Lipoproteins of the egg perivitelline fluid of Pomacea canaliculata snails (Mollusca: Gastropoda)

The lipid and protein composition of the perivitelline fluid of the eggs of Pomacea canaliculata was investigated. Two lipoproteins (PV 1 and PV 2) and one lipoprotein fraction (PV 3) were detected for the first time in gastropods. They represent 57.0, 7.5, and 35.5% of the egg total proteins, respectively. PV 1 is a glyco-carotene-protein complex with characteristics of a very high-density lipoprotein (VHDL). It has 0.33% lipids, mainly free sterols and phospholipids. The particle has a MW of 300 Kd and is composed of three subunits of 35, 32, and 28 Kd, respectively. PV 2 particle is a VHDL of 400 Kd and 3.75% lipids. The major lipid classes are free sterols and phospholipids and also have significant quantities of energy-providing triacylglycerides and free fatty acids. It is composed of two apoproteins of 67 and 31 Kd. PV 3 density corresponds to a high-density lipoprotein (HDL). It was fractionated into two subfractions "h" and "p". Fraction "h" contains 5.16% lipids, mainly free sterols, phospholipids, and free fatty acids, and two particles of 100 and 64 Kd. Dissociating electrophoresis showed two subunits of 34 and 29 Kd. Fraction "p" is composed of a single particle of 26 Kd that contains 9.5% lipids, which represents 30% of the total egg lipids. It has high levels of a carotenoid pigment. Besides it contains free fatty acids, hydrocarbons, sterified sterols, and triacylglycerides. These three fractions are probably the major supply of lipids and amino acids for the developing embryo.     

Preparation and properties of partially purified pulmonary cytochrome P-450 from rabbits

Cytochrome P-450 from rabbit pulmonary microsomes was purified approximately 32-fold. The purification method involved solubilization of microsomes using sodium cholate, and recovery of cytochrome P-450 in the precipitate formed between 25 to 42% saturation of the digested microsomes with ammonium sulfate in the absence of glycerol. Further purification was achieved by chromatography on DEAE-cellulose and hydroxylapatite using Emulgen 913 as an eluent. Partially purified preparations containing up to 7.4 nmol of cytochrome P-450 per mg of protein were essentially free of NADPH-cytochrome c reductase activity and cytochromes b5 and P-420. However, epoxide hydrase was found to co-purify with cytochrome P-450. The CO-difference spectrum of dithionite-reduced purified cytochrome showed the expected peak at 450 nm. However, the magnitude of the peak was dependent on added microsomal lipid fraction in the assay medium. Purified pulmonary cytochrome P-450 formed typical types I and II substrate difference spectra with benzphetamine and pyridine, respectively. Sodium dodecyl sulfate-gel electrophoresis of partially purified cytochrome P-450 gave two major bands when stained with Coomassie blue. The faster moving band which contained peroxidase activity had an estimated molecular weight of 49,000 +/- 1,200. The cytochrome P-450 fraction, when combined with solubilized pulmonary microsomal NADPH-cytochrome c reductase and lipid fractions, was active in the O-deethylation of 7-ethoxycoumarin and the N-demethylation of benzphetamine.     

Chromatographic assay and peptide substrate characterization of partially purified farnesyl- and geranylgeranyltransferases from rat brain cytosol

The dose-response relationships for DNA fragmentation (assessed by pulsed-field gel electrophoresis, PFGE) and for viability (evaluated by measuring the reduction of MTT dye which can be accomplished by viable cells only) were investigated in order to discriminate between genotoxicity and cytotoxicity in the pathogenesis of DNA double-strand breaks (DSB). Cultured human lung epithelial cells (A549) were treated with the DNA-intrastrand crosslinker cisplatin, the DNA-interstrand crosslinker melphalan and the topoisomerase II inhibitor etoposide. The cytotoxic mode of DSB induction was investigated by using the mitochondrial respiratory chain toxin potassium cyanide (KCN) and the detergent Triton X-100. gamma-Irradiation induced a linear dose response for DSB which were efficiently repaired and did not cause reduction in cell survival over a period of 72 h. With etoposide and melphalan a significant increase in DSB was seen 8 h after treatment initiation with concentrations that did not affect cell survival, implicating genotoxicity as the causal event. In contrast, induction of DSB by KCN and Triton X-100, and also by cisplatin, was seen only after cell viability was reduced to less than about 60%, indicating that DSB were the consequence of extragenomic damage. This mechanistic distinction of the two classes was supported by DNA fragment length analysis. In line with a genotoxic mechanism and absence of additional cytotoxic effects, the DNA fragments generated by gamma-irradiation as well as by etoposide and melphalan displayed a distribution between 1 and 4 Mbp with a peak around 2 Mbp. In contrast, DNA fragments induced by Triton X-100 and KCN peaked below 0.5 Mbp, implicating activation of DNA-degrading enzymes. This type of investigation is suggested for the study of chemicals for potential DNA interstrand crosslinking, an important promutagenic type of DNA damage. To avoid false positive results in genetic toxicity testing it is suggested that all assays include a dose-response relationship for both genotoxicity and viability.     

Biochemical properties of a novel 28KDA protein tyrosine kinase partially purified from the particulate fraction of rat spleen

In this report we present some of the biochemical properties of the enzyme, here called pp28(PTK), isolated from particulate fraction of rat spleen (1). The kinase is very susceptible for polyions as regulators of the enzymatic activity. The polyanions like dextran sulfate or heparin inhibited, and polycations such as spermidin, protamin, poly-L-lysine and some random polypeptides containing tyrosine besides a basic amino acid, stimulated the enzyme markedly. The kinase showed high sensitivity towards class IA salts. In the casein phosphorylation reaction the apparent Km value for ATP was 4 microM. An unusual property is associated with autophosphorylation which leads to a reduced activity towards external substrates. Some kinase inhibitors described in the literature were tested for their potency.     

Binding of insulin analogs to partially purified insulin receptor from rat liver membrane (author's transl)

The insulin binding fraction was solubilized from rat liver membrane vesicles by triton and partially purified up to the specific binding activity of 2.1 pmol/mg. A further characteristic of the partially purified soluble receptor is a decrease in irreversible binding to 0.36% (+/- 0.28) with regard to total iodine insulin and to (+/- 1.8) in comparison to reversible binding. From Scatchard plots a high affinity binding site (KD = 5 X 10(-10)M) with low capacity (5 pmol/mg) and a low affinity binding site (KD = 3 X 10(-8) M) with high capacity (30 pmol/mg) can be seen. With carefully prepared liver membrane vesicles, the dissociation constant of the high affinity binding site from Scatchard plot is only 2 X 10(-9)M. With liver membrane vesicles, isolated for preparative purification procedure, the high affinity binding site could not be demonstrated. Displacement studies with insulin analogs were performed with [A1-D-alanine] insulin, [A1-L-alanine] insulin, [des-Gly-A1, NB1, NB29-(Msc)2]-insulin, proinsulin and [desoctapeptid B23-B30]-insulin. Results of binding measurements are presented in half-maximal iodo-insulin binding, in determination of inhibitor- and dissociation constants from Dixon-, Scatchard- and Lineweaver-Burk plots. There are equal relative binding potencies of analogs, observed with crude membrane vesicles and partially purified soluble receptor, although there is a 50-fold difference in specific binding activity. Biologically active insulins are characterized by strong binding to the high affinity binding site. The binding to the low affinity binding site is not correlated to the biological activity of the insulin analog. With insulin and biologically responsive analogs a non-linear curve in the double-reciprocal Lineweaver-Burk plot can be observed. Analogs with low biological activity show a linear dependency. Functional interactions of insulin with the receptor can be demonstrated in a high affinity binding with the first binding site of the Scatchard plot and in a non-linear hyperbolic Lineweaver-Burk plot.     

Membrane potential at the low pH in neurons from the snail, Lymnaea stagnalis L

In the snail, the pH 6.7-6.8 decreased the neuron membrane potential in 10% of cases and increased it in 30%. The hyperpolarisation was equal to 9(5 mV, the effect being abolished by addition of 5-10 M strophantidine. The findings suggest that low pH activates sodium conduction, the hyperpolarisation being caused by activation of the K+/Na(+)-pump.     

Growth factor receptor expression during in vitro differentiation of partially purified populations containing murine stem cells

The Word Accentuation Test assesses the accentuation of 30 infrequent Spanish words written without the accentuation mark and is an easy-to-use tool for estimating premorbid intelligence of Spanish-speaking people. Its intraobserver (0.97) and interobserver (0.93) reliabilities and its correlation with the Wechsler Adult Intelligence Scale (.837) and Raven's Progressive Matrices (.655) are high, offering a good prediction of general intelligence. It is resistant to mental deterioration; 20 demented and 40 controls matched by sex, age, and education obtained similar scores. The discrepancies between current and predicted scores in Raven's scale can diagnose mild-moderate dementia with 0.79 accuracy (sensitivity, 0.78; specificity, 0.82).     

A new glycosaminoglycan from the giant African snail Achatina fulica

A new glycosaminoglycan has been isolated from the giant African snail Achatina fulica. This polysaccharide had a molecular weight of 29,000, calculated based on the viscometry, and a uniform repeating disaccharide structure of -->4)-2-acetyl,2-deoxy-alpha-D-glucopyranose (1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->. This polysaccharide represents a new, previously undescribed glycosaminoglycan. It is related to the heparin and heparan sulfate families of glycosaminoglycans but is distinctly different from all known members of these classes of glycosaminoglycans. The structure of this polysaccharide, with adjacent N-acetylglucosamine and 2-sulfo-iduronic acid residues, also poses interesting questions about how it is made in light of our current understanding of the biosynthesis of heparin and heparan sulfate. This glycosaminoglycan represents 3-5% of the dry weight of this snail's soft body tissues, suggesting important biological roles for the survival of this organism, and may offer new means to control this pest. Snail glycosaminoglycan tightly binds divalent cations, such as copper(II), suggesting a primary role in metal uptake in the snail. Finally, this new polysaccharide might be applied, like the Escherichia coli K5 capsular polysaccharide, to the study of glycosaminoglycan biosynthesis and to the semisynthesis of new glycosaminoglycan analogs having important biological activities.     

Chronic exposure of rat fat cells to insulin enhances lipolysis and activation of partially purified hormone-sensitive lipase

The activity of adipose tissue hormone-sensitive lipase in animals with hyperinsulinemia has been reported to be increased compared with that in control animals. We examined whether this results from a direct effect of insulin on the tissue and whether it is accompanied by alteration in the regulation of lipolysis. When rat epididymal fat pads are incubated in culture medium with bovine serum albumin for 2-4 h with 2 ng/ml or 50 microU/ml of insulin, hormone-sensitive lipase activity in the postmicrosomal supernatant fraction after acid precipitation and activation with ATP-Mg2+ increases significantly compared with preparations from tissues incubated with the vehicle. The specific activities of hormone-sensitive lipase in sonicates of adipocytes after primary culture with insulin at concentrations from 10 to 4000 ng/ml (250 microU to 100 mU/ml) increase in an insulin-dose-related manner. Lipolysis in response to 10(-7) M isoproterenol also increases in an insulin-dose-dependent manner. Enhancement of isoproterenol-mediated lipolysis is not attributable to a difference in the triglyceride content of the cells. Lipolysis caused by the beta-agonist could be completely blocked by the simultaneous presence of insulin in both control and insulin-treated cells reflecting normal responsiveness of both types of cells to the acute effect of insulin. Although an increase in lipolysis is seen with norepinephrine and growth hormone after insulin treatment, other lipolytic agents such as ACTH, thyrotropin, and glucagon evoke similar responses in insulin-treated and control cells. The simultaneous presence of growth hormone and insulin during the 16-h culture results in additive effects on the subsequent response of the cells to 10(-7) M isoproterenol compared with the responses of the cells cultured with each hormone alone. beta-Agonist-mediated cAMP accumulation in the presence of Ro-20.1724, a specific phosphodiesterase inhibitor, is significantly higher in cells cultured in the presence of insulin than in control cells. Forskolin (1-25 microM) increases the lipolytic responses of insulin-treated cells compared with control cells, but the maximal response of the insulin-treated cells to forskolin is lower than that to isoproterenol. We conclude that changes produced by chronic insulin treatment involve more than one site along the lipolytic cascade.     

Properties of purified papain-solubilized rat AgB antigens and reactivity of a xenoantiserum against the isolated antigens

Rat AgB transplantation antigens were isolated after papain digestion of spleens from the inbred strain Hooded Lister. Both subunits of the AgB antigens were present in the purified material. Some physical characteristics of the antigens have been determined. An antiserum, raised in a rabbit, against the purified material reacted exclusively with AgB antigens on splenocytes but detected novel structures on both adult and embryonic fibroblasts. These structures, antigenically related to AgB antigens, were not detected on plasmacytoma or hepatoma cells, nor did they display any antigenic similarity with rat beta 2-microglobulin. Radioimmunoassays specific for the AgB antigen heavy chain and for beta 2-microglobulin, respectively, were used to estimate the contents of these antigens in several tissues. Spleen and thymus exhibit the largest density, while brain is almost devoid of these antigens.     

Population studies on Oncomelania quadrasi, the snail intermediate host of Schistosoma japonicum, in the Philippines. 3. Data transformation for significance test of snail density

For the comparative study of densities of Oncomelania quadrasi, the snail intermediate host of Schistosoma japonicum in the Philippines, the survey data should be transformed to stabilize the variance in determining the significant difference of densities among populations and to evaluate control measures by the difference, since the snail is distributed unevenly in the field. Comparison was made for the efficiency of stabilizing variance among 7 transformation formulas so far reported, and 2 new additional formulas in the present study. As a result, a simple transformation by y=log (x+0.01) was found to be most reliable. This nonparametric transformation can be applied to any snail population irrespective of the type of distribution and the degree of clumping. By this formula, there is no need to discard snail-free samples which are important to evaluate control measures. Using this transformation, snail survey data were analyzed in the areas where control measures were undertaken during the past several years. And significant reduction of snail densities was proven using the t-test.     

Phase I study of VRCTC-310, a purified phospholipase A2 purified from snake venom, in patients with refractory cancer: safety and pharmacokinetic data

A phase I study was performed to evaluate the maximum tolerated dose (MTD), safety profile and pharmacokinetic data with VRCTC-310, a natural product derived from purified snake venom fractions, with phospholipase A2 activity and inhibitory effects against human and murine tumor cell lines. Fifteen patients with refractory malignancies were entered after providing written informed consent. VRCTC-310 was administered as an intramuscular injection daily for 30 consecutive days. Doses were escalated from 0.0025 to 0.023 mg/kg. Toxicities included local pain at the injection site, eosinophilia, reversible diplopia and palpebral ptosis. Dose escalation was stopped at 0.023 mg/kg, when two patients had developed anaphylactoid reactions. Both cases had high VRCTC-310-specific IgG by EIA. MTD was 0.017 mg/kg and the recommended dose for phase II studies is 0.017 mg/kg. Stabilization was found in six patients.     

Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca

The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit. In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.     

The reconstitution of microtubules from purified calf brain tubulin

The in vitro reconstitution of calf brain tubulin, purified by the method of Weisenberg et al. [(1968), Biochemistry 7, 4466-4479; (1970), Biochemistry 9, 4110-4116] as modified by Lee et al. [(1973), J. Biol. Chem. 248, 7253-7262], was successful in a medium consisting of 10(-2) M sodium phosphate, 10(-4) M GTP, and concentrations of magnesium ions ranging from 0.5 to 16 X 10(-3) M at 37 degrees. Filaments resembling native microtubules were formed. The filaments are in equilibrium with the associating species of tubulin and the equilibrium can be shifted to depolymerization by lowering the temperature to 20 degrees. Filament formation is inhibited by calcium ions which also cause disassembly of the formed filaments. The effects of calcium ion can be reversed by the addition of [ethylenebis-oxyethylenenitrilo)]tetraacetic acid. The formation of filaments is favored by the presence of 3.4 M glycerol; only twisted abnormal filaments are observed in the presence of 1 M sucrose. The high molecular weight components observed in the sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of many tubulin preparations were shown not to be essential for the formation of the filaments.     

A gene apparently determining the extent of sialylation of lysosomal alpha-mannosidase in mouse liver

Organ-specific electrophoretic heterogeneity of lysosomal alpha-mannosidase has been observed within individual strains of inbred mice. Polymorphism between C57BL/6J and CBA/J for liver lysosomal alpha-mannosidase is determined by a single genetic locus on chromosome 5 and appears to be the result of differences in sialylation of the lysosomal enzyme. Two different patterns of expression of development of the liver electrophoretic forms have been observed.     

Ultrastructural study of the shell-repair membrane in the snail, Helix pomatia L.

The shell-repair membrane of the snail, Helix pomatia, has been studied with the transmission electron microscope (TEM). The ultrastructure of the repair membrane, in the initial stages of calcification, revealed the presence of a fibrillar protein, proteoglycan granules, osmiophilic vesicles, and cytoplasmic dense bodies of different size and structure. The involvement of the cell constituents in the formation of calcifying centra and initial crystal formation is discussed. The amoebocytes present within the repair membrane appeared to be involved in three different functions: (1) phagocytosis, (2) release of granules, vesicles and dense bodies, and (3) secretin of a fibrillar protein. The possible lytic function of the amoebocytes is mentioned. The common features in the minearlizing process of the shell-repair membrane of the snail and the epiphyseal cartilage of the mammals were noted.     

Slime-trail tracking in the predatory snail, Euglandina rosea.

Euglandina rosea, a predatory land snail, tracks prey and mates by following slime trails. Euglandina follow slime trails more than 80% of the time, following trails of their own species, but not those of prey snails, in the direction that they were laid. The attractive elements of prey slime are small, water-soluble compounds detected by specialized lip extensions. Although olfaction plays no role in trail following, strong odors disrupt tracking. Inhibition of nitric oxide synthase also disrupts slime trail following, suggesting a role for nitric oxide in neural processing of slime trail stimuli. Euglandina can be conditioned to follow novel trails of glutamate or arginine paired with feeding on prey snails. These experiments demonstrate that slime-trail tracking in Euglandina is a robust, easily measured behavior that makes a good model system for studying sensory processing and learning in a novel modality. (PsycINFO Database Record (c) 2010 APA, all rights reserved)