Regulation of protein kinase C activity by gangliosides

The activity of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the presence of phosphatidylserine and its physiological regulator, diacylglycerol, could be suppressed by a mixture of brain gangliosides. Half-maximal inhibition was observed at 30 microM and was nearly complete at 100 microM. Inhibition was observed at all concentrations of Ca2+ between 10(-8) and 10(-4) M. Inhibition of protein kinase C activity could not be reversed by increasing the concentration of diacylglycerol or the substrate, histone. Inhibition was also observed when myelin basic protein or a synthetic myelin basic protein peptide was used as substrate. Among the individual gangliosides, the rank order of potency was GT1b greater than GD1a = GD1b greater than GM3 = GM1. Our results suggest that gangliosides may regulate the responsiveness of protein kinase C to diacylglycerol.     

Purification and characterization of p93fes- and p60src-related tyrosine protein kinase activities in differentiated HL-60 leukemia cells

Two tyrosine protein kinase activities have been identified previously to be present in HL-60 leukemia cells during induction of granulocytic and monocytic differentiation with a variety of differentiating agents. We have copurified a membrane-associated tyrosine kinase (p93) and an activity associated with both the cytosol and membrane fractions (p60). Triton X-100 extracts from HL-60 cells treated with dimethyl sulfoxide were subjected to tyrosine-agarose chromatography, polypropyl aspartamide high performance liquid chromatography (HPLC), and HPLC using an antiphosphotyrosine IgG-derivatized column. Overall purification was 2700-fold for p93 and 1800-fold for p60. p60 and p93 are phosphorylated exclusively on tyrosine residues and can use poly(Glu,Tyr)4:1, histone H1 and vasoactive intestinal peptide as substrates. Poly(Glu,Tyr)1:1 and poly(Glu,Ala,Tyr)6:3:1 were less effective substrates for p60 and p93. The activity of p93 was dependent on Mg2+ or Mn2+, whereas p60 was dependent on Mg2+; however, the activity of p60 was stimulated in a synergistic manner by the presence of both Mg2+ and Mn2+, whereas the activity of p93 was not enhanced further by the combination of divalent ions. Both p60 and p93 were immunoprecipitated by an anti-v-src monoclonal antibody but only p93 was immunoprecipitated by an anti-v-fps/fes antibody. V8 protease digestion of p60 revealed one major proteolytic fragment containing phosphotyrosine, whereas V8 protease digestion of p93 produced two major peptides that were phosphorylated on tyrosine residues. These results suggest that, although p93 and p60 may possess some epitopic similarities, they have distinguishing phosphorylation sites. Moreover, p93, in contrast to p60, appears to be strictly associated with granulocytic/monocytic differentiation and related to the cellular fps/fes protooncogene.     

Characterization of tumor-associated fucogangliosides from PC 12 pheochromocytoma cells

PC 12h pheochromocytoma cells were subcutaneously transplanted into rat. We found the transplanted tumors accumulated some fucogangliosides associated with PC 12 cells. These gangliosides were isolated and purified by DEAE-Sephadex A-25 and Iatrobeads column chromatographies. Their structures were determined by fast atom bombardment mass spectrometry, proton nuclear magnetic resonance spectrometry, permethylation study, and sequential degradation using various exoglycosidases and mild acid hydrolysis. Two tumor-associated fucogangliosides were found to possess the blood group B determinant as follows: G6: IV2Fuc alpha, IV3Gal alpha, II3NeuAc, GgOse4Cer; G11: IV2Fuc alpha, IV3Gal alpha, II3 (NeuAc)2, GgOse4Cer. A ganglioside with the similar structure as ganglioside G6 was isolated from rat hepatoma cells (Holmes, E.H., and Hakomori, S-I. (1982) J. Biol. Chem. 257, 7698-7703). However, ganglioside G11 has not previously been reported in the literature. These fucogangliosides reacted with the monoclonal antibody prepared by immunizing mice with PC 12h cells. Other fucogangliosides were also found to accumulate in the transplanted tumor tissues. They were identified as fucosyl-GM1 and fucosyl-GDlb. These fucogangliosides did not react with the monoclonal antibody against PC 12h cells.     

Properties of bovine heart mitochondrial cytochrome b560

A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome b-c1 particles is achieved by a procedure involving Triton X-100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome b560/mg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 mumol of succinate oxidized per min per mg of QPs protein at 23 degrees C. Although cytochrome b560 in isolated QPs is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome b560 is shown to be physically associated with succinate dehydrogenase by the following observations. The dithionite reduced form of cytochrome b560 in isolated QPs has a symmetrical alpha-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b560 in succinate-ubiquinone reductase. Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome b560 in the QPs becomes insensitive to carbon monoxide treatment. The redox potential of cytochrome b560 in QPs is -144 mV which is higher than that of cytochrome b560 in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome b560 in QPs preparation becomes identical to that of cytochrome b560 in succinate-ubiquinone reductase. Cytochrome b560 in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome b560 in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome b560, indicating that cytochrome b560 is indeed a unique cytochrome b and not a denatured product of cytochrome b562 or b565.     

脑室注射P物质对大鼠血压、心率及交感传出活动的影响

给乌拉坦麻醉六鼠侧脑室注射P物质(SP)10μg,引起动脉血压、心率和内脏交感神经放电增加。同样剂量的SP静脉注射后却引起血压降低。阿托品预处理不影响SP的开心率作用。预先脑室注射0.25,4,64μg阿片受体拮抗剂纳洛酮,对SP的升压效应有剂量依赖式对抗作用。以上说明脑室注射SP 引起的血压升高是交感神经活动增强,导致心率加快及外周血管紧张性增加的结果,并提示SP的中枢升压效应可能与脑内释放内源性阿片样物质有关。     

Development of immunoassay methods by use of liposomes

In order to develop liposomes for use in an immunoassay system, the preparation of immune liposomes and their characterization have been investigated. Liposomes have potential use in extremely sensitive analytical immunoassays, in addition to serving as an attractive drug delivery system. This liposome immunoassay system is based on membrane immunochemistry and an enzymatic reaction. An intense yellow color, easily detectable with the naked eye, was produced quite rapidly by the lysis of bovine serum albumin (BSA)-labeled, alkaline phosphatase-entrapped liposomes in the presence of anti-BSA rabbit serum and active complement under alkaline conditions. Sensitive detection is possible because of the antigen-antibody complex reaction, which leads to liposome lysis and an enzymatic reaction. The liposome immunoassay method offers a rapid, simple, and sensitive testing procedure which can quantitatively and qualitatively determine the presence or absence of antigenic materials and antibodies.     

Detection of luteinizing hormone beta messenger ribonucleic acid (RNA) in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated complementary RNA probe

Patterns of gonadotropin storage in individual gonadotropes change with alterations in the physiological state. After castration in the male rat, there is a 2.5-fold increase in the percentage of gonadotropes and an increase in the proportion of gonadotropes storing both LH and FSH. In addition, there are 6- to 8-fold increases in the pituitary concentrations of LH beta subunit mRNAs. In order to determine whether these changes are due to increases in the number of gonadotropes containing subunit mRNA, or the amount of mRNA per cell or both, an in situ hybridization technique using a photobiotinylated rat LH beta cRNA probe (bio-LH beta-cRNA) was applied to detect LH beta mRNA in fixed whole rat pituitary cells from intact or castrated rats. After hybridization, the bio-LH beta-cRNA was localized with either avidin-biotin peroxidase complex or the fluorescent streptavidin phycoprobe methods. The cells containing LH beta mRNA were then counted and the amount of mRNA per cell was measured by video microdensitometry. Ten percent of the anterior pituitary cells from intact animals contained LH beta mRNA. After castration (2-4 weeks) this percentage rose to 19-24.5%. Image and microdensitometric analyses showed that castration produced a 1.9-fold increase in the amount of LH beta mRNA per cell, and a 2.2-fold increase in the area of cells containing LH beta mRNA. Hence, castration resulted in an increase in the level of LH beta mRNA per cell as well as the number of LH beta mRNA-containing cells. When in situ hybridization was followed by immunocytochemistry in cells from intact rats, 83% of gonadotropes that stained for LH beta and 80% of gonadotropes that stained for FSH beta contained LH beta mRNA whereas after castration 99% of LH-storing and 93% of FSH-storing cells contained LH beta mRNA. This new in situ hybridization protocol is rapid and allows quantification of mRNA within individual gonadotropes. In addition, since the hybridization protocol does not apparently alter the gonadotropin antigens, the hormone content of the same gonadotrope may be defined by immunocytochemistry.     

Influence of thyroidal status on pituitary content of thyrotropin beta- and alpha-subunit, growth hormone, and prolactin messenger ribonucleic acids

Thyroidectomized rats were used to study the effects of a single injection of T3 on pituitary mRNA synthesis and hormone secretion. T3 was injected ip at doses of 0, 0.2, 1, or 5 micrograms/100 g body weight, and and animals were killed 24 h later. T3 caused a significant decrease in serum TSH, but caused no significant change in either serum GH or PRL. Pituitary mRNA was quantified by slot blot hybridization with cDNA probes specific for alpha-TSH, beta-TSH, PRL, and GH. We found that both the alpha and beta mRNA subunits decreased, that PRL mRNA remained relatively unchanged, and that GH mRNA increased with increasing T3 dose. The data show that a single dose of T3 can profoundly influence mRNA levels in the anterior pituitary; the lowest dose of T3 caused maximum inhibition of alpha-TSH mRNA while beta-TSH mRNA declined further in a dose-dependent manner.     

Molecular cloning of an endoglucanase gene from an alkalophilic Bacillus sp. and its expression in Escherichia coli.

One of the cellulase genes from alkalophilic Bacillus sp. strain N-4 was cloned in pBR322. A recombinant plasmid, pYBC107, expressing carboxymethyl cellulase (CMCase) was isolated, and the size of the cloned HindIII fragment was found to be 5.5 kilobases. The restriction map of pYBC107 showed a different pattern from those of pNKI and pNKII (N. Sashihara, T. Kudo, and K. Horikoshi, J. Bacteriol. 158:503-506, 1984). When the HindIII fragment from pYBC107 was subcloned into pYEJ001, there was a 3.8-fold increase in CMCase activity over that observed with pYBC107. Plasmid pYBC108 constructed by treatment of pYBC107 with HindIII and EcoRI expressed the CMCase activity, although to a limited extent. To verify the originality of cloned pYBC107 from Bacillus sp., we analyzed the restriction digest by Southern blotting.