Catabolite repression of the formation of precursors of electron transfer complexes III and IV in adapting bakers' yeast: dependence upon a mitochondrially translated repressor.

When bakers' yeast cells which had been grown anaerobically in galactose were aerated in the presence of 10% glucose, they showed a 40% decrease in $\text{in}\text{vivo}$ [¹⁴C]-leucine incorporation into a washed mitochondrial membrane fraction compared with cells which had been aerated in a low glucose medium. The observed catabolite repression of membrane protein synthesis was primarily due to a decrease in cytoplasmic translational activity, but this repression was entirely dependent upon concomitant mitochondrial translation. The inductions of reduced coenzyme Q cytochrome $\text{c}$ reductase (complex III) and of cytochrome $\text{c}$ oxidase (complex IV) activities were repressed 30 and 60%, respectively, by aeration of the cells for 8 hours in 10% glucose. The catabolite repression of the formation of these two inner membrane complexes was again shown to be dependent upon concomitant mitochondrial translation. Both the amino acid incorporation and enzyme induction data suggest that catabolite repression of both cytoplasmically and mitochondrially translated mitochondrial membrane proteins is mediated through a mitochondrially translated repressor.     

Functional efficiency of mitochondrial membrane of rats with hepatic chronic iron overload

The effect of $\text{in}\text{vivo}$ hepatic iron overload, induced by two different amounts of iron, on the energy-transducing efficiency of the mitochondrial membrane has been examined. It has been found that when the epatic iron concentration is up to a threshold value mitochondria present an anomalous membrane potential. Addition of oligomycin fully restitutes it. A low content of intramitochondrial K⁺ is connected with this pathological condition. A relative lack of antioxidant capability is parallely exhibited by these mitochondria. A possible involvement of lipid peroxidation process $\text{in}\text{vivo}$ in causing the membrane potential drop and the net efflux of intramitochondrial K⁺ is suggested.     

The effect of long chain fatty acyl CoA esters on the adenine nucleotide translocase and myocardial metabolism.

The adenine nucleotide translocase, the transport protein for ADP and ATP, located in the inner mitochondrial membrane is an important site for the regulation of cell metabolism. Inhibition of the adenine nucleotide translocase by long chain fatty acyl CoA esters demonstrated $\text{in}$$\text{vitro}$ may also occur $\text{in}$$\text{vivo}$ when the complete oxidation of fatty acids by the myocardium has been compromised during ischemia. Reversal of this biochemical lesion may be of benefit in the preservation of the ischemic myocardium.     

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.     

Myocardial phospholipid metabolism in alloxan diabetic rats

Alloxan diabetes in rats was found to decrease the level of phospholipids in the heart. Measurement of specific phosphatides showed that the decrease was restricted only to phosphatidylethanolamine and lysophosphatidylcholine. Study of $\text{in}$$\text{vivo}$ incorporation of ³²Pi indicated an impairment of phosphatidylethanolamine synthesis and conversion of phosphatidylcholine into lysophosphatidyl choline in the heart of diabetic rats. Treatment of diabetic rats with insulin restored the levels of phosphatidylethanolamine and lysophosphatidylcholine and incorporation of ³²Pi into these phosphatides to almost normal.     

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.     

Sulfate transport in brush border membrane vesicles prepared from human placental syncytiotrophoblast

Isolated brush-border membrane vesicles prepared from human placenta are known to transport amino acids via a Na⁺-dependent mechanism akin to that found in gut and kidney vesicle preparations. We studied sulfate transport in placental vesicles and failed to identify any Na⁺-dependent uptake mechanism. Rather, uptake is a non-electrogenic process that is trans-stimulated by outwardly directed anion flux which is independent of cation. If anion exchange is tightly coupled $\text{in}\text{vivo}$, the net transfer of sulfate from mother to the growing fetus may be driven by the continuous flux of bicarbonate in the opposite direction.     

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.     

Phosphorylation of high mobility group proteins 14 and 17 by nuclear protein kinase NII in rat O6 glioma cells

High mobility group (HMG) proteins 14 and 17 of rat C₆ glioma cells are phosphorylated $\text{in}\text{vivo}$ on both serine and threonine. In HMG 14 about 60% of the total [³²P]phosphate was identified as phosphoserine and 40% as phosphothreonine. In HMG 17, there was 88% phosphoserine and 12% phosphothreonine. Glioma cell nuclear protein kinase NII phosphorylates HMG 14 and 17 $\text{in}\text{vitro}$ on serine as well as threonine and the relative percentages of [³²P]phosphoamino acid are similar to those seen $\text{in}\text{vivo}$. Nuclear protein kinase NI and the type I and II cAMP-dependent protein kinases exhibit only minor phosphorylating activity towards HMG 14 and 17. We conclude that nuclear protein kinase NII is responsible for the phosphorylation of HMG 14 and 17 $\text{in}\text{vivo}$.     

Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.     

Biosynthesis of 5-methylaminomethyl-2-thiouridylate. I. Isolation of a new tRNA-methylase specific for 5-methylaminomethyl-2-thiouridylate

A new enzyme, which catalyzes the transfer of a methyl group to tRNA to form 5-methylaminomethyl-2-thiouridylate, was isolated from $\text{E.}$$\text{coli}$ by a procedure including affinity chromatography. The purified enzyme was nearly homogeneous upon disc electrophoresis. Using methyl-deficient tRNA^Glu of $\text{E.}$$\text{coli}$ as substrate, the 5-methylaminomethyl-2-thiouridylate residue synthesized was mostly found in the anticodon loop, showing a coincidence of the modification site $\text{in}$$\text{vitro}$ with that $\text{in}$$\text{vivo}$.     

Effect of removing the zona pellucida on development of hamster and bovine embryos invitro and invivo

Uterine stage embryos collected from the hamster (8-cell) and cow (morula, early blastocyst) were monitored for development $\text{in}$$\text{vitro}$ (embryo culture) and $\text{in}$$\text{vivo}$ (embryo transfer) following premature removal of the zona pellucida.Removal of the zona pellucida did not significantly affect $\text{in}$$\text{vitro}$ development to the blastocyst stage of (1) 8-cell hamster embryos (zonae removed by a combined enzymic-mechanical procedure), (2) bovine morulae (zonae removed by mechanical means only) (3) early bovine blastocysts (zonae removed by the enzymic-mechanical technique).Zona-free hamster embryos formed significantly fewer viable fetuses than did zona-intact embryos. The lower incidence of fetal development observed following transfer of zona-free 8-cell hamster embryos may have resulted in part from the formation of chimeras by fusion of these embryos $\text{in}$$\text{utero}$. Such fusion was observed to occur $\text{in}$$\text{vitro}$ between zona-free embryos placed in close proximity. The proportion of pregnancies resulting from transfer of bovine blastocysts cultured from zona-free morulae was similar to that of zona-intact embryos.In this study we have demonstrated that (1) enzymic and mechanical procedures used to remove zonae pellucidae from uterine-stage hamster and bovine embryos do not adversely affect subsequent development of these embryos $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ and (2) zonae pellucidae are not required for normal development of these embryos. These findings have implications for microsurgery of mammalian embryos and for embryo transfer.     

Altered restriction of nuclear RNA during incubation in vitro

Nuclei were isolated from rat liver and incubated $\text{in}\text{vitro}$ in two commonly employed RNA transport assays. Released [¹⁴C] RNA was isolated and hybridized with filter-bound DNA in the presence of competing cytoplasmic RNA. A significant portion of RNA which was transported to either medium was not represented in cytoplasmic RNA. These results indicate that the restriction of some sequences to the nucleus $\text{in}\text{vivo}$ is not maintained $\text{in}\text{vitro}$.     

Mass spectrometric identification of various molecular forms of dynorphin in bovine adrenal medulla

Deliberate miscompartmentalization of liver outer mitochondrial membrane (OMM) proteins and liver mitochondrial proteins has been achieved by polyethylene-glycol mediated OMM vesicle-hepatocyte or mitochondrial-hepatocyte fusion. Reductively methylated OMM and mitochondrial proteins (³H) are destroyed at rates remarkably similar to those for OMM ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 60–70 h) or mitochondrial proteins ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 84–104 h) in liver $\text{in vivo}$ when studied over 4–5 days in hepatocyte monolayers cultured in conditions giving stabilized endogenous protein catabolic rates mimicking endogenous $\text{in vivo}$ rates. Destruction of transplanted OMM proteins is partially sensitive to chloroquine, supporting some lysosomally mediated autophagic destruction of long-lived transplanted OMM proteins in hepatocyte monolayers.     

Synexin protein is non-selective in its ability to increase Ca2+-dependent aggregation of biological and artificial membranes

Synexin, a soluble protein which increases the specificity of Ca²⁺ to aggregate isolated bovine chromaffin granules was prepared from bovine adrenal medullary tissue by the method of Creutz, Pazoles and Pollard (J. Biol. Chem. $\text{253}$, 2858–2866, 1978). We also find that synexin increases both the initial rate and final amplitude of Ca²⁺-promoted aggregation of granule membranes. This effect is Ca²⁺-specific. However in contrast to Creutz $\text{et}\text{al}$, we find that synexin also potentiates aggregation of adrenal medulla and liver mitochondria and microsomes as well as phosphatidylserine vesicles. This lack of membrane specificity argues against the suggestion of Creutz $\text{et}\text{al}$ that synexin specifically binds the granule to the plasma membrane prior to exocytosis $\text{in}\text{vivo}$.     

Metabolic sequestration of putrescine in Neurospora crassa

The metabolic fate of putrescine labelled $\text{in}\text{vivo}$ was investigated after administration of a trace (10^?7 M) of L-[¹⁴C]ornithine to exponentially growing mycelia of $\text{Neurospora}\text{crassa}$, followed by a large chase (2 mM) of L-[¹²C]ornithine. The specific radioactivities of putrescine and spermidine were determined during the chase period by reaction with [³H]dansyl chloride of known specific radioactivity and isolation of the dansyl-derivatives by thin-layer chromatography. Radioactivity remained in the putrescine pool for over 2 h during the chase period. This suggests that putrescine is largely sequestered (80% or more) $\text{in}\text{vivo}$. The metabolic sequestration of polyamines may be a significant factor in the regulation of polyamine synthesis.     

DDT inhibition of Ca-Mg ATPase from peripheral nerves and muscles of lobster, Homarus americanus

Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, $\text{Homarus}\text{,}\text{americanus}$, to DDT was studied. The CaMg ATPase found in SR with the high Ca²⁺ affinity is sensitive to DDT while the portion of ATPase related to the low Ca²⁺ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and $\text{in}\text{,}\text{vivo}$ poisoning symptoms of the nervous system is discussed.     

Absorbance and fluorescence properties of protochlorophyllide in etiolated bean leaves

Primary leaves of 7-to-9 day-old etiolated bean seedlings contain a species of protochlorophyllide which is not transformed to chlorophyllide by light; this pigment species exhibits an absorption peak at 631nm $\text{in}\text{vivo}$ at ?196° and a fluorescence emission peak at 639nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of etiolated leaves converts the phototransformable protochlorophyllide holochrome to a pigment species with $\text{in}\text{vivo}$ absorption and fluorescence peaks identical to those of endogenous nontransformable protochlorophyllide. Administration of δ-amino-levulinic acid to etiolated leaves causes the synthesis of non-transformable protochlorophyllide with an absorption peak also at 631nm $\text{in}\text{vivo}$ at ?196° but with a fluorescence emission peak at 643nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of such leaves does not affect the position of these bands. The results indicate that protochlorophyllide which is derived from exogenous δ-amino-levulinic acid is in a physically different state from other forms of protochlorophyllide in the leaf.     

The synthesis of proteolipid protein by isolated rat liver mitochondria

About 15% of the total (³H)leucine incorporated into protein by isolated rat liver mitochondria $\text{in}\text{vitro}$ could be extracted by chloroform:methanol. This incorporation was inhibited by chloramphenicol and carbomycin, both specific inhibitors of mitochondrial protein synthesis. SDS-gel electrophoresis of the mitochondrial membrane revealed 6–7 labeled bands. Label in the proteolipid fraction was present mainly in a band of 40,000 molecular weight. Several labeled bands observed in gels of the mitochondrial membrane were not removed or changed by extraction with chloroform:methanol suggesting that some, but not all, of the proteins synthesized by rat liver mitochondria are proteolipids.