Effects of 3′-deoxyadenosine triphosphate on in vitro RNA synthesis of plant RNA-dependent RNA polymerases

3′-deoxyadenosine triphosphate inhibited $\text{in}\text{vitro}$ [³H]UMP incorporation by RNA-dependent RNA polymerases from tobacco and cowpea plants. The inhibition of [³H]UMP incorporation could be reversed by simultaneous addition of higher ATP concentrations but not with increasing concentrations of UTP or when excess ATP was added 10 min after the inhibitor. These results suggest 3′-deoxyadenosine triphosphate competes specifically with ATP in reaction mixtures and results in premature termination of RNA synthesis $\text{in}\text{vitro}$ by RNA-dependent RNA polymerase.     

Nuclear 115cadmium: uptake and disappearance correlated with cadmium-binding protein synthesis.

The intracellular distribution of ¹¹⁵cadmium was determined following a pulsed exposure to the metal. The uptake and disappearance of label from rat liver nuclei was correlated with the appearance of a cytoplasmic Cd-binding protein. By coupling $\text{in}\text{vivo}$ - $\text{in}\text{vitro}$ experiments it was shown that unspecifically bound cadmium is free to enter the nucleus while specifically bound cadmium remains in the cytoplasm.     

Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of inner membrane.     

Serotonin action on short-circuit current and ion transport across isolated rabbit ileal mucosa.

Serotonin has previously been implicated as the cause of the diarrhea associated with carcinoid syndrome and the amine has been shown by others to be an intestinal secretagogue in preparations of intestinal loops $\text{in}$$\text{vivo}$. In the present paper the action of serotonin on isolated segments of rabbit ileal mucosa stripped of muscle layers was studied $\text{in}$$\text{vitro}$. Serotonin (10^?4M) caused an abrupt significant rise in short-circuit current (Isc) across the mucosal epithelial cell layer but this effect was transient. No change was observed in tissue conductance. In this preparation, serotonin did not alter ²²Na, ³⁶Cl or residual ion fluxes across the mucosa. High blood serotonin levels for a period of several days also did not alter ion fluxes or Isc in isolated rabbit ileum. Therefore, it is concluded that serotonin must cause its secretory activity observed $\text{in}$$\text{vivo}$ by some mechanism other than a direct action on epithelial cell transport mechanisms.     

Loss of hepatoma ribosomal RNA during warfarin therapy

40–50% decreases in cytoplasmic ribosomal RNA were observed in mouse hepatoma implants, but not livers, after 4–5 daily warfarin injections. Similar treatment greatly depressed rates of $\text{in}\text{vivo}$¹⁴C-orotate incorporation into hepatoma ribosomal RNA in the cytoplasm. Labelling of mature 18S and 28S RNA in the nucleus appeared to be unaffected. Possible mechanisms for this warfarin effect are briefly discussed.     

Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum.

The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both $\text{in}$$\text{vivo}$ (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and $\text{in}$$\text{vitro}$ (1–100μM). $\text{in}$$\text{vitro}$, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS $\text{in}$$\text{vivo}$ did not alter dopamine-induced stimulation of AC activity. MS, $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10⁻³M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.     

Androgen-induced gene activation in the rat prostate

The effect of androgens on gene activation in the rat prostate has been investigated by examining precursor incorporation into RNA, by DNA-RNA hybridization of RNA transcribed $\text{in}\text{vitro}$ from prostate chromatin, and by thermal denaturation of prostatic chromatin. The results show a selective synthesis of nuclear RNA and a changed thermal melting profile of prostatic chromatin as a result of testosterone administration. Further, the $\text{in}\text{vitro}$ synthesized RNA transcribed from prostatic chromatin of androgen-treated rats contained new RNA species that were not transcribed from chromatin of untreated castrated controls. The data provide direct evidence for an activated state of the prostatic chromatin stimulated by androgens.     

5' Cap methylation of homologous poly A(+) RNA by a RNA (guanine-7) methyltransferase from Neurospora crassa.

RNA (guanine-7) methyltransferase, partially purified from $\text{N.}\text{crassa}$ mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both $\text{N.}\text{crassa}$ poly A(+) RNA and reovirus unmethylated mRNA. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated poly A(+) RNA from $\text{N.}\text{crassa}$ yielded the “cap” structures m ⁷G(5′)pppAp and m ⁷G(5′)pppGp in a ratio of 2:1 respectively. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated reovirus mRNA yielded m ⁷G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in $\text{N.}\text{crassa}$ mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. $\text{187}$, 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate.     

Isolation of an androgen acceptor from salt extract of rat prostatic chromatin

The soluble androgen acceptor has been isolated from 0.35 M NaCl extract of rat prostatic chromatin by affinity chromatography on DNA-cellulose. The acceptor activity was assayed by interaction with 5α-dihydrotestosterone-receptor. Native DNA enhances this interaction. Polyacrylamide gel electrophoresis of the acceptor under denaturing conditions reveals a single polypeptide of molecular weight of 14,000. Amino acid analysis shows that the acceptor protein contains a higher content of acidic amino acid residues than basic amino acid residues. In an $\text{in}\text{vitro}$ RNA synthesizing system catalyzed by rat RNA polymerase II, addition of the acceptor stimulates RNA synthesis. Based on incorporation of [γ-³²P]ATP and [γ-³²P]GTP, the stimulation by the acceptor is mainly on the initiation of RNA chains.     

Differences in aminoacylation in vivo and in vitro of lysine isoaccepting tRNAs from virus-transformed cells

When murine sarcoma virus-transformed cells are labeled with [³H]lysine $\text{in}\text{vivo}$ for various periods, 5 of 6 isoaccepting lysine tRNAs separable by RPC-5 chromatography are aminoacylated in 1 hr to the same extent that they are aminoacylated $\text{in}\text{vitro}$. The sixth isoacceptor, tRNA₆^Lys, is not aminoacylated $\text{in}\text{vivo}$ to a measurable extent in 1 hr, although it is present in the tRNA prepared from the cells. All six isoacceptors are aminoacylated with [³H]lysine $\text{in}\text{vivo}$ when the labeling period is 2 or 3 hr. These results further show that $\text{in}\text{vitro}$ correlations of the amount of tRNA₄^Lys with cell division accurately reflect the situation $\text{in}\text{vivo}$. Results of differential centrifugation indicate that tRNA₆^Lys occurs in mitochondria.     

The estrogenic activity of DDT: Invivo and invitro induction of a specific estrogen inducible uterine protein by o,p'-DDT

The pesticide o,p'-DDT stimulates the production of a specific uterine protein, the so-called induced protein or IP, normally associated with an estrogenic response of the uterus. $\text{In}$$\text{vivo}$ stimulation of IP production is observed 1 hour after the administration of 250 mg/kg of o,p'-DDT to immature rats. $\text{In}$$\text{vitro}$ stimulation of IP production is observed after a 1 hour incubation of uteri with 100 μM o,p'-DDT. This $\text{in}$$\text{vitro}$ response is blocked by Actinomycin D. In contrast to o,p'-DDT, which binds to the cytoplasmic estrogen receptor and stimulates IP production, p,p'-DDT which does not bind well to the estrogen receptor does not stimulate IP production $\text{in}$$\text{vitro}$. These findings represent the first report of an estrogenic effect of o,p'-DDT in a completely $\text{in}$$\text{vitro}$ system.     

Unscheduled DNA synthesis in isolated hepatic nuclei after treatment of rats with methylnitrosourea in vivo

Administration of the carcinogenic methylating agent, methylnitrosourea, to rats caused a significant increase in endogenous DNA synthesis assayed subsequently in isolated hepatic nuclei $\text{in}\text{vitro}$. DNA synthesis was related directly to the dose of carcinogen and inversely to the interval between treatment and isolation of nuclei. This synthesis appears to represent the continuation $\text{in}\text{vitro}$ of unscheduled, reparative DNA synthesis initiated in damaged cells $\text{in}\text{vivo}$.