Properties of epinephrine-induced activation of cardiac adenosine 3′,5′-monophosphate-dependent protein kinase

The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (—cyclic AMP/+cyclic AMP) of 12 000 × g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 μg/kg) resulted from an increase in independent protein kinase activity (—cyclic 2 AMP) without a change in total protein observ activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 μg/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.     

RNA-DNA hybridization analyses of tRNA-Val-3b in Drosophila melanogaster

Summary Transfer RNA was extracted from 50–300 mg of adult flies and specifically labeled in vitro. The level of individual isoacceptors was quantitated by efficient annealing to Drosphila tRNA genes carried on recombinant DNA plasmids immobilized on nitrocellulose filters. The level of tRNA _3b ^Val in the tRNA isolated from flies deficient in the major tRNA _3b ^Val loci has been examined. The results show that deletion of the major tRNA _3b ^Val loci resulted in a reduction of approximately 50% in the level of tRNA _3b ^Val but did not produce the Minute phenotype; furthermore the effects of deficiencies at two loci were approximately additive.     

Restriction endonuclease mapping of the colicin Ib plasmid

Summary A cleavage site map of the colicin Ib plasmid (ColIb) has been determined for the enzymes Sall, XhoI, and HindIII by analysis of partial digests, double digests, DNA-DNA hybridization, and Tn5-induced insertion mutants. The site of the colicin gene has been determined by probing with cloned DNA coding for colicin production, as well as by analysis of a colicin negative ColIb:Tn5.     

Chromium oxalate: A new spin label broadening agent for use with thylakoids

Potassium tris(oxalato)chromate(III) trihydrate (chromium oxalate) has been shown to be a more useful broadening agent than potassium ferricyanide for the spin label 2,2,6,6-tetramethylpiperidine-N-oxy-4-amine (Tempamine) in thylakoid suspensions. Our data show that chromium oxalate is less permeable than ferricyanide, does not inhibit thylakoid electron transport or photophosphorylation, and is not photoreduced by thylakoids.     

SYNAPTIC VESICLES ISOLATED FROM RAT HEART: l-[3H]NOREPINEPHRINE UPTAKE PROPERTIES1

A fraction containing synaptic vesicles was isolated from rat heart by differential centrifugation, and the uptake of l-[³H]norepinephrine was studied in vitro., Uptake was highly dependent upon time and temperature, and was linear for 6 min at 30° or 4 min at 37°C. About 80% of the measured uptake required both ATP and Mg²⁺ and was inhibited by nanomolar concentrations of reserpine; no inhibition was obtained with cocaine. These properties are characteristic of storage vesicle uptake as opposed to synaptic membrane uptake. Uptake of norepinephrine was saturable and displayed a single K_m value of 2 μM. The uptake was completely stereospecific, as unlabeled dl-norepinephrine was less than half as effective as unlabeled l-norepinephrine in reducing uptake of l-[³H]norepinephrine. Norepinephrine uptake could be inhibited by various phenethylamines and indoleamines following the rank order: reserpine > harmaline > 5-hydroxytryptamine > dopamine > norepinephrine. The vesicle preparation also incorporated [³H]5-hydroxytryptamine and [³H]dopamine. 5-Hydroxytryptamine uptake displayed a K_m of 0.5 μM and a maximal uptake equivalent to that seen with norepineph-rine; dopamine uptake followed complex kinetics. Administration of reserpine in vivo or destruction of sympathetic neurons by long-term guanethidine treatment both eliminated the ability of the preparation to take up norepinephrine. Synaptic vesicles of cardiac sympathetic neurons thus resemble vesicles prepared from other central and peripheral catecholaminergic tissues; this method may be used readily to examine drug effects on rat heart synaptic vesicle function.     

Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cm^r, Tc^r and Em^r determinants that are expressed in E. coli. Only the Em^r determinant is expressed in S. sanguis. Both the Cm^r and the Tc^r of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras in to S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345–353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.     

Zinc levels in zinc-stabilized insulin are inhibitory to the growth of cells in vitro

Summary Adult human prostatic epithelium was cultured in a defined medium consisting of RPMI 1640 supplemented with transferrin, insulin, epidermal growth factor, dexamethasone, and vitamin A. In the presence of insulin, stabilized with zinc, maximum epithelial multiplication was obtained at an insulin concentration of 0.03 to 0.1 U/ml, corresponding to a zinc concentration of 1.4×10⁻⁷ M. At higher insulin concentrations, growth stimulation declined. Zinc-free insulin, on the other hand, stimulated cell multiplication with an optimum concentration of 0.3 to 1.0 U/ml. At this concentration the maximum growth was twice that obtained with zinc-stabilized insulin. Results demonstrate that growth inhibition caused by zinc limits the concentration of zinc-stabilized insulin, which can be used in serum-free, defined culture media. This work was supported by the Division of Cancer Cause and Prevention, National Cancer Institute, DHHS Grant No. CA-28279 to Webber.     

Pleiotropic effects of a null mutation in the c-fos proto-oncogene.

The c-fos proto-oncogene has been implicated as a central regulatory component of the nuclear response to mitogens and other extracellular stimuli. Embryonic stem cells targeted at the c-fos locus have been used to generate chimeric mice that have transmitted the mutated allele through the germline. Homozygous mutants show reduced placental and fetal weights and significant loss of viability at birth. Approximately 40% of the homozygous mutants survive and grow at normal rates until severe osteopetrosis, characterized by foreshortening of the long bones, ossification of the marrow space, and absence of tooth eruption, begins to develop at approximately 11 days. Among other abnormalities, these mice show delayed or absent gametogenesis, lymphopenia, and altered behavior. Despite these defects, many live as long as their wild-type or heterozygous littermates (currently 7 months). These data indicate that c-fos is not required for the growth of most cell types but is involved in the development and function of several distinct tissues.     

An analog of ACTH/MSH (4–9), ORG-2766, reduces cerebral uptake of morphine

The uptake of morphine was significantly reduced in most regions of the brains of conscious, unrestrained rats within 10 minutes after treatment with an analog of ACTH/MSH (4–9), ORG-2766. The effect was most obvious in regions with significant densities of enkephalin receptors, namely basal ganglia, hippocampus and cortex. The results explain, in part, how some fragments and analogs of ACTH/MSH may antagonize behavioral actions of morphine, even though some of these peptides lack significant opiate receptor binding properties. We believe that this effect of ORG-2766 is related to an action on the permeability characteristics of the brain microvasculature. The underlying mechanism is unknown.