Modulation of platelet responsiveness through selective phosphorylation of plasma membrane proteins: Antagonistic eeffects of cyclic AMP and cyclic GMP

³²P phosphorylation of plasma membranes from human blood platelets, under conditions that closely resemble physiological ones (endogeneous phosphate donors and intact platelets in homologous plasma), result in the incorporation of the label mainly in a membrane glycoprotein of apparently high molecular weight (greater than 400 000). Dibutyryl cyclic AMP, an inhibitor of platelet aggregation, specifically increases the degree of phosphorylation of this glycoprotein. Moreover, it has been found that prostaglandin E₁ one of the most potent inhibitors of platelet aggregation which also increases phosphorylation of the same glycoprotein, is significantly more effective than cyclic AMP.Cyclic GMP does not have any apparent effect on platelet aggregation. However, incubation of platelet-rich plasma with both cyclic GMP and cyclic AMP results in a partial recovery of the platelet responsiveness towards ADP-induced aggregation. Coincidently, the degree of phosphorylation of the high molecular weight glycoprotein under these conditions, although still higher than in controls (no nucleotides added), is significantly decreased as compared with cyclic AMP-treated cells. Furthermore, cyclic GMP inhibits the cyclic AMP-dependent protein kinase activity in isolated platelet plasma membranes.These results suggest a central role for this membrane phosphoglycoprotein in the triggering of platelet aggregation and, furthermore, suggest that modulation of its degree of phosphorylation may be exerted through some cyclic AMP/cyclic GMP relationship, which in the basal state might be critical for platelet responsiveness.     

Coprophilous fungi of the horse

A total of 1267 microfungi, including 35 Myxomycetes, were recorded from the fecal samples of the 60 horses; of these 395 were found on 20 saddle-horse feces, 363 on 20 race-horses and 509 on 20 working-horses. Eighty two species representing 53 genera were recorded; of these 7 were Zygomycetes, 18 Ascomycetes, 1 Basidiomycetes and 25 Fungi Imperfecti: 2 Myxomycetes. Common coprophilous fungi are in decreasing orderPilobolus kleinii, Saccobolus depauperatus, Mucor hiemalis, Lasiobolus ciliatus, Podospora curvula, Petriella guttulata, M. circinelloides, Coprinus radiatus, Dictyostelium mucoroides, Sordaria fimicola, C. miser, C. stercorarius, Acremonium sp., Coprotus granuliformis, Graphium putredinis, Iodophanus carneus, Chaetomium murorum, Podospora communis, P. inaequalis, P. setosa, Saccobolus versicolor andCladosporium cucumerinum. Species ofMyrothecium verrucaria, Actinomucor elegans, Kernia nitida, Spiculostilbella dendritica andMucorparvispora were found exclusively in working-horses feces.Badhamia sp., Anixiopsis stercoraria, Echinobotryum state ofD. stemonitis, Geotrichum candidum andOidiodendron sp. were found only in saddle-horses feces.Chlamidomyces palmarum andPhilocopra sp. were found exclusively in race-horses feces.Notes on infrequent or interesting fungi includeThamnostylum piriforme, Phialocephala dimorphospora, Rhopalomyces elegans andSpiculostilbella dendritica.     

Properties of a major α-globulin of rice endosperm

Globulin was isolated from milled rice (Oryza sativa, line IR480-5-9) by 5% NACl extraction and was precipitated by (NH₄)₂SO₂ or by dialysis against water. The extract was purified by repeated isoelectric precipitation at pH 4.5. The major globulin fraction (40%) exhibited one band by electrophoresis at pH 4.5 but two bands at pH 8.3. Similarly, one sharp peak was shown by sedimentation corresponding to 1.41S (α-globulin) in acetic acid (pH 2) and NaOH (pH 11.7) but a broad asymmetric peak was obtained at pH 6.7, 8.3 and 8.9. Gel filtration of the α-globulin at pH 6.5 exhibited 2 proteins with MW 20 000 and 98 000. The results suggest a pH dependent aggregation phenomenon. The two proteins could not be separated by DEAE-cellulose chromatography. SDS-polyacrylamide electrophoresis of α-globulin revealed one subunit with MW 18 000. This α-globulin is poorer in lysine and histidine but richer in cystine, methionine, arginine, tyrosine and glutamic acid than whole milled rice proteinfa]Taken part from the M.S. thesis of AAP from the University of the Philippine at Los Baños (1977).     

Protein-lipid interactions and differential scanning calorimetric studies of bacteriorhodopsin reconstituted lipid-water systems

Bacteriorhodopsin has been reconstituted at various molar concentrations into liposomes of dimyristoyl- and also of dipalmitoylphosphatidylcholine. Differential scanning calorimetry indicates that as the protein concentration within the lipid bilayer increases, the cooperativity of the lipid phase transition is reduced, i.e. the transition is broadened, while the midpoint transition temperature remains virtually unchanged. Freeze-fracture electron microscopy of our preparation shows, in agreement with previous data from other laboratories, that extensive protein aggregation occurs when the liposome is cooled below the T_c transition temperature of the lipid. Laser flash photolysis measurements of protein rotation of the bacteriorhodopsin show, especially in the case of protein-rich recombinants, that protein aggregates exist even above T_c. The perturbation caused by the presence of bacteriorhodopsin in the lipid bilayer is similar to that produced by other intrinsic proteins. The difficulty of correlating the observed calorimetric enthalpy data with a simple concept of a ‘boundary lipid layer’ based upon consideration of a single isolated protein is discussed in view of the occurrence of protein aggregates both above and below T_c. It is concluded that the reduction of enthalpy is related to the number of lipids which solvate the protein aggregates within the protein-lipid patches and are thereby removed from the cooperative melting and enthalpy of the remaining regions of pure lipid.     

Fructose 1,6-bisphosphatase: Dissociation of AMP cooperativity and AMP inhibition by carbamylation

Selective treatment of pig kidney fructose 1,6-bisphosphatase with potassium cyanate leads to the formation of an active carbamylated enzyme that has lost the cooperative interactions among AMP sites, but retains sensitivity to inhibition of catalytic activity by the regulator AMP. Incorporation data on [¹⁴C]KNCO indicate that the loss of enzyme cooperativity at the AMP sites is related to selective carbamylation of four lysine residues per mole of tetrameric enzyme. Exhaustive carbamylation suggests that a second lysine residue per subunit is essential for AMP inhibition.     

Transforming growth factor type beta 1 (TGF-beta 1) down-regulates interleukin-2 production and up-regulates interleukin-2 receptor expression in a thymoma cell line

Transforming growth factor type beta 1 (TGB-beta 1) belongs to a family of polypeptides with regulatory effects on growth and differentiation of a variety of cell types. TGB-beta 1 plays an important role in regulation of immune response by acting as a negative control signal for T cell proliferation through still unknown mechanisms. In this study we have analysed the effects of TGB-beta 1 on EL 4-6.1, a variant of the murine EL 4 thymoma, which can be induced by phorbol 12-myristate 13-acetate (PMA) and/or interleukin 1 (IL-1) to secrete interleukin 2 (IL-2) and express IL-2 receptors (IL-2R). Using this defined model system, we show that TGB-beta 1 simultaneously down-regulates IL-2 expression and up-regulates the number of both high and low affinity IL-2R. These changes correlate with changes at the mRNA level, suggesting an effect at the pre-translational level. The specificity of both TGF-beta 1 effects was demonstrated using a neutralizing antiserum to TGF-beta 1. Our data also suggest that TGF-beta 1 does not interfere with early activation signals of PMA and/or IL-1. This model might be useful for elucidating the complex role of TGF-beta 1 in the regulation of T cell responses.     

Progressive changes in the structure and dynamics of the phytoplankton community along a pollution gradient in a lowland river — a multivariate approach

The phytoplankton of the River Lujan (Buenos Aires, Argentina) was studied for a period of 18 months, together with physical and chemical variables, in relation to a pollution gradient. 167 taxa were recorded within a seasonal succession characterized by dominance of diatoms with a brief summer green algae facies. A combination of several biotic indices and multivariate analysis was employed to assess the impact of pollution on the phytoplankton community. The biotic indices used were species diversity and richness, algal quotients (green algae/diatom ratio, Centrales/Pennales ratio) and the SD succession rate index. Multivariate procedures included cluster analysis and ordination by PCA of both species and samples, stepwise discriminant analysis and multiple discriminant analysis of variance (MANOVA). Results indicate that community dynamism is attenuated at the more polluted sites, concomitant with an increased predominance of a broad-tolerance algal assemblage, co-dominated by Cyclotella meneghiniana and Nitzschia stagnorum. The changes in the community structure and dynamics described herein involved alterations in the distribution and relative proportions of the algae, rather than modifications in the basic species composition. These changes may not be readily detectable by methods which over-simplify the ecological information, such as systems of indicator species and biotic indices, designed to assess the degree of pollution. The suitability of multivariate analysis and biotic indices in river phytoplankton studies is further discussed.     

Postnatal Changes in the Activity Ratio of Specific and Nonspecific Cholinesterases from Neuronal Perikarya

Abstract: A soluble fraction from rat brain neuronal perikarya was shown to contain both the specific and nonspecific forms of the enzyme acetylcholines-terase (EC's 3.1.1.7. and 3.1.1.8., respectively). The ratio of the enzyme activities varied along the course of brain development: the nonspecific form being predominant from 1 to 15 days of age and the specific one showing the pattern of rising activity from day 15 onward. We suggest a possible relationship between this changing in cholinesterase activities and the establishment of synapses within the rat cerebral cortex.     

A gel-forming (1→3)-β-D-glucan isolated from cyttaria harioti fischer

An alkali-soluble glucan, [α]_D + 11° (M potassium hydroxide) having a degree of polymerization of 220, has been isolated from the fruit bodies of the tree fungus Cyttaria harioti Fischer. Periodate oxidation and methylation analysis show that it consists of a highly branched β-D-(1→3)-linked backbone. Hydrolysis of the methylated polysaccharide yielded 2,3,4,6-tetra-O-methyl- (24.5 mol%), 2,4,6-tri-O-methyl-(39.4 mol%), 2,3,4-tri-O-methyl- (8.6 mol%), and 2,4-di-O-methyl-D-glucose (27.5 mol%). Periodate-oxidation results substantiate the methylation studies. The general structural features of the glucan are discussed.