Entamoeba histolytica: Molecular cloning and characterization of a novel neutral sphingomyelinase

A novel neutral sphingomyelinase (nSMase) was characterized in Entamoeba histolytica trophozoites. SMase, a sphingomyelin-specific form of phospholipase C, catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Three amebic putative nSMase genes were found to be actively transcribed. Mg²⁺-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn²⁺ and partially inhibited by Zn²⁺. nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5 mM Mn²⁺, and abolished by 5 mM Zn²⁺. A dose-dependent inhibition of rEhnSM1 was observed with scyphostatin, a specific inhibitor of nSMases. The EhnSM1 and EhnSM3 were detected in the soluble fraction of the amebic lysate as 35-37 kDa proteins by western blot analysis. Immunofluorescence assay showed that the overexpressed HA-tagged EhnSM1 and EhnSM3 were localized to the cytosol. The biological role of these novel E. histolytica nSMases described in this work remains to be determined.     

Entamoeba histolytica: ultrastructural localization of Ca2+-dependent nucleotidases

Localization of nucleotidases dependent on Ca²⁺ was investigated cytochemically in axenically cultivated trophozoites of Entamoeba histolytica, strain HM-1:IMSS, with an electron microscope. Ca²⁺-dependent ATPase (EC 3.6.1.3) activity was found on the plasma membrane and on the inner surface of the limiting membrane of a few cytoplasmic vacuoles. Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase, and acid phosphatase (EC 3.1.3.2) activities were detected on the inner surface of the limiting membrane of most of the cytoplasmic vacuoles but not on the plasma membrane. Cytoplasmic vacuoles with these enzymatic activities seemed similar in morphological characteristics. Moreover, the reaction product formed by Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase and acid phosphatase was demonstrable on the inner surface of the limiting membrane of vacuoles containing ingested red blood cells. The reaction product formed by these enzymes was also observed on the periphery of ingested red blood cells. The findings suggest that cytoplasmic vacuoles with these enzymatic activities are lysosomal in nature, probably phagolysosomes; therefore, the enzymes appear to be at least partially associated with primary lysosomes of E. histolytica.     

Activation of kidney guanylate cyclase by cobalt.

Guanylate cyclase activity and cyclic nucleotide content were studied in individual slices from guinea pig kidneys. Basal guanylate cyclase activity, assayed in homogenates or in particulate fractions (100,000g × 1 h), and the tissue content of cGMP and cAMP were greater in the inner than in the outer (entirely cortical) slices. The fraction of guanylate cyclase activity recovered in the supernatant was greater in the cortex. Taurodeoxycholate increased activity of the particulate cyclase but decreased that of the supernatant enzyme. Activity of the particulate was increased ca. 200% and that of the supernatant >500% by 1 mm NaN₃. Supernatant activity was markedly increased by 0.1 mm Co²⁺, which had no effect on the particulate enzyme. (Incubation of kidney slices with 2 mm Co²⁺ did not alter their cGMP content, but caused a small increase in the cAMP content of slices containing medullary tissue.) Basal guanylate cyclase activity in fresh supernatants increased linearly with pH from 5.9 to 9, whereas in the presence of Co²⁺ there was a clear maximum at pH 7.3 to 7.5. Incubation of fresh supernatant fractions at 37 °C for 3 h increased guanylate cyclase activity and abolished Co²⁺ activation. The relationship between Co²⁺ activation and that resulting from incubation remains to be defined. It seems probable, however, that these phenomena reflect regulatory properties of the supernatant guanylate cyclases of kidney and other tissues.     

The membrane ATPase of Escherichia coli. II. Release into solution,allotopic properties and reconstitution of membrane-bound ATPase

Membrane-bound ATPase of Escherichia coli was released in a soluble form by decreasing the Mg²⁺ concentration to 0.05 mM. The particulate fraction left behind was depleted by more than 90% from its initial ATPase activity.Soluble ATPase exhibits a number of different properties as compared with membrane-bound ATPase. These are a 2-fold increased K_m toward ATP, a shift of 1–1.5 pH units in the pH-dependence curve, a greatly increased resistance to inhibition by N,N′-dicyclohexylcarbodiimide (DCCD) and a stimulation by Dio 9 instead of an inhibition.Upon mixing the soluble fraction and the depleted membrane fraction, the initial properties of native membrane-bound ATPase reappear. This reconstitution requires Mg²⁺ and results in the physical binding of the activity to sedimentable material.Soluble ATPase and depleted membrane can be titrated against each other until an equivalence point is reached, beyond which the component in excess keeps its previous characteristics.During the release procedure, DCCD remains associated with the particulate fraction with conservation of the ATPase-binding sites.Such DCCD-treated depleted membranes behave as a specific inhibitor of soluble ATPase.     

Characterization of a cold-adapted and salt-tolerant esterase from a psychrotrophic bacterium Psychrobacter pacificensis

An esterase gene, est10, was identified from the genomic library of a deep-sea psychrotrophic bacterium Psychrobacter pacificensis. The esterase exhibited the optimal activity around 25 °C and pH 7.5, and maintained as high as 55.0 % of its maximum activity at 0 °C, indicating its cold adaptation. Est10 was fairly stable under room temperatures, retaining more than 80 % of its original activity after incubation at 40 °C for 2 h. The highest activity was observed against the short-chain substrate p-nitrophenyl butyrate (C4) among the tested p-nitrophenyl esters (C2–C16). It was slightly activated at a low concentration (1 mM) of Zn²⁺, Mg²⁺, Ba²⁺, Ca²⁺, Cu²⁺, Fe³⁺, urea and EDTA, but was inhibited by DTT and totally inactivated by PMSF. Interestingly, increased salinity considerably stimulated Est10 activity (up to 143.2 % of original activity at 2 M NaCl) and stability (up to 126.4 % after incubation with 5 M NaCl for 6.5 h), proving its salt tolerance. 0.05 and 0.1 % Tween 20, Tween 80, Triton X-100 and CHAPS increased the activity and stability of Est10 while SDS, CTAB had the opposite effect. Est10 was quite active after incubation with several 30 % organic solvents (methanol, DMSO, ethanediol) but exhibited little activity with 30 % isopropanol, ethanol, n-butanol and acetonitrile.     

Invertases of pollen of Haemanthus albiflos

Soluble and insoluble invertase occurs in dormant pollen of Haemanthus albiflos, with pH optima of 5·7 and 5·5 respectively. At their pH optima the activity of the soluble enzyme is 3·5-fold higher. After 2 hr germination the pH optimum of the insoluble invertase is increased to 6·0 and the activity is increased 2-fold while the activity of the soluble invertase is decreased by 26%.     

Characterization of arginase activity from mouse epidermis and its relation to ornithine decarboxylase induction by the tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate

Arginase, which catalyzes the cleavage of l-arginine to urea and ornithine, was detected in both soluble and particulate fractions of mouse epidermis. In a typical experiment, about 75 and 25% of the total arginase activity was associated with the soluble (100 000 × g supernatant) and the washed particulate fraction, respectively. Both soluble and particulate enzymes required the presence of divalent Mn²⁺ for activity. Arginase activity was increased by about 50% in the particulate fraction, but not in the soluble fraction, by preheating the fractions at either 50 or 55°C in the presence of 15 mM MnCl₂. Enzyme activity in both fractions, in the absence of 15 mM MnCl₂, dropped precipitously during heating. A comparison of the nature of arginases in the soluble and particulate fractions revealed similar K_m values (13 mM) and pH optima (9.5) and identical heat denaturation curves. Application of 10 nmol of 12-O-tetradecanoylphorbol-13-acetate to mouse skin did not increase arginase activity in either fraction over a period of 24 h. In contrast, there was a large increase in ornithine decarboxylase activity in the soluble fraction 4.5 h after treatment. Mouse epidermal ornithine decarboxylase activity was much less than arginase activity and was predominantly localized in the soluble fraction. These results indicate that the normal level of arginase activity is not a limiting factor for the stimulation of polyamine biosynthesis by TPA. High arginase activity in mouse epidermis may play a role in providing ornithine for polyamine biosynthesis and in the production of glutamate and proline as well as in the production of keratinous proteins.     

GUANYLATE CYCLASES IN CNS: ENZYMATIC CHARACTERISTICS OF SOLUBLE AND PARTICULATE ENZYMES FROM MOUSE CEREBELLUM AND RETINA

Guanylate cyclase activity is present in both soluble and particulate fractions of homogenates of mouse cerebellum and retina. Soluble guanylate cyclases in cerebellum and retina have an apparent K_m for GTP of approx 40 and 70 μM, respectively; are stimulated by Ca²⁺ and Mg²⁺ in the presence of low Mn²⁺; and do not respond to NaN₃, NH₂OH or detergent. The particulate guanylate cyclase found in brain has an apparent K_m GTP of 237 7mu;M, is not stimulated by Ca²⁺ or Mg²⁺ in the presence of low Mn²⁺, but is stimulated by NaN₃, NH₂OH, and detergent. In particulate fractions of normal retina, guanylate cyclase has two apparent K_m GTP values (42 and 225 μM); has higher activity at low concentrations of Mn²⁺ (0.5 mM) than at high concentrations (5.0 mM); is inhibited by Ca²⁺; and does not respond to NaN₃, NH₂OH, or detergent. Retinas essentially devoid of photoreceptor cells (from mice with photoreceptor dystrophy) have soluble guanylate cyclase activity which is similar to that in normal retina, but have only 4% as much particulate guanylate cyclase activity. This residual particulate guanylate cyclase has an apparent K_m GTP value of 392 μM and other properties similar to particulate guanylate cyclase from brain. These data indicate the presence of three distinguishable guanylate cyclases in CNS: (1) a soluble enzyme present in both brain and retina: (2) a particulate enzyme which is also present in brain and in the inner or neural retina: and (3) another particulate enzyme which is apparently unique and confined to retinal photoreceptor cells.     

Photoregulation of the Carotenoid Biosynthetic Pathway in Albino and White Collar Mutants of Neurospora crassa

The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme fractions obtained from light-treated and dark-grown wild type, albino-1, albino-2, albino-3, and white collar-1 strains were mixed in various combinations, and the activity for conversion of [1-¹⁴C]isopentenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that: (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo light treatment increases the enzyme activity in the particulate fraction; (d) this light effect occurs in wild type, albino-1, and albino-3 strains; and (e) enzyme activity is present in the particulate fraction obtained from the white collar-1 mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity, [¹⁴C]geranylgeranyl pyrophosphate was used as a substrate. This compound, which is not available commercially, was synthesized enzymically using extracts of pea cotyledons. Particulate enzyme fractions obtained from wild type, albino-1, and albino-3 strains incorporate [¹⁴C]geranylgeranyl pyrophosphate into phytoene, and this activity is higher in extracts obtained from light-treated cultures. The particulate fraction obtained from the white collar-1 mutant also incorporates [¹⁴C]geranylgeranyl pyrophosphate into phytoene, but the in vivo light treatment does not cause an increase in this activity. No incorporation occurs when particulate fractions obtained from either dark-grown or light-treated albino-2 cultures are assayed. The soluble enzyme fraction obtained from the albino-3 mutant was shown to be almost totally defective in enzyme activity required for the biosynthesis of [¹⁴C]geranylgeranyl pyrophosphate from [1-¹⁴C]isopentenyl pyrophosphate. An in vivo light treatment increases the level of this activity in wild type, albino-1, albino-2, and albino-3 strains, but not in the white collar-1 mutant. A model is presented to account for all of the results obtained in this investigation. It is proposed that the white collar-1 strain is a regulatory mutant blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains are each defective for a different enzyme in the carotenoid biosynthetic pathway.     

Phenol β-glucosyltransferase and β-glucosidase activities in the tobacco hornworm larva Manduca sexta (L.): Properties and tissue localization

Phenol β-glucosyltransferase (PGT; EC 2.4.1.35) was studied in feeding fifth stadium larvae of the tobacco hornworm, Manduca sexta, using reversed-phase HPLC with absorbance detection to separate and quantify both the model substrate, p-nitrophenol (PNP), and the product, p-nitrophenyl β-D-glucopyranoside (PNP-Glc). About 90% of total PGT activity in tissue homogenates was associated with the particulate fraction (15,000g), with the remainder in the microsomal fraction. PGT activity was observed in all tissues, with highest activities in the labial gland and fat body. Appreciable activity occurred in midgut and hindgut tissue but none was found in hemolymph. PGT activity was also observed in eggs and larval fat body at different times during development. Activity was optimal at pH 7.5–9 and was highest with UDP-Glc as a glucose donor. However, appreciable PGT activity was observed with dTDP-Glc or GDP-Glc in place of UDP-Glc. The divalent cations Ca²⁺, Co²⁺, Mg²⁺, and Mn²⁺ stimulated activity, whereas Zn²⁺ and Hg²⁺, as well as pretreatment with the detergent Triton X-100, were inhibitory. Endogenous β-glucosidase in PGT-enriched fractions, especially from the midgut, antagonized the β-glucossylation process, and interference was minimized with higher pH and the addition of D-gluconic acid lactone to the incubation mixtures. The possible role of transglucosylation in the detoxication of phenolic xenobiotics and biotransformation of endogenous phenolic compounds in insects is discussed. Comparisons of PGT with some glycosyltransferases involved in endogenous and xenobiotic conjugation in insects and other organisms are reviewed. © 1992 Wiley-Liss, Inc.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

A monophenol oxidase activity in extracts of sorghum

A p-hydroxycinnamic acid oxidase activity was present in enzyme preparations from first internodes of Sorghum vulgare variety Wheatland milo when incubated in phosphate buffer at pH 7.5. This preparation had no classical polyphenolase activity but had both peroxidase and catalase activities. Since horseradish preparations catalyzed the same reaction, the oxidation probably is another example of a peroxidase-oxidase reaction. A second substrate was p-hydroxyphenylpyruvic acid. Ferulic acid was slightly active at low concentrations and inhibitory at higher ones. Diphenols such as caffeic and chlorogenic acids were inactive and inhibitory to p-hydroxycinnamic acid oxidation. A variety of monophenols such as tyrosine and cinnamic acid were inactive. An active substrate must have a free monophenolic group and para to this a C₃ side chain with a double bond and probably a free terminal acid group. A sulfhydryl reducing agent at the 5 millimolar level such as mercaptoethanol, reduced glutathione, or dithiothreitol was obligatory. Products were varied and were found in both the ethyl acetate-soluble and insoluble fractions after acidification of the incubation mixtures. With internode extracts, about 1 micromole of O₂ was consumed per micromole of p-hydroxycinnamic acid that disappeared in the presence of mercaptoethanol. Tetrahydrafolic acid plus mercaptoethanol were required for a second step oxidation or a parallel reaction; about 2 micromoles of O₂ were consumed per micromole of p-hydroxycinnamic acid that disappeared. Potassium cyanide, diethyldithiocarbamate, ascorbic acid, and ethylenediaminetetraacetate were inhibitory. A similar mercaptoethanol-dependent monophenol oxidase was present in preparations from green shoots that also contained a classical polyphenolase activity. The activity was present in both soluble and particulate (500 to 100,000 gravity) fractions of internodes. Preliminary studies were made of enzyme complexes in the particulate fractions capable of converting phenylalanine and tyrosine to the level of ferulic acid when the above p-hydroxycinnamic acid oxidase was blocked with ascorbic acid. The ratelimiting step was the hydroxylation of p-hydroxycinnamic acid.     

Hexokinases of Tobacco Leaves: Changes in the Cytosolic and Non-Cytosolic Isozyme Complexes Induced by Tobacco Mosaic Virus Infection

Changes in the cytosotic (soluble) and the non-cytosolic (particulate) isozyme composition of hexokinases and in their properties were studied by ion exchange chromatography on DEAE cellulose after the subcellular fractionation both in the healthy and the tobacco mosaic virus (TMV) infected tobacco leaves. Three main isozyme complexes were obtained: one particulate fraction (the particulate hexokinase phosphorylating both glucose and fructose, EC 2.7.1.1), and two soluble fractions (the soluble hexokinase phosphorylating both the glucose and the fructose, and the soluble fructokinase, which phosphorylates primarily fructose, EC 2.7.1.4). The total fructokinase activities were nearly twice higher than the total glucokinase activities (188.6 % of glucokinase activity in healthy plants and 181.3 % in infected plants). The total particulate glucokinase activity was increased to 120.6 % and the fructokinase to 118.9 % in TMV infected tissue when compared with healthy control. The similar pattern of activity was observed for soluble hexokinase isozymes - the sum of soluble glucokinase activity was increased to 175.4 % and of fructokinase activity to 131.2 % in TMV infected tissue. The isozymes isolated both from the healthy control and TMV-infected leaves had the similar elution profiles, displayed Michaelis-Menten kinetics, showed the identical profiles of pH optima and were Mg²⁺ dependent with the highest enzyme activity at equimolar Mg²⁺ and ATP concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Guanylate cyclase in the accessory gland of the cricket, Acheta domesticus

Guanylate cyclase (E.C. 4.6.1.2.) was investigated in the accessory reproductive gland of the male house cricket, Acheta domesticus, which is known to accumulate exceptionally high levels of guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Accessory gland guanylate cyclase activity was linear with time for at least one hour, and with enzyme concentration to about 5 mg soluble protein per ml. Activity was dependent on Mn²⁺ and was maximal at pH 7.3 to 8.0. Sodium fluoride had no effect on activity, but sodium azide was slightly stimulatory. About 80% of the activity was sedimentable at 16,000 g, and both soluble and particulate activities were increased slightly in the presence of Triton X-100. Kinetic analysis indicated half-maximal velocity at 85 μM GTP in the presence of excess Mn²⁺, and reciprocal plots were concave upward. Changes in activity during maturation of the gland were small, and did not provide evidence for a regulatory role of guanylate cyclase in the accumulation of accessory gland cyclic GMP. The regulation and rôle of cyclic GMP in the accessory gland are discussed.     

Purification and characterization of an intracellular esterase from a Fusarium species capable of degrading dimethyl terephthalate

Esterase is the key enzyme involved in microbial degradation of phthalate esters (PAEs). In this study, an intracellular esterase was purified from a coastal sediment fungus Fusarium sp. DMT-5-3 capable of utilizing dimethyl terephthalate (DMT) as a substrate. The purified enzyme is a polymeric protein consisting of two identical subunits with a molecular mass of about 84 kDa. The enzyme showed a maximum esterase activity at 50 °C and was stable below 30 °C. The optimal pH was 8.0 and the enzyme was stable between pH 6.0 and 10.0. The esterase activity was inhibited by Cr³⁺, Hg²⁺, Cu²⁺, Zn²⁺, Ni²⁺, and Cd²⁺. Substrate specificity analysis showed that the enzyme was specific to DMT hydrolysis, but had no effect on other isomers of dimethyl phthalate esters (DMPEs) or monomethyl phthalate esters (MMPEs). These findings suggest that the phthalate esterase produced by Fusarium sp. DMT-5-3 is inducible and distinctive esterases involved in hydrolysis of the two carboxylic ester linkages of DMPEs.     

Studies On Cyclic Nucleotide Metabolism In Tetrahymena Pyriformis: Partial Characterization of Cyclic Amp- and Cyclic Gmp-Dependent Phosphodiesterases*

SYNOPSIS. Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in Tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction: the activities were optimal at pH 8.0–9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg²⁺ or Mn^2-. A kinetic analysis of the properties of the enzymes yielded 2 apparent K_III values ranging in concentration from 0.5 to 50 μM and from 0.1 to 62 μ M for cyclic AMP and GMP. respectively. A Ca²⁺-dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, Tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca²⁺, although this activator did not stimulate the phosphodiesterase. the results suggested that Tetrahymena might contain 2 types of Ca²⁺-dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Activation of the renal cortical and hepatic guanylate cyclase-guanosine 3′,5′-monophosphate systems by nitrosoureas divalent cation requirements and relationship to thiol reactivity

Streptozotocin, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and N-methyl nitrosourea, compounds with both oncogenic and cytotoxic properties, increased guanylate cyclase activity in the 100 000 × g soluble fractions of rat renal cortex and liver 35- to 65-fold over basal values. Particulate enzyme activities of these tissues were increased 2- to 4-fold by a maximally effective concentration of the nitrosoureas. In the presence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, maximally effective concentrations of these nitrosoureas increased cyclic GMP accumulation of hepatic and renal cortical slices to peak levels 7- to 10-fold over control in 30 min. By contrast, with the structurally related carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) peak increases occurred in 5–10 min and were 40- to 70-fold over control levels in renal cortex and liver, respectively. Unlike the Ca²⁺-dependent actions of cholinergic stimuli on cyclic GMP, the nitrosoureas and MNNG increased cyclic GMP in either the presence or absence of extracellular Ca²⁺. Moreover, while basal soluble guanylate cyclase of renal cortex was highly Mn²⁺-dependent and decreased 85% when either Mg²⁺ or Ca²⁺ was employed as sole divalent cation in reaction mixtures, the actions of nitrosoureas on enzyme activity were well expressed with either Mn²⁺ or Mg²⁺, but not with Ca²⁺, as sole divalent cation. Improved utilization of Mg²⁺ by guanylate cyclase in the presence of nitrosoureas would favor enhanced enzyme activity under cellular conditions where Mg²⁺ is abundant. In the presence of maximally stimulatory concentrations of streptozotocin or BCNU, high concentrations of Mg²⁺ or Mn²⁺ further increased soluble guanylate cyclase, suggesting important differences in metal and nitrosourea stimulation of enzyme activity.Preincubation of supernatant fractions with nitrosoureas plus dithiothreitol inhibited the action of the N-nitroso compounds to increase renal cortical guanylate cyclase. Glutathione and cysteine were also inhibitory, but less effective than dithiothreitol. Initial incubation of nitrosoureas with dithiothreitol in buffer alone similarly suppressed the subsequent action of the N-nitroso compounds on guanylate cyclase, and implicated direct chemical interactions. Prior incubation of renal cortical supernatant fractions with the SH blockers N-ethylmaleimide or maleimide significantly suppressed guanylate cyclase activation mediated by streptozotocin or BCNU. Direct drug interactions seemed unlikely, since effects of the inhibitors were optimally expressed by initial exposure of the supernatant fraction of tissue to the SH blockers and were not potentiated by a 30 min preincubation of the SH blockers and nitrosoureas in buffer alone.Thus, nitrosoureas activate and alter the metal requirements of soluble guanylate cyclase and increase cellular cyclic GMP in the presence or absence of extracellular Ca²⁺. Activation of soluble guanylate cyclase by nitrosoureas may involve an interaction of these agents with tissue SH groups, and possibly SH to SS transformation. Stimulation of the guanylate cyclase system by nitrosoureas could be related to the oncogenic actions of these agents.     

Conditions of cultivation,composition, and biological activity of mycelium of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> (Fr.) P. Karst

A study is made on a strain of higher basydiomycete Flammulia velutipes (Fr.) P. Karst. The conditions of maximum biomass production by Flammulia velutipes were studied. Soluble and insoluble fractions were isolated from mycelium. The composition of cultured mycelium and aqueous extracts from mycelium were investigated. These objects mainly contained carbohydrates (65.3 and 84.0% in insoluble and soluble fractions, respectively, and 56% mycelium), proteins (7.5–10.0% in fractions and 17.5% in mycelium), as well as an insignificant amount of mineral substances. The main carbohydrate component of fractions was glucose (53.6–78.8%); galactose and mannose were also present, as well as fucose and xylose in insignificant amounts. The aqueous extracts from mycelium demonstrated immunomodulating activity. They rendered a stimulating effect on the functional activity of macrophages—central cells of the reticluoendothelial system. The soluble fraction had a more pronounced effect than the insoluble fraction.     

Structural and functional diversity of Entamoeba histolytica calcium-binding proteins

Entamoeba histolytica (E. histolytica) is an etiological agent of human amoebic colitis, and it causes a high level of morbidity and mortality worldwide, particularly in developing countries. Ca²⁺ plays a pivotal role in amoebic pathogenesis, and Ca²⁺-binding proteins (CaBPs) of E. histolytica appear to be a major determinant in this process. E. histolytica has 27-EF-hand containing CaBPs, suggesting that this organism has complex Ca²⁺ signaling cascade. E. histolytica CaBPs share (29–47%) sequence identity with ubiquitous Ca²⁺-binding protein calmodulin (CaM); however, they do not show any significant structural similarity, indicating lack of a typical CaM in this organism. Structurally, these CaBPs are very diverse among themselves, and perhaps such diversity allows them to recognize different cellular targets, thereby enabling them to perform a range of cellular functions. The presence of such varied signaling molecules helps parasites to invade host cells and advance in disease progression. In the past two decades, tremendous progress has been made in understanding the structure of E. histolytica CaBPs by using the X-ray or NMR method. To gain greater insight into the structural and functional diversity of these amoebic CaBPs, we analyzed and compiled all the available literature. Most of the CaBPs has about 150 amino acids with 4-EF hand or EF-hand-like sequences, similar to CaM. In a few cases, all the EF-hand motifs are not capable of binding Ca²⁺, suggesting them to be pseudo EF-hand motifs. The CaBPs perform diverse cellular signaling that includes cytoskeleton remodeling, phagocytosis, cell proliferation, migration of trophozoites, and GTPase activity. Overall, the structural and functional diversity of E. histolytica CaBPs compiled here may offer a basis to develop an efficient drug to counter its pathogenesis.     

Boophilus microplus: strain differences of the cholinesterase system.

Cholinesterase (EC 31.1.7 and EC 31.1.8) activity was determined for homogenates of two Argentinian strains of larval ticks, Boophilus microplus. One strain (strain A) was sensitive to organophosphate acaricides. The other strain (strain G) was insensitive to coumaphos and to a lesser degree to other acaricides. For both strains maximal activity was found at 8 × 10^?3M substrate, showing inhibition by excess substrate. Maximal activity was recorded at pH 6.7 and 7.8, the latter peak being more evident for strain G. Values of Q₁₀ of 1.15 and 1.25 were obtained for strains A and G, respectively. Centrifugation at 27,000g resulted in the separation of the total cholinesterase activity into two fractions, one soluble and one particulate; the soluble fraction in strain G showed marked resistance to inhibition by an organophosphate ester and to heat inactivation.