Variable stabilities and recoveries of rat-liver RNA polymerases A and B according to growth status of the tissue.

1. The effect of growth status on the relative levels and recoveries of DNA-dependent RNA polymerase in rat liver nuclei was determined by two independent procedures: (a) measurement of RNA polymerase A and B activities in fraction IV [Roeder, R. G. and Rutter, W. J. (1970) Proc. Natl Acad. Sci. U.S.A. 65, 675--682] in the presence and absence of low concentrations of alpha-amanitin; (b) DEAE-Sephadex chromatography of fraction IV to resolve RNA polymerases A and B (and possibly other forms of the enzyme). 2. Growth was arrested in young rats (less than 100 g body weight) by hypophysectomy and stimulated by the administration of growth hormone or triiodothyronine. Under these conditions the rate of RNA synthesis in vivo or in isolated nuclei is known to be markedly depressed or stimulated relatively soon after hypophysectomy or hormone administration, respectively. RNA polymerases were obtained from animals under different growth conditions. There were no differences in the activities of nuclear RNA ploymerases per se, when these were separated from their endogenous template and assayed with heterologous denatured DNA. These reports contrast with earlier reports [Smuckler, E. A. and Tata, J. R. (1971) Nat. New Biol. 234, 37--39; Sajdel, E. M. and Jacob, S. T. (1971) Biochem. Biophys. Res. Commun. 45, 707--715]. 3. The discrepancy was resolved when a 'balance sheet' of enzyme recovery was established. Cessation of growth by hypophysectomy led to a marked reduction in the recovery of both forms A and B of the enzyme (less than 20% of the input RNA polymerase activity in fraction iv) following chromatography on DEAE-Sephadex. This effect was reversed within a short time after the administration of growth hormone (3--9 h) or triiodothyronine (18--24 h), leading to a doubling of the enzyme recoveries. These alterations which were more marked for RNA polymerase A, resulted in different elution profiles for RNA polymerases A and B upon chromatography. 4. It is concluded that the use of DEAE-Sephadex chromatography to compare the levels of RNA polymerases A and B isolated from tissues of different growth rate can give rise to over-estimates of apparent changes in their relative activities and that the measurement of enzyme activity in fraction IV is a better index of RNA polymerase levels. The relationship between growth rate of cells, the stability of RNA polymerases, and the importance of determining enzyme recoveries upon chromatography, are discussed.     

Cell death: Are new proteins synthesized during hormone-induced tadpole tail regression?

The possibility that tadpole tail regression might be initiated by thyroid hormone-induced synthesis of new proteins was investigated. Changes in the newly-synthesized proteins of cultured Xenopus laevis tadpole tails treated with 1.5 × 10^?7 M tri-iodothyronine (T₃) were studied, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate for protein separation. After initial studies of unfractionated tail proteins, fractionated mixtures of [³H]methionine and [³⁵S]methionine labelled proteins derived from control and hormone-treated tails respectively were examined for hormone-induced changes. Using a new procedure developed to allow effective analysis of small differences in distribution of two isotopes within gel slices, it was shown that no significant changes in synthesis of fractionated tail proteins are induced by the hormone during the first 3–4 days in culture. The average detection limit was approx. 0.02% of total tail protein synthesis. Although no changes in the synthesis of the tissue structural or enzymic proteins are induced by the hormone this study still leaves open the possibility of changes in the synthesis of regulatory proteins. Based on the known method of activation of the tadpole tail collagenase (which is shown here directly for the first time to be involved in T₃-induced tail regression), it is suggested that many of the initial hormone-induced changes might result from T₃-induced activation of proteolytic “cascades”.     

Characterization of polysomes from Xenopus liver synthesizing vitellogenin and translation of vitellogenin and albumin messenger RNA's in vitro.

1. Conditions are described for the isolation of polysomes from the liver of Xenopus laevis. The method involves homogenization of liver in 0.2 M Tris-HCl pH 8.5, treatment with 2% Triton X-100 and subsequent sucrose density gradient fractionation of polysomes from a 10000 X g supernatant. 2. Vitellogenin synthesis was induced in male Xenopus liver by oestradiol treatment. Polysomes were isolated and vitellogenin-synthesizing polysomes characterized by their association with monospecific 125 I-labelled rabbit anti-vitellogenin antibody and by reaction with rabbit anti-vitellogenin immunoglobulins followed by indirect immunoprecipitation with goat anti-rabbit antibody. 3. Changes in liver polysome content following oestrogen treatment of male Xenopus are correlated with the appearance of vitellogenin synthesis using an organ culture assay. 4. RNA extracted from livers of oestradiol-treated male Xenopus and from purified polysomes is shown to code for the synthesis of vitellogenin-specific immunoprecipitable polypeptides in a rabbit reticulocyte cell-free protein-synthesizing system, a major component having a molecular weight of 210000. Xanopus liver RNA is also shown to code for the synthesis of an albumin-specific immunoprecipitable polypeptide of 74000 molecular-weight which coelectrophoresed with Xenopus albumin.     

Acceleration Workspace of Cooperating Multi-Finger Robot Systems

We present a mathematical method for acceleration workspace analysis of cooperating multi-finger robot systems using a model of point-contact with friction. A new unified formulation from dynamic equations of cooperating multi-finger robots is derived considering the force and acceleration relationships between the fingers and the object to be handled. From the dynamic equation, maximum translational and rotational acceleration bounds of an object are calculated under given constraints of contact conditions, configurations of fingers, and bounds on the torques of joint actuators for each finger. Here, the rotational acceleration bounds can be applied as an important manipulability index when the multi-finger robot grasps an object. To verify the proposed method, we used a set of case studies with a simple multi-finger mechanism system. The achievable acceleration boundary in task space can be obtained successfully with the proposed method and the acceleration boundary depends on the configurations of fingers.     

Differential expression and localization of calmodulin-dependent phosphodiesterase genes during ontogenesis of chick dorsal root ganglion

The level and characteristics of 3'-5'-cyclic nucleotide phosphodiesterase (PDE) activity in chick dorsal root ganglion (DRG) extracts of 5-day posthatching chicken (P5) and E10 and E18 embryos were studied. At all stages, PDE activity is stimulated by calcium and calmodulin. A 5-fold increase in basal cAMP and cGMP PDE activity is evident from E10 to E18, while from E18 to P5 basal PDE activity remains constant. Ion exchange chromatography elution profile indicates that PDE1 isoforms represent the bulk of the PDE activity present. Inhibition studies were performed in order to distinguish the activity due to PDE1A, B and C. Western blot analysis using anti-mammalian PDE1A, B and C specific antibodies was also performed. Densitometric analysis of the stained bands reveals that PDE1B and PDE1C display a prominent increase between day 10 and day 18 of development (eight- and 3.6 fold, respectively) while a more limited increase (1.6- and 1.5-fold) is observed between E18 and P5; on the other hand PDE1A shows continuously increasing levels throughout development. Immunohistochemical analysis was performed with isoform specific antibodies used for western blot analysis. PDE1A immunoreactivity is found in the cytoplasm and fibers of several neurons differing in size and distributed throughout the ganglion. PDE1B staining is evident on all neurons, however, fibers appear very faintly labelled. All neurons appear stained by PDE1C antibody, although the intensity of immunostaining is always heterogeneous in different neuronal populations: no staining was evident on fibers or in non-neural cells. The distinct spatial and temporal expression patterns of PDE1 isoforms may indicate their different physiological roles in developing and mature chick DRG.