Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

Identification of up-regulated genes in human uterine leiomyoma by suppression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.     

Choice in humans: Functional properties of reinforcers established by instruction

Adult human subjects chose between schedules containing stimuli (indicator lights) that the subjects were instructed to consider pleasurable. The schedules differed in amount of reinforcement (period of illumination) or delay (interval between a choice response and light onset). Although subjects preferred large to small amounts of reinforcement, they were essentially indifferent between immediate and delayed reinforcement. In contrast, previous data on video game reinforcement demonstrated preferences for both immediate and large amounts of reinforcement. The instructed reinforcer was thus partially effective in controlling choice but was not equivalent to a reinforcer that presumably had intrinsic value.     

Hyperthermia Induces the Synthesis of a Heat Shock Protein by Polysomes Isolated from the Fetal and Neonatal Mammalian Brain

Abstract: Analysis of the cell-free translation products of polysomes isolated from fetal brain and other organs indicates that elevation of maternal body temperature induces the synthesis of a heat shock protein of molecular weight 74,000 (74K). The newborn mammal is particularly sensitive to induction of the 74K protein. As early postnatal development proceeds, higher body temperatures are required to induce synthesis of the 74K heat shock protein.     

Molecular phylogeny of Heritiera Ait on (Sterculiaceae), a tree mangrove: variations in RAPD markers and nuclear DNA content

Random amplified polymorphic DNA (RAPD) markers are used to estimate interspecific variation among mangrove and non-mangrove Heritiera fomes, H. littoralis and H. macrophylla. All the species have 2n = 38 chromosomes, with minute structural changes distinguishing the karyotype of each species. Significant variation of 4C DNA content occurs at the interspecific level. Interspecific polymorphism ranged from 14.09% between H. fomes and H. littoralis to 52.73% between H. fomes and H. macrophylla. H. macrophylla showed wide polymorphism in the RAPD marker with H. littoralis (51.23%) and H. fomes (52.73%). Two distinct RAPD products obtained from OPA-10 (1000 bp) and OPD-15 (900 bp) found characteristic molecular markers in H. macrophylla , a species from a non-mangrove habitat. H. macrophylla was more distantly related to H. fomes [genetic distance (1-F) = 0.305] than to H. littoralis [genetic distance (1-F) = 0.273]. H. littoralis was of a closer affinity to H. fomes [genetic distance (1-F) = 0.218] than to H. macrophylla.     

Subcellular distribution of enzymes in the yeast Saccharomycopsis lipolytica, grown on n-hexadecane,with special reference to the ω-hydroxylase

The subcellular localization of the ω-hydroxylase of Saccharomycopsis lipolytica was assessed by the analytical fractionation technique, originally described by de Duve C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F., and hitherto little, if at all, applied to yeast. Protoplasts were separated in six fractions by differential centrifugation. Some of these fractions were further fractioned by density gradient centrifugation. The distribution of ω-hydroxylase and 15 other constituents chosen as possible markers of its subcellular membranes has been established. ω-Hydroxylase resulted in being bound to a membrane that containes also cytochrome P-450 and NADPH-cytochrome c reductase. This membrane clearly differs from five other subcellular entities. (1) Mitochondria were characterized by particulate malate dehydrogenase, particulate Antimycin A-insensitive NADH-cytochrome c reductase, oligomycin-sensitive and K⁺-stimulated ATPase pH 9. (2) Most if not all of the catalase and urate oxidase is peroxisomal. (3) Free ribosomes account for most RNA. (4) Nucleoside diphosphatase is for the first time reported in a yeast and appears to belong to an homogeneous population of small membranes. (5) The soluble compartment contains magnesium pyrophosphatase, alkaline phosphatase, 5′-nucleotidase and part of the NADH-cytochrome c reductase. Latent arylesterase and ATPase pH7 have an unspecific distribution. Alkaline phosphodiesterase I has not been detected.     

Validation of the first step of the heuristic refinement method for the derivation of solution structures of proteins from NMR data

A new method for the analysis of NMR data in terms of the solution structure of proteins has been developed. The method consists of two steps: first a systematic search of the conformational space to define the region allowed by the initial set of experimental constraints, and second, the narrowing of this region by the introduction of additional constraints and optional refinement procedures. The search of the conformational space is guided by heuristics to make it computationally feasible. The method is therefore called the heuristic refinement method and is coded in an expert system called PROTEAN. The paper describes the validation of the first step of the method using an artificial NMR data set generated from the known crystal structure of sperm whale carbon monoxymyoglobin. It is shown that the initial search procedure yields a low-resolution structure of the myoglobin molecule, accurately reproducing its main topological features, and that the precision of the structure depends on the quality of the initial data set.     

Cascade of autophosphorylation in the beta-subunit of the insulin receptor

Insulin stimulated autophosphorylation of the beta-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the beta-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C-terminus containing Tyr-1316 and Tyr-1322. In the presence of antiphosphotyrosine antibody (alpha-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that alpha-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1151. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of alpha-PY, which contained Tyr(P)-1146 and either Tyr(P)-1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the beta-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the beta-subunit was mainly (greater than 80%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the beta-subunit (White et al.: Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.