Identification of a hypertrehalosemic factor in Spodoptera exigua

Trehalose is a major blood sugar in insects with a range of physiological functions, including an energy source and a cryoprotectant. Hemolymph trehalose concentrations are tightly regulated according to physiological conditions. An insulin‐like peptide, SeILP1, downregulates hemolymph trehalose concentrations in Spodoptera exigua. Here, we identified a factor that upregulates hemolymph trehalose concentration in S. exigua. Hemolymph trehalose concentrations were significantly increased after immune challenge or under starvation in a time‐dependent manner. To determine endocrine factors responsible for the upregulation, stress‐associated mediators, such as octopamine, serotonin, or eicosanoids were injected, but they did not upregulate hemolymph trehalose. On the other hand, injection with Schistocerca gregaria adipokinetic hormone (AKH) significantly increased hemolymph trehalose concentration in S. exigua. During upregulation of hemolymph trehalose by AKH injection, trehalose degradation appeared to be inhibited because expression of trehalase and SeILP1 were significantly suppressed while that of trehalose phosphate synthase was not significantly changed. Interrogation of a Spodoptera genome database identified an S. exigua AKH‐like gene and its expression was confirmed. During starvation, its expression concentrations were increased, although RNA interference specific to the AKH‐like hypertrehalosemic factor (SeHTF) gene significantly prevented the upregulation of hemolymph trehalose concentrations during starvation. A synthetic peptide of SeHTF was prepared and injected into S. exigua larvae. At nanomolar concentration, the synthetic SeHTF peptide effectively upregulated hemolymph trehalose concentrations. Here we report a novel hypertrehalosemic factor in S. exigua (SeHTF).     

Proton gradient mediates hemolymph trehalose influx into aphid bacteriocytes

Aphids harbor proteobacterial endosymbionts such as Buchnera aphidicola housed in specialized bacteriocytes derived from host cells. The endosymbiont Buchnera supplies essential amino acids such as arginine to the host cells and, in turn, obtains sugars needed for its survival from the hemolymph. The mechanism of sugar supply in aphid bacteriocytes has been rarely studied. It also remains unclear how Buchnera acquires its carbon source. The hemolymph sugars in Acyrthosiphon pisum are composed of the disaccharide trehalose containing two glucose molecules. Here, we report for the first time that trehalose is transported and used as a potential carbon source by Buchnera across the bacteriocyte plasma membrane via trehalose transporters. The current study characterized the bacteriocyte trehalose transporter Ap_ST11 (LOC100159441) using the Xenopus oocyte expression system. The Ap_ST11 transporter was found to be proton-dependent with a K_m value ≥700 mM. We re-examined the hemolymph trehalose at 217.8 mM using a fluorescent trehalose sensor. The bacteriocytes did not obtain trehalose by facilitated diffusion along the gradient across cellular membranes. These findings suggest that trehalose influx into the bacteriocytes depends on the extracellular proton-driven secondary electrochemical transporter.     

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.     

Preface

The vector of Chagas' disease, Rhodnius prolixus, feeds exclusively on blood. The blood meals are slowly digested, and these insects wait some weeks before the next meal. During the life of an insect, energy‐requiring processes such as moulting, adult gonadal and reproductive growth, vitellogenesis, muscular activity, and fasting, lead to increased metabolism. Carbohydrates are a major source of energy and their mobilization is important. We determined the amounts of glycogen, trehalose, and glucose present in the fat body and/or hemolymph of adult males of R. prolixus and recorded the processes of accumulation and mobilization of these carbohydrates. We also tested our hypothesis that these processes are under endocrine control. The amount of glycogen in the fat body progressively increased until the fourth day after feeding (from 9.3±2.2 to 77. 3±7.5 µg/fat body), then declined to values around 36.3±4.9 µg/fat body on the fifteenth day after the blood meal. Glycogen synthesis was eliminated in decapitated insects and head‐transplanted insects synthesized glycogen. The amount of trehalose in the fat body increased until the sixth day after feeding (from 16. 6±1.7 to 40. 6±5.3 nmol/fat body), decreased abruptly, and stabilized between days 7 and 15 at values ranging around 15–19 nmol/fat body. Decapitated insects did not synthesize trehalose after feeding, and this effect was reversed in head‐transplanted insects. The concentration of trehalose in the hemolymph increased after the blood meal until the third day (from 0.07±0.01 to 0.75±0.05 mM) and at the fourth day it decreased until the ninth day (0.21±0.01 mM), when it increased again until the fourteenth day (0.79±0.06 mM) after the blood meal, and then declined again. In decapitated insects, trehalose concentrations did not increase soon after the blood meal and at the third day it was very low, but on the fourteenth day it was close to the control values. The concentration of glucose in the hemolymph of untreated insects remained low and constant (0.18±0.01 mM) during the 15 days after feeding, but in decapitated insects it progressively increased until the fifteenth day (2.00±0.10 mM). We recorded the highest trehalase activity in midgut, which was maximal at the eighth day after feeding (2,830±320 nmol of glucose/organ/h). We infer that in Rhodnius prolixus, the metabolism of glycogen, glucose, and trehalose are controlled by factors from the brain, according to physiological demands at different days after the blood meal. © 2009 Wiley Periodicals, Inc.     

Simultaneous Extraction of RNA and DNA from Rat Liver with Cupric Sulfate

Homogenization of fresh rat livers in 0.5mM cupric sulfate and 0.5% sodium dodecyl sulfate yielded both RNA and DNA in the aqueous phase after treatment vith phenol at 0–4°C. Effective deproteinization was achieved by three additional phenol treatments. Nucleic acids vere freed from Cu²⁺by repeated precipitation with ethanol in the presence of EDTA. The final yield was 6–7 mg/g of liver, of which about 20% was DNA and 11% tRNA. Physicochenical studies showed that pure tRNA, undegraded rRKA (30S, 18S) and native DNA (s°_{20, w} = 24.4S) were isolated by this procedure.     

Changes in lipid and carbohydrate metabolism during starvation in adult Manduca sexta

Summary Adult Manduca sexta feed very irregularly in the laboratory, and many adult males never feed. Feeding adults live longer and feeding females lay many more eggs; however, in both feeding (sugar water) and starving adults a decrease of metabolic reserves is observed. Carbohydrates disappear from hemolymph and from fat body. Fat body lipid also decreases, while hemolymph lipid concentration increases strongly in starving adults. The activity of fat body glycogen phosphorylase increases strongly in starving adult M. sexta. The activity of glycogen phosphorylase is correlated inversely with hemolymph sugar concentration. Injected trehalose inactivates glycogen phosphorylase within 2 h, and lowers the hemolymph lipid level within 6 h. In starving adult M. sexta, neither the activation of glycogen phosphorylase nor the increase of hemolymph lipid concentration depends on adipokinetic hormone, since cardiacectomy does not prevent the activation of glycogen phosphorylase nor the increase of hemolymph lipid level.Abbreviations AKH adipokinetic hormone - EDTA ethylenediamine tetraacetate Present address: Department of Biochemistry and Center for Insect Science, The University of Arizona, Tucson, AZ 85721, USA     

In vitro bioassay for hypertrehalosemic hormone-dependent trehalose biosynthesis by fat body from adult Blaberus discoidalis cockroaches

An in vitro bioassay suitable for routine use to investigate hypertrehalosemic hormone (HTH)-dependent trehalose biosynthesis was developed for the cockroach fat body. Blaberus discoidalis fat bodies were isolated and divided so that eight matched pieces from a single tissue could be compared for multiple control and experimental treatments. Optimum incubation conditions and the properties of HTH-dependent trehalose synthesis were determined. Dose-response studies determined an EC₅₀ of 0.044 nM HTH for male fat body and 0.16 nM HTH for female tissue. HTH increased trehalose production by male fat body 3-fold compared to only a 67% maximum increase by the female tissue, and only the male tissue was used in subsequent studies. Fat body required only 5-min exposure to HTH for maximum trehalose production for 1 h. Trehalose synthesis was inhibited by ≥ 15 mM trehalose in the incubation medium. The fat body showed a developmental increase in trehalose synthesis in vitro that was reflected by hemolymph trehalose in vivo. Basal and HTH-related trehalose synthesis were low between days 0 and 10, increased 3-fold by day 20, and were high thereafter. These studies have established baseline data for future investigations to identify the signal transduction mechanisms involved in HTH regulation of fat body metabolism. © 1995 Wiley-Liss, Inc.     

Production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae

The production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae from sheep were studied. D. flagrans was grown in liquid medium with glucose, casein, bibasic potassium phosphate (K₂HPO₄), magnesium sulfate (MgSO₄), zinc sulfate (ZnSO₄), ferrous sulfate (FeSO₄), and copper sulfate (CuSO₄). The proteolytic activity was measured within varied pHs and temperatures. To determine the thermostability, the crude extract was incubated at 28°C for 72 h. To study the effect of different chemical compounds on the activity of the crude extract, the samples were incubated in solutions containing (10 mM): calcium chloride (CaCl₂), copper II sulfate (CuSO₄), zinc sulfate (ZnSO₄), magnesium sulfate (MgSO₄), inhibitor phenylmethylsulfonyl fluoride (PMSF), and 0.5% SDS. Results showed that the highest activity obtained (79.23 U/mL) was at pH 9.0, while the optimum temperature was 60°C (119.6 U/mL). The thermostability analysis demonstrated that after 72 h the activity was maintained or increased. It was found that the CuSO₄, ZnSO₄, and PMSF strongly inhibited the proteolytic activity. Moreover, the MgSO4 and SDS, caused a weak inhibition of the proteolytic activity. There was a significant (P<0.01) reduction in number of treated L₃ when compared to control (94.2%). The results suggest that the crude extract produced by D. flagrans (AC001) in liquid medium exerted larvicidal activity on trichostrongilid L₃ and therefore may contribute to a large-scale industrial production.     

The potential of trehalose to replace insect hemolymph in artificial media for Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae)

Insect additives have been shown to improve the value of artificial media for Trichogramma species, but at the same time maintain dependence on parallel cultures of host insects. In the present study, Trichogramma dendrolimi Matsumura was reared in vitro from egg to adult on artificial media with different contents of pupal hemolymph of Chinese oak silkmoth Antheraea pernyi (Guérin‐Méneville) and with supplements of distilled water, or of trehalose dissolved in water or in Grace's insect medium. The results indicated insect hemolymph was the key component of artificial medium. Developmental parameters, including rates of parasitism, pupation, adult emergence and normal adults, and numbers of produced adults, were increased on media supplemented with trehalose even when the proportion of pupal hemolymph was reduced. Two artificial media, the first containing 30% hemolymph and 10% trehalose in water with 98.9% parasitism rate, 77.7.0% pupation rate, 77.2% emergence rate, 80.0% normal adult rate and 333 produced adults, and the second containing 25% hemolymph and 15% trehalose in Grace's insect medium with 97.8% parasitism rate, 91.0% pupation rate, 85.2% emergence rate, 76.1% normal adult rate and 757 produced adults, were believed to hold potential to mass produce T. dendrolimi. The use of trehalose to partially replace pupal hemolymph in artificial medium of this and other Trichogramma species may contribute to a significant reduction in their production cost and may as such help to evade problems related to short supplies of lepidopteran eggs, which currently constitute the main factitious host for the mass rearing of the parasitoids.     

Orcadian Rhythm of Trehalose in the Face Fly Musca Autumnalis De Geer

Trehalose levels were determined over two 24 hr spans in groups of face fly adults 3–4 days after emergence from the puparium. Face fly pupae were placed in rearing chambers at 27° C in a staggered light-dark regimen, LD 16:8, so that at a given clock hour, samples could be obtained at several different hours after lights on (HALO). Trehalose was determined in hemolymph collected from a puncture in the intersegmental membrane of the abdomen. Treated hemolymph samples were passed through a Bio-Rad Amino S-S disaccharide column and a Waters 410 refractive index detector was used to differentiate among sugars. The circadian acrophase derived by cosinor analysis in hemolymph trehalose (when the values were 25.49 and 26.86μg/μ¹ on the first and second days respectively) occurred at -226° (ca 15 HALO) and the bathyphase at 24 HALO. The mesor = 11.82μg/μ¹ trehalose, the amplitude = 8.57/μg/μ¹ trehalose and the P-value for presence of a rhythm was 0.003. Based on these data, differences between control and test flies in a bioassay of hypertrehalosemic activity would be most easily observed at 0-8 HALO, while exogenous hypotrehalosemic activity would be best assayed at 12-20 HALO.     

Determination of relative bioavailability of zinc in a petit suisse cheese using weight gain and bone zinc content in rats as markers

The aim of the study was to determine the relative bioavailability of zinc gluconate stabilized with glycine in a Petit Suisse cheese from an infant dessert. Weight gain and bone zinc content were the nutritional responses evaluated for the diets of different zinc content: 2 ppm (basal) and 5, 10, and 30 ppm from zinc gluconate stabilized with glycine and zinc sulfate. Nonlinear regression analysis of the fitted curves for weight gain determined a relative zinc bioavailability of 100% for the Y _max ratio and 96% for Y _max/t _1/2 ratio for zinc gluconate stabilized with glycine (R ²=0.7996 for zinc sulfate and 0.8665 for zinc gluconate stabilized with glycine). The slope ratio analysis from linear regression of femur zinc determined a relative zinc bioavailability of 93% for zinc gluconate stabilized with glycine (R ²=0.8693 for zinc sulfate and 0.8307 for zinc gluconate stabilized with glycine). Zinc gluconate stabilized with glycine has similar bioavailability as zinc sulfate in a Petit Suisse cheese nutritional matrix, with the advantage that the stabilized compound does not modify the sensorial characteristics of the fortified cheese.     

Intensification of surfactant synthesis in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 cultivated on hexadecane

Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n-hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP⁺-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 μmol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5–1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with n-hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3–5-fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.     

Diphenols in hemolymph and cuticle during development and cuticle tanning of Periplaneta americana (L.) and other cockroach species

Diphenolic compounds in cockroach hemolymph and cuticle were extracted with 1.2 N HCI, partially purified by alumina adsorption, and analyzed by liquid chromatography. Dopamine (DA) is the major catecholamine in hemolymph of Periplaneta americana, Blatta orientalis, Blattella germanica, Gromphadorhina portentosa, and Blaberus craniifer at adult ecdysis, while N-acetyldopamine (NADA) predominates in hemolymph of Leucophaea maderae. In P. americana, NADA is the second most abundant catecholamine, while N-β-alanyldopamine (NBAD), norepinephrine (NE), 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylethanol, 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxybenzoic acid occur in lesser quantities. Catecholamines occur mainly as acid labile conjugates in hemolymph. Dopamine, conjugated primarily as the 3-sulfate ester, increases in hemolymph from 0.1 to 0.8 mM during the last instar. Concentrations decrease by 75% in pharate adults, partially because of an increase in hemolymph volume. A second smaller peak of DA sulfate occurs after ecdysis followed by a rapid disappearance as the cuticle tans. A conjugate of catechol (o-dihydroxybenzene) is also present in relatively high concentrations at all ages examined. In cuticle, N-β-alanylnorepinephrine accumulates during the early period of adult tanning, while NBAD, NADA, N-acetylnorepinephrine, and DA increase more slowly. The N-β-alanyl and N-acetyl derivatives of DA and NE occure in relatively high concentrations in tanned cutical of P. americana and probably play an important role in the stablization process.     

Purification and properties of α-glucose 1-phosphate-forming trehalose phosphorylase from a basidiomycete,Pleurotus ostreatus

Pleurotus ostreatus produced a high activity of α-glucose 1-phosphate (α-Glc 1-P) forming trehalose phosphorylase in vegetative mycelia and fruit-bodies. The enzyme was purified to homogeneity from the fruit-bodies by a procedure involving ammonium sulfate fractionation, DEAE-cellulose column chromatographies and cellulose phosphate column chromatographies. The enzyme catalyzes both the phosphorolysis of trehalose to produce α-Glc 1-P and glucose, and the synthesis of trehalose. It was not active toward other α- or β-glucosyl disaccharides and polysaccharides. The optimum pH was 7.0 for phosphorolysis and 6.4 for synthesis of trehalose. The Km values for trehalose and Pi in phospholytic reaction were 75 mM and 4.2 mM, respectively. Those for glucose and α-Glc 1-P in synthetic reaction were 505 mM and 38 mM, respectively. The estimated molecular mass by the sedimentation equilibrium method using an ultracentrifuge was 120 kDa. The molecular mass of the subunit (61 kDa) by SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a dimer of two identical subunits. The addition of glycerol higher than 25% into the enzyme solution stabilized its activity. The removal of phosphorus ions from the enzyme solution, by means of dialysis or electrophoresis, caused inactivation of the enzyme, probably by dissociation of the holoenzyme into the subunit proteins.     

Sensitivities to juvenile hormone and ecdysteroid in the diapause larvae of Omphisa fuscidentalis based on the hemolymph trehalose dynamics index

The final instar larva of the bamboo borer, Omphisa fuscidentalis, is in diapause for 9 months from September to the following June. Trehalose and ecdysteroid concentrations in hemolymph were measured through the larval diapause period and in the pupal stage. The ecdysteroid concentration remained low until November, followed by a gradual increase to about 30 ng/ml in May. The trehalose concentration remained at levels ranging between 40-50 mM until May, and decreased to an almost undetectable level after pupation. Since a juvenile hormone analogue (JHA), methoprene, is capable of terminating diapause by stimulating larval prothoracic glands, we examined its effects on ecdysteroid and trehalose concentrations in larvae in December and February. The hemolymph ecdysteroid increased more quickly in February than in December, indicating that the sensitivity of the prothoracic glands to JHA increased towards the end of diapause termination. Similarly, hemolymph trehalose in February decreased within a few days after JHA application, while in December the decrease occurred in the third week. Exogenous 20-hydroxyecdysone (20E) caused a decrease in trehalose concentration in a dose-dependent manner. The effective dose of 20E, however, did not change from January until April, implying that the sensitivity of tissue(s) to 20E may not change until the end of diapause. Taken together, our results suggest that the sensitivities of tissues to JH and 20E do not increase simultaneously with the progress of diapause development and that termination of larval diapause is not associated simply with the restoration of hormone deficiencies.     

Carbohydrate metabolism during starvation in the silkworm Bombyx mori

The effect of starvation on carbohydrate metabolism in the last instar larvae of the silkworm Bombyx mori was examined. Trehalose concentration in the hemolymph increased slightly during the first 6 h of starvation and decreased thereafter, whereas glucose concentration decreased rapidly immediately after diet deprivation. Starvation-induced hypertrehalosemia was completely inhibited by neck ligation, suggesting that starvation stimulates the release of a hypertrehalosemic factor(s) from the head. The percentage of active glycogen phosphorylase in the fat body increased within 3 h of starvation and its glycogen content decreased gradually. These observations suggest that production of trehalose from glycogen is enhanced in starved larvae. However, hypertrehalosemia during starvation cannot be explained by the increased supply of trehalose into hemolymph alone, as similar changes in phosphorylase activity and glycogen content in the fat body were observed in neck-ligated larvae, in which hemolymph trehalose concentration did not increase but decreased gradually. When injected into larvae, trehalose disappeared from hemolymph at a rate about 40% lower in starved larvae than neck-ligated larvae. The hemolymph lipid concentration increased during starvation, suggesting that an increased supply of lipids to tissues suppresses the consumption of hemolymph trehalose and this is an important factor in hypertrehalosemia.     

Purification and properties of trehalase in Frankia ArI3

Trehalase was purified from cultures of Frankia strain ArI3 grown on media with or without NH₄Cl. The purified enzyme was specific for trehalose, exhibited a broad pH optimum of pH 4.5 to 5.3 and had a K _m for trehalose of 4.2 mM. The trehalase was inhibited in vitro completely by sucrose, glucose and mannose and partially by mannitol and sorbitol. In addition to the specific trehalase, a mixture of non-specific - and -glucosidases which exhibited some activity with ,-trehalose as a substrate were also partially purified in Frankia extracts made from nitrogen-fixing cells. These enzymes were not detected in the purifications of crude extracts made from non-nitrogen-fixing cells (grown on media supplemented with NH₄Cl). Trehalase activity in crude extracts increased over time when cells were induced to fix nitrogen, and the maximum specific activity of trehalase from nitrogen-fixing cultures was 4 times the maximum activity from non-fixing cultures. Trehalase activity was also examined in crude extracts made from Frankia vesicle clusters isolated from Alnus rubra nitrogen-fixing nodules infected with ArI3. The maximum activity of trehalase in these clusters was 6–7 times greater than in the nitrogenfixing pure cultures of ArI3 and 26–33 times greater than the non-fixing pure cultures.Abbreviations pcv packed cell volume - DTE dithioerythritol - PMSF phenylmethylsulphonylfluoride - EDTA sodium ethylenediaminetetraacetate     

Isolation of rapidly labeled RNA from mouse L-cells using cupric sulfate.

Cultured mouse L-cells, pulse labeled for 5 min with 3H-uridine, were gently suspended in 0.5mM cupric sulfate and 0.5% sodium dodecyl sulfate at 4 degrees C and treated with cold phenol. Only the RNA, containing less than 1% DNA, was extracted by this procedure. The rapidly labeled ribosomal RNA precursors (44S, 34S) and cytoplasmic 8S RNA showed specific activities higher than that of tRNA and were present in the RNA fraction insoluble in 2M NaCl.     

Sublethal effects of cupric ion activity on the phototaxis of three calanoid copepods

Using wavelengths near maximal photosensitivity, phototactic responses of two estuarine calanoid copepods (Acartia tonsa, Acartia hudsonica) and one nearshore, neritic copepod (Temora longicornis) were measured after 24 h exposures to sublethal concentrations of free cupric ions. A nitrilotriacetate-trace metal ion buffer system was used to control the free cupric ion activity (pCu = negative log of the free cupric ion activity), which determines organismic response. All three species exhibited positive phototaxis at pCu 13.0 reported for unpolluted surface sea waters and estuarine waters. As cupric ion activity increased, percent positive phototactic response decreased, indicating a strong sublethal effect of free cupric ions on photobehavior. Changes in photobehavior occurred at cupric ion activities that have been reported for many estuaries and coastal waters near urban and industrialized areas. Temora longicornis was much less phototactically sensitive than the two estuarine species. It also exhibited phototactic sign switching as pCu changed.     

Characteristics of the dephosphorylated form of phosphorylase purified from rat liver and measurement of its activity in crude liver preparations.

The phosphorylated form of liver glycogen phosphorylase (alpha-1,4-glucan : orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1) (phosphorylase a) is active and easily measured while the dephosphorylated form (phosphorylase b), in contrast to the muscle enzyme, has been reported to be essentially inactive even in the presence of AMP. We have purified both forms of phosphorylase from rat liver and studied the characteristics of each. Phosphorylase b activity can be measured with our assay conditions. The phosphorylase b we obtained was stimulated by high concentrations of sulfate, and was a substrate for muscle phosphorylase kinase whereas phosphorylase a was inhibited by sulfate, and was a substrate for liver phosphorylase phosphatase. Substrate binding to phosphorylase b was poor (KM glycogen = 2.5 mM, glucose-1-P = 250 mM) compared to phosphorylase a (KM glycogen = 1.8 mM, KM glucose-1-P = 0.7 mM). Liver phosphorylase b was active in the absence of AMP. However, AMP lowered the KM for glucose-1-P to 80 mM for purified phosphorylase b and to 60 mM for the enzyme in crude extract (Ka = 0.5 mM). Using appropriate substrate, buffer and AMP concentrations, assay conditions have been developed which allow determination of phosphorylase a and 90% of the phosphorylase b activity in liver extracts. Interconversion of the two forms can be demonstrated in vivo (under acute stimulation) and in vitro with little change in total activity. A decrease in total phosphorylase activity has been observed after prolonged starvation and in diabetes.