Transgenic animals as a tool for studying the effect of the c-myc proto-oncogene on cardiac development

Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis.     

Decrease in insulin-containing secretory granules and mitochondrial gene expression in mouse pancreatic islets maintained in culture following streptozotocin exposure

We have previously described a preferential reduction in the secretory response to nutrient secretagogues in pancreatic mouse islets maintained in culture after in vitro exposure to streptozotocin (SZ). This reduction was associated with an impaired substrate metabolism at the mitochondrial level. To further clarify this issue, mouse pancreatic islets were exposed in vitro to 2.2 mM SZ for 30 min. At 4 h after SZ treatment ultrastructural changes were apparent in the endoplasmic reticulum and Golgi areas of the B-cells. However, 2 and 6 days following SZ exposure the B-cells appeared well preserved, except for a marked decrease in the number of insulin-containing secretory granules. A morphometric analysis of the B-cells 6 days after SZ exposure showed a normal B-cell size and a normal volume fraction of B-cell mitochondria. However, there was a decrease in total islet size and a 13% decrease in the volume fraction of B-cells in the islets. These mouse islets exhibited a decreased content of the mitochondrial DNA-encoded cytochrome b mRNA, as evaluated by dot-blot analysis. As a whole, the data obtained indicate that SZ treatment does not induce a decrease in the number of mitochondria or long-lasting ultrastructural damage to this organelle. However, there is a clear decrease in the cytochrome b mRNA, suggesting that SZ can induce damage to the mitochondrial DNA.     

Molecular cloning and characterization of the trpC gene from Penicillium chrysogenum

Summary We cloned the Penicillium chrysogenum trpC gene from a genomic library by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. The gene ecodes a 2.7 kb poly(A)⁺ RNA. We localized the gene by sequence analysis in a 2.9 kb DNA insert found in the smallest plasmid selected from the library. Sequence data strongly suggest that the organization of the gene is similar to that described in other Ascomycetes. We found that a DNA fragment which codes only for the carboxy-terminal protion of the polypeptide is sufficient for complementation of the E. coli trpC9830 mutation.     

An Examination of the Involvement of Phospholipases A2 and C in the α-Adrenergic and γ-Aminobutyric Acid Receptor Modulation of Cyclic AMP Accumulation in Rat Brain Slices

Experiments were undertaken to define the role of two calcium-associated enzyme systems in modulating transmitter-stimulated production of cyclic nucleotides in rat brain. Cyclic AMP (cAMP) accumulation was examined in cerebral cortical slices using a prelabeling technique. The enhancement of isoproterenol-stimulated cAMP production by alpha-adrenergic and gamma-aminobutyric acid-B (GABAB) agonists was reduced by exposing the tissue to EGTA, a chelator of divalent cations, or quinacrine, a nonselective inhibitor of phospholipase A2. Likewise, chronic (2 weeks) administration of corticosterone decreased the alpha-adrenergic and GABAB receptor modulation of second messenger production. Neither cyclooxygenase nor lipoxygenase inhibitors selectively influenced the facilitating response of alpha-adrenergic and GABAB agonists. Other experiments revealed that although norepinephrine and 6-fluoronorepinephrine stimulated inositol phosphate (IP) production in cerebral cortical slices with potencies equal to those displayed in the cyclic nucleotide assay, selective alpha 1-adrenergic agonists were less efficacious on IP formation and were without effect in the cAMP assay. Conversely, a selective alpha 2-adrenergic receptor agonist facilitated the cAMP response to a beta-adrenergic agonist without affecting IP formation. The rank orders of potency of a series of alpha-adrenergic antagonists suggest that IP accumulation is mediated solely by alpha 1-adrenergic receptors, whereas the augmentation of cAMP accumulation is regulated by a mixed population of alpha-adrenergic sites. The results suggest that the alpha-adrenergic and GABAB receptor-mediated enhancement of isoproterenol-stimulated cAMP formation appears to be more closely associated with phospholipase A2 than phospholipase C and may be mediated by arachidonate or some other fatty acid.     

雄激素对大鼠储精囊分泌蛋白mRNA的调节作用

本文报道了应用DNA重组和分子克隆技术研究雄激素与大鼠储精囊分泌蛋白基因的相互作用。大鼠储精囊总mRNA在逆转录酶作用下合成dsc-DNA,并克隆于pBR322/X1776中。经原位杂交法筛选含有互补于雄激素调节的mRNA的三个克隆株,其中二株经信使选择杂交翻译法证实它们是编码54K和16.6K道尔顿的分泌蛋白质的基因。同时应用后者的cDNA作为探针进一步研究雄激素对处于不同生理态状下的大鼠储精囊mRNA水平的影响。     

Effect of temperature and cell density on cellular protein content in temperature-sensitive mutants of rat 3Y1 diploid fibroblasts

Summary We examined cellular protein content in four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) under various conditions of culture that affect cell proliferation. When proliferation of the ts mutants was inhibited at a nonpermissive temperature (39.8°C) in the G₁ phase, prominent accumulation of cellular protein occurred in three mutants (3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) but not in 3Y1tsD123. The over-accumulation of protein at 39.8°C in the former three mutants was inhibited at high cell densities. At low cell densities there was an upper limit in the protein accumulation at 39.8°C. When the three mutants, proliferation-arrested at high cell densities at 33.8°C, were replated sparsely in fresh medium and shifted to 39.8°C, proliferation was completely inhibited whereas over-accumulation of protein occurred. These results indicating dissociation of protein accumulation and cell proliferation suggest that the two events are regulated by different mechanisms. This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (1984) to K. Y. from the Ministry of Education, Science, and Culture, Japan.     

Translation and functional expression of cell-cell channel mRNA inXenopus oocytes

Summary mRNA from estrogen-stimulated rat myometrium, a tissue known to upregulate cell-cell channels in response to this hormone, was microinjected intoXenopus laevis oocytes. The oocytes had been freed from covering layers of follicle cells and vitelline to allow direct cell membrane interactions when paired. About 4 hours after the mRNA injection, paired oocytes become electrically coupled. This coupling was due to the presence of typical cell-cell channels characterized by size-limited intercellular tracer flux, the presence of gap junctions at the oocyte-oocyte interface, and the reversible uncoupling that occurred in the presence of carbon dioxide. The induction of new cell-cell channels in the oocyte membrane was observed against a zero background or a low level of endogenous coupling, depending on the maturation stage of the oocytes. The time course of development of cell-cell coupling after the microinjection of mRNA was determined. The mRNA capable of inducing cell-cell coupling was confined to an intermediate size class when fractionated on a sucrose gradient.     

Carotenoid composition in milo (Sorghum vulgare) shoots as affected by phytochrome and chlorophyll

The composition of coloured carotenoids in the milo shoot was investigated quantitatively (high performance liquid chromatography) during light-mediated plastidogenesis, including the time span of photodelay as caused by medium and high light fluxes. It was found that as long as only the far-red-absorbing form of phytochrome operates, the carotenoid pattern remains virtually the same as in complete darkness (violaxanthin and lutein as major constituents, traces of -carotene). On the other hand, the pattern changes dramatically in white or red light with increasing amounts of chlorophyll (lutein and -carotene dominate, -carotene showing the strongest relative increase). Photodelay during the early phase of plastidogenesis affects the carotenoid composition strongly. Increase of neoxanthin, violaxanthin and -carotene contents are diminished while lutein accumulation proves resistant towards chlorophyll-mediated photoinhibition. The photodelay can be diminished by an appropriate light pretreatment. The data indicate that light-mediated control over carotenoid accumulation is exerted at three levels: i) a coarse control through phytochrome, ii) fine tuning in connection with chlorophyll accumulation, iii) stabilization of holocomplexes against photodecomposition.Abbreviations GG14 high fluence rate green-yellow light - HPLC high-performance liquid chromatography - Chl chlorophyll - WL_w weak white light (1200 lx) - WL_m medium flux white light (12000 lx)