mediated by at least one (or both) of its hydrolytic properties.     

Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1

A thermophilic microorganism, Bacillus thermoleovorans ID-1, isolated from hot springs in Indonesia, showed extracellular lipase activity and high growth rates on lipid substrates at elevated temperatures. On olive oil (1.5%, w/v) as the sole carbon source, the isolate ID-1 grew very rapidly at 65 degrees C with its specific growth rate (2.50 h(-1)) and its lipase activity reached the maximum value of 520 U l(-1) during the late exponential phase and then decreased. In addition to this, isolate ID-1 could grow on a variety of lipid substrates such as oils (olive oil, soybean oil and mineral oil), triglycerides (triolein, tributyrin) and emulsifiers (Tween 20, 40). The excreted lipase of ID-1 was purified 223-fold to homogeneity by ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. As a result, the relative molecular mass of the lipase was determined to be 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed optimal activity at 70-75 degrees C and pH 7.5 and exhibited 50% of its original activity after 1 h incubation at 60 degrees C and 30 min at 70 degrees C and its catalytic function was activated in the presence of Ca(2+) or Zn(2+).     

The effects of acclimation on thermal tolerance, desiccation resistance and metabolic rate in Chirodica chalcoptera (Coleoptera: Chrysomelidae)

Despite much focus on species responses to environmental variation through space and time, many higher taxa and geographic areas remain poorly studied. We report the effects of temperature acclimation on thermal tolerance, desiccation rate and metabolic rate for adult Chirodica chalcoptera (Coleoptera: Chrysomelidae) collected from Protea nerifolia inflorescences in the Fynbos Biome in South Africa. After 7 days of acclimation at 12, 19 and 25 degrees C, critical thermal maxima (mean+/-s.e.: 41.8+/-0.2 degrees C in field-fresh beetles) showed less response (<1 degrees C change) to temperature acclimation than did the onset of the critical thermal minima (0.1+/-0.2, 1.0+/-0.2 and 2.3+/-0.2 degrees C, respectively). Freezing was lethal in C. chalcoptera (field-fresh SCP -14.6 degrees C) and these beetles also showed pre-freeze mortality. Survival of 2 h at -10.1 degrees C increased from 20% to 76% after a 2 h pre-exposure to -2 degrees C, indicating rapid cold hardening. Metabolic rate, measured at 25 degrees C and adjusted by ANCOVA for mass variation, did not differ between males and females (2.772+/-0.471 and 2.517+/-0.560 ml CO2 h(-1), respectively), but was higher in 25 degrees C-acclimated beetles relative to the field-fresh and 12 degrees C-acclimated beetles. Body water content and desiccation rate did not differ between males and females and did not respond significantly to acclimation. We place these data in the context of measured inflorescence and ambient temperatures, and predict that climate change for the region could have effects on this species, in turn possibly affecting local ecosystem functioning.     

Division of labour during incubation in a woodpecker Colaptes auratus with reversed sex roles and facultative polyandry

The contribution of males to incubation has rarely been studied in altricial birds because the pursuit of extra mating opportunities is believed to conflict with incubation. Woodpeckers show reversed sex roles in parental care with males doing most of the nest construction, incubating and brooding of the young while females may be polyandrous. I investigated incubation by each sex at 71 monogamous and four polyandrous nests of the Northern Flicker Colaptes auratus , predicting that males would contribute to incubation according to their energy reserves (body condition) whereas females would contribute based on alternate reproductive opportunities. Nest attendance was 99% with males contributing a mean of 66% of the total incubation including nocturnal incubation. The length of daytime bouts averaged about 2 h and did not differ between the sexes. Consistent with predictions of investment strategies, structurally larger males and those in poorer body condition incubated less than smaller males, perhaps because they required more recess time to forage or to conserve energy. Older females contributed less incubation than young females and polyandrous females contributed less incubation at their secondary nests than monogamous females. Incubation period, nest depredation rate and hatching success were not influenced by bout length, number of bouts or relative contribution of the sexes. Hatching success was 86% at nests of both monogamous and polyandrous females because males compensated for reduced female participation. Because incubation of the sexes is compensatory and not additive, incubation pattern did not influence short-term reproductive success. I conclude that males invest in incubation according to their energy needs, and females may adjust their contributions based on alternate reproductive tactics.     

Purification and properties of lipase from Penicillium simplicissimum.

A Penicillium simplicissimum strain has been found to produce an inducible extracellular lipase. Triolein was the best inducer for the enzyme production with the highest activity being achieved after 48 h of incubation. The purified lipase showed a molecular weight of 56,000 by SDS-PAGE. The enzyme exhibited a high ratio of apolar amino acids. The lipase was stable in the pH range of 5-7 and at 50 degrees C for 15 min. The optimum assay conditions were 37 degrees C and pH 5.0. The enzyme showed a high stability in water immiscible organic solvents. Lipase from P. simplicissimum is nonspecific and hydrolyses each of the three bonds of triacylglycerols.     

Enhancement of misonidazole chemopotentiation by mild hyperthermia (41 degrees C) in vitro and selective enhancement in vivo

Experiments were designed to test the hypothesis that mild heat treatment would selectively increase misonidazole (MISO) chemopotentiation of CCNU toxicity in hypoxic versus aerobic cells in vitro and in tumours in vivo via an augmentation of nitroreduction. EMT-6 cells were exposed to CCNU +/- 1.0 mM MISO under aerobic or hypoxic conditions for 4 h either at a constant 37 degrees C or at 41 degrees C for the first hour followed by 37 degrees C for the remaining 3 h. Chemopotentiation was not observed under aerobic conditions and heat treatment did not modify CCNU toxicity. Co-incubation with MISO and CCNU under hypoxic conditions resulted in enhanced toxicity (i.e. chemopotentiation) with either incubation protocol; however, the magnitude of the enhancement was significantly larger (P less than 0.025) when 41 degrees C incubation was included. Systemic heat treatment produced a similar enhancement of chemopotentiation in KHT tumours in C3H/HeN mice treated with MISO (0.5 mg g-1) and whole body hyperthermia (41 degrees C, 1 h) prior to administration of CCNU (15 mg kg-1). Heating had no effect on CCNU response but doubled the median growth delay produced by the CCNU-MISO combination. Heat treatment did not enhance myelosuppression of the combination. Both the in vitro and in vivo data indicate that mild hyperthermia can selectively enhance the magnitude of MISO chemopotentiation.     

Frequency of micronuclei induced in peripheral lymphocytes by trimethyltin chloride

The clastogenicity of trimethyltin chloride was evaluated in human peripheral blood lymphocytes with micronucleus counts (MNC) as the endpoint. Two concentrations (0.5 micrograms and 1.0 microgram) of trimethyltin chloride were added to lymphocytes of healthy male and female subjects of different age groups, in mitogen-stimulated and serum-supplemented culture medium (RPMI 1640, Gibco) for 48 h at 37 degrees C. A significant increase in micronucleus counts was observed with both doses, which was more pronounced with the lower dose. ANOVA in male and female donors revealed significant differences between age groups (P less than 0.001), chemical concentrations (P less than 0.001) and for the interaction of these 2 factors (P less than 0.05 in females only). However, no regular increase or decrease in MNC frequencies was observed with the donor's age. Higher frequencies of MNC enhancement were observed in male individuals than in females.     

链霉菌Z94-2碱性脂肪酶产生条件及酶学性质

在１５２株脂肪酶产生菌中,链霉菌Ｚ９４－２产脂肪酶活力为５９６ｕ／ｍＬ,其最适培养基（ｇ／Ｌ）为：糊精１０、黄豆饼粉３０、尿素１０、Ｋ２ＨＰＯ４０．５、ＭｇＳＯ４０．５、ＮａＣｌ１和ＡＥＯ９０．５,产酶的最适条件为：初始ｐＨ９．５～１０．０,在２６℃培养４８ｈ。用ＰＶＡ橄榄油乳化系统测定该酶的最适ｐＨ９．８,最适温度３７℃,在ｐＨ８．６～１０．２于５℃存放２４ｈ,酶活力不变。０．１４ｍｏｌ／Ｌ的氯     

Characterization of the lipoprotein lipase in the functional pool of rat heart by immunoblotting

Rat hearts were perfused with heparin for 2 min at 4 degrees C. The lipoprotein lipase activity in the perfusate was inhibited by antiserum to rat adipose tissue lipoprotein lipase. By immunoblotting, the lipoprotein lipase derived from the functional pool of the heart was found to be a protein with an apparent Mr of 69 000. After incubation of the perfusate at 37 degrees C for 24 h an immunologically reactive protein with an apparent Mr of 28 000 was found. This protein is not a physiological derivative of the enzyme but a degradation product.