Characterization and Biosynthesis of Cyclic-AMP-Binding Proteins in the Rat Central Nervous System

Cyclic-AMP-binding proteins in membrane and soluble fractions from rat forebrain were compared; membrane fractions included smooth and rough microsomes and a plasma membrane fraction enriched in synaptic membranes. Protein fractions were treated with 8-azido-[32P]cyclic AMP and ultraviolet irradiation to covalently tag cyclic-AMP-binding proteins. Labeled proteins were then analyzed by two-dimensional gel electrophoresis (2DGE) and fluorography. The soluble CNS proteins contained two major cyclic-AMP-binding species at 48K (48K 5.5 and 48K 5.45), differing slightly in their isoelectric points. Another protein was seen at 54K (54K 5.3) adjacent to the beta-tubulin subunits in the 2D electrophoretogram. The analysis of the smooth microsome and plasma membrane fractions differed from the soluble fraction in that there were two cyclic-AMP-binding proteins adjacent to the beta-tubulin region (54K 5.3 and 52K 5.3) differing slightly in apparent molecular weight. The membrane fractions also contained a cyclic-AMP-binding protein at 54K 5.8. The 52K 5.3 and 54K 5.8 species were unique to the membrane fractions. The rough microsomes did not contain detectable amounts of cyclic-AMP-binding proteins. Free polysomes were isolated from brain tissue, and translation products were analyzed by cyclic AMP affinity chromatography and immunopurification with antibodies to the brain specific type II regulatory subunit. The translation products that were found to bind cyclic AMP Sepharose are as follows: 48K 5.5, 48K 5.45, 52K 5.3, and 54K 5.8. These species comigrated with proteins that were photoaffinity-labeled in cytosol and membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new species of Kanahia (Asclepidiaceae) with a reconsideration of the genus

A new species, K. carlsbergiana , is described in what is currently considered a monotypic genus. The new species is only known from permanent streams in the Arssi and Bale regions of S Ethiopia. The delimitation and position of the genus is reconsidered in light of the additional information provided by the new species. The distinctiveness of the genus is reconfirmed whilst no new clues to possible relationships with other genera were observed. The taxonomy of the other species, K. laniflora (Forssk.) R. Br., is also briefly reconsidered.     

Biosynthesis of the third component of complement (C3) by the human monocytic-cell line U-937. Induction by phorbol myristate acetate.

Phorbol myristate acetate (PMA)-stimulated human monocyte-like cells (U-937) were found to synthesize the third component of complement (C3), as shown by enzyme-linked immunosorbent assay and immunoprecipitation from [35S]methionine-labelled culture supernatants. C3 synthesis occurred at a rate of about 160 ng of C3/24 h per 10(6) cells on day 7 after addition of PMA; it was blocked by cycloheximide treatment and was restored after removal of the inhibitor. SDS/polyacrylamide-gel-electrophoretic analysis of the immunoprecipitated protein showed that the size and subunit structure of the newly synthesized C3 were identical with those of plasma C3, and that a single-chain intracellular precursor was present in the cell lysates. Haemolytic assays showed that the synthesized C3 fully expressed functional activity in early culture within 4 h. After longer culture, a loss of haemolytic activity was observed. The possibility that newly secreted C3 is cleaved by U-937 cells themselves was suggested.     

Detection of gonadotrophic hormones on isolated granulosa cells of the domestic hen, Gallus domesticus, by an immunohistochemical method

Two immunohistochemical methods were used to detect the presence of luteinizing hormone (LH) on the cells of chicken granulosa. Using a peroxidase-labelled anti-rabbit serum together with anti-chicken LH serum raised in rabbits, a strong positive response was obtained with granulosa cells from small and large pre-ovulatory follicles obtained from the mid-cycle. Similarly, by using an available antiserum to human follicle stimulating hormone (FSH), a slightly weaker response was obtained with cells from both large and small follicles. After incubating cells with ovine LH, ovine FSH and ovine prolactin, there was no detectable difference with the method used in reaction with their respective antisera to cells which had received no incubation, implying that chicken gonadotrophins were displaced only partially from their receptors by mammalian gonadotrophic hormones. Pre-incubation of the cells with human chorionic gonadotrophin gave negative results with anti-hCG serum. Using a fluorescent-labelled antibody method, similar results were obtained except that the distribution of the receptors on the granulosa cell for LH or FSH appeared to be different. With the LH, the fluorescence formed a halo around the cell in contrast to the overall fluorescence with FSH.     

Antimicrobial activity and physico-chemical properties of some alkyldimethylbenzylammonium chlorides

A series of alkyldimethylbenzylammonium chlorides have been synthesized with n-alkyl chain lengths of C1 leads to C18. Octanol/water partition coefficients were determined and the antimicrobial activity assessed as the minimum growth inhibitory concentrations towards twelve strains of micro-organisms, representative of Gram-negative and Gram-positive bacteria, yeasts and fungi. The data were subjected to a numerical analysis. Antimicrobial activity of the compounds was found to be a parabolic function of their lipophilicity and maximized with n-alkyl chain lengths of between C12 and C16. The data fit to quadratic functions estimated for low (C1-C7) and high (C8-C16) alkyl chain length compounds was better than for a single quadratic describing the activity of the complete series (C1-C18). These maximized at log P values corresponding to alkyl-chain lengths of approximately C7 and C14 respectively, and were suggestive of low and high affinity binding sites upon the cell surface. The data analysis allowed the chain lengths of compounds with optimal activity towards the various groups of organisms to be determined. Generally yeasts and fungi were most sensitive towards C12, Gram-positive bacteria towards C14, and the Gram-negative bacteria towards C16. Gram-negative cells were the most resistant towards all the compounds and Gram-positive cells the least.     

Dominance status and certain operants in a communal colony of rhesus macaques

This study was designed to test the hypothesis that in some species of primates individual differences in responsiveness to certain situations is related to dominance status. During the first phase of the study, the existence of a linear dominance hierarchy was confirmed by ratings of agonistic interactions. In the second phase, bar-pressing behavior was recorded on a cumulative recorder while the experimenter simultaneously rated, at 30-second intervals, all animals present in the research setting. Results indicated that dominance status was systematically related both to rate of bar-pressing and to duration of response blocks, with the more dominant animals bar-pressing at slower rates for longer blocks of time. The finding that individual differences in rate did not vary with social context suggests that dominance-related differences in responsiveness may be quite stable. Certain dominancerelated trends in the variation of social context in the research setting were also noted.     

The isolation of cholest-5-ene-3β,26-diol from human brain (Short Communication)

Cholest-5-ene-3beta,26-diol, isolated from human brain, was further characterized by oxidation to 3-oxocholest-4-en-26-ol and to 3-oxocholest-4-en-26-oic acid. Identification was achieved by comparison (by t.l.c., g.l.c. and g.l.c.-mass spectrometry) with corresponding reference compounds derived from kryptogenin.     

The quantitative assay of 5''-nucleotidase in brain by histophotometry

Synopsis A method for the photometric assay of 5-nucleotidase in histochemical preparations of brain is described. The method, which is based upon the determination of the specular density under the microscope of the final reaction product, is shown to be valid statistically because of the correlation of the values obtained by it with those obtained by biochemical assay of the enzyme in adjacent sections. Correlation of the specular density with the amount of lead in the final reaction product is also shown. The accuracy of the method of assay is good as judged by a coefficient of variation of 9.1% in a series of replicate measurements. The assay of the amount of enzymic activity by this method shows that it is linearly proportional to the incubation time for periods of up to 2 hr, and also for sections ranging in thickness from 5 to 20 .