Developmental changes in the activity and cellular localization of hepatic casein kinase II in the rat

Abstract The activity and cellular localization of hepatic casein kinase II(CKII) was examined during late fetal development in the rat. Cultured fetal hepatocytes displayed constitutive CKII activity which was not further activated by growth factor exposure. Similarly, fetal liver CKII showed approximately fivefold greater activity than adult liver. The fetal hepatic activity was, to a large degree, localized to a nuclear fraction. Postnuclear cytosol preparations from fetal and adult liver showed similar CKII activity. In all cases, FPLC ion exchange chromatography followed by Western immunoblotting showed that immunoreactive CKII coincided with kinase activity. However, Parallel determinations of CKII activity and immunoreactive CKII levels showed a higher(five-to sixfold) CKII specific activity in nuclear extracts compared to cytosol. In summary, fetal hepatic CKII demonstrates coincident nuclear localization and activation. We hypothesize that the regulation of hepatic CKII is relevant to the mitogen-independent proliferation displayed by fetal rat hepatocytes. © Wiley-Liss, Inc.     

A familial paracentric inversion: A short review of the current status

Summary We present here a familial case of a paracentric inversion in man with a short review of the literature. A paracentric inversion of chromosome 10(q11q26) was found in the amniocytes drawn for advanced maternal age. The presence of the inversion was investigated in 35 family members in three generations. No recombinants were recognized. The significance of these data for appropriate genetic counselling and possible reproductive risks is discussed.Genetic Nurses, Department of Health and Welfare     

New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

Summary We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 1100 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictivetemperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.     

Assortative mating and gene flow in the Lesser Snow Goose: A modelling approach

Nonrandom mating with respect to color is well documented in the dimorphic Lesser Snow Goose. A set of mathematical models and data from the long-term study at La Pérouse Bay, Manitoba, Canada are used to examine various hypotheses advanced to explain the mechanisms behind the assortative mating. Among the factors considered are mate choice based on familial color, accidental formation of genetically unrelated families, and nonuniform distribution of colors in the region where mate selection occurs.     

Dephosphorylation and activation of exogenous glycogen synthase by adipose-tissue phosphatase

Exogenous purified rabbit skeletal-muscle glycogen synthase was used as a substrate for adipose-tissue phosphoprotein phosphatase from fed and starved rats in order to (1) compare the relationship between phosphate released from, and the kinetic changes imparted to, the substrate and (2) ascertain if decreases in adipose-tissue phosphatase activity account for the apparent decreased activation of endogenous glycogen synthase from starved as compared with fed rats. Muscle glycogen synthase was phosphorylated with [gamma-(32)P]ATP and cyclic AMP-dependent protein kinase alone, or in combination with a cyclic AMP-independent protein kinase, to 1.7 or 3mol of phosphate per subunit. Adipose-tissue phosphatase activity determined with phosphorylated skeletal-muscle glycogen synthase as substrate was decreased by 35-60% as a consequence of starvation. This decrease in phosphatase activity had little effect on the capacity of adipose-tissue extracts to activate exogenous glycogen synthase (i.e. to increase the glucose 6-phosphate-independent enzyme activity), although there were marked differences in the activation profiles for the two exogenous substrates. Glycogen synthase phosphorylated to 1.7mol of phosphate per subunit was activated rapidly by adipose-tissue extracts from either fed or starved rats, and activation paralleled enzyme dephosphorylation. Glycogen synthase phosphorylated to 3mol of phosphate per subunit was activated more slowly and after a lag period, since release of the first mol of phosphate did not increase the glucose 6-phosphate-independent activity of the enzyme. These patterns of enzyme activation were similar to those observed for the endogenous adipose-tissue glycogen synthase(s): the glucose 6-phosphate-independent activity of the endogenous enzyme from fed rats increased rapidly during incubation, whereas that of starved rats, like that of the more highly phosphorylated muscle enzyme, increased only very slowly after a lag period. The observations made here suggest that (1) changes in glucose 6-phosphate-independent glycogen synthase activity are at best only a qualitative measure of phosphoprotein phosphatase activity and (2) the decrease in glycogen synthase phosphatase activity during starvation is not sufficient to explain the differential glycogen synthase activation in adipose tissue from fed and starved rats. However, alterations in the phosphorylation state of glycogen synthase combined with decreased activity of phosphoprotein phosphatase, both as a consequence of starvation, could explain the apparent markedly decreased enzyme activation.     

DEVELOPMENTAL CHANGES IN LEVELS AND FORMS OF CHOLINESTERASES IN MUSCLES OF NORMAL AND DYSTROPHIC CHICKENS

Abstract— Acetylcholinesterase (AChE) and pseudocholinesterase (°ChE) were studied in vivo and during the first several months of development of pectoral and posterior latissimi dorsi (PLD) muscles in normal and dystrophic chickens. Muscle extracts were prepared in a high ionic strength-nonionic detergent medium in the presence of protease inhibitors, in order to obtain complete solubilization and to prevent degradation of intrinsic molecular forms of both enzymes. In both normal and dystrophic pectoral muscles levels of AChE and °ChE increase rapidly in vivo, °ChE accounting for 5–10% of total cholinesterase activity. In the normal pectoral muscle the concentration of both enzymes drops rapidly after hatching with increasing muscle mass; total AChE per muscle remains relatively constant for 30 days post-hatch. In the dystrophic pectoral muscle both AChE and °ChE accumulate after hatching, resulting in greatly elevated levels (approx 10–25-fold) of both enzymes throughout the period studied. Multiple molecular forms of AChE and °ChE are observed in the pectoral muscle by sucrose gradient centrifugation. Four principal forms are distinguished: two light (L₁, L₂), one medium (M), and one heavy (H₂). The °ChE forms are 0.5–1.0 S units lighter than the corresponding AChE forms. L₂ is the predominant light form of AChE, whereas L₁ is the major light °ChE form detected. The lighter forms of AChE predominate in normal and dystrophic embryonic pectoral muscle at day 14, being replaced by the H₂ form by day 19. H₂ is the major °ChE form detected at day 19. After hatching, H₂ AChE is the predominant form found in both of the normal muscles studied. In the dystrophic pectoral muscle, progressive accumulation of the L₂ form of AChE is detected as early as day 4 post-hatch; this form eventually becomes predominant, although the heavier forms are also elevated. In PLD muscle the same phenomenon occurs, but with a slower time course. In dystrophic pectoral muscle a similar rise in the L₁ form of °ChE is first observed by day 4, with heavier forms also elevated in the mature muscle. Thus the alteration in the control of these two enzymes in dystrophic fast-twitch muscles results in an accumulation of the light forms of AChE and °ChE.     

Detection of the Viral Sarcoma Gene Product in Cells Infected with Various Strains of Avian Sarcoma Virus and of a Related Protein in Uninfected Chicken Cells

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.     

Pathway of Succinate and Propionate Formation in Bacteroides fragilis

Cell suspensions of Bacteroides fragilis were allowed to ferment glucose and lactate labeled with (14)C in different positions. The fermentation products, propionate and acetate, were isolated, and the distribution of radioactivity was determined. An analysis of key enzymes of possible pathways was also made. The results of the labeling experiments showed that: (i) B. fragilis ferments glucose via the Embden-Meyerhof pathway; and (ii) there was a randomization of carbons 1, 2, and 6 of glucose during conversion to propionate, which is in accordance with propionate formation via fumarate and succinate. The enzymes 6-phosphofrucktokinase (pyrophosphate-dependent), fructose-1,6-diphosphate aldolase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, and methylmalonyl-coenzyme A mutase could be demonstrated in cell extracts. Their presence supported the labeling results and suggested that propionate is formed from succinate via succinyl-, methylmalonyl-, and propionyl-coenzyme A. From the results it also is clear that CO(2) is necessary for growth because it is needed for the formation of C4 acids. There was also a randomization of carbons 1, 2, and 6 of glucose during conversion to acetate, which indicated that pyruvate kinase played a minor role in pyruvate formation from phosphoenolpyruvate. Phosphoenolpyruvate carboxykinase, oxaloacetate decarboxylase, and malic enzyme (nicotinamide adenine dinucleotide phosphate-dependent) were present in cell extracts of B. fragilis, and the results of the labeling experiments agreed with pyruvate synthesis via oxaloacetate and malate if these acids are in equilibrium with fumarate. The conversion of [2-(14)C]- and [3-(14)C]lactate to acetate was not associated with a randomization of radioactivity.