Mitochondrial protein synthesis in a mammalian cell-line with a temperature-sensitive leucyl-tRNA synthetase.

The temperature-sensitive Chinese hamster ovary cell mutant tsH1, has been shown previously to contain a temperature-sensitive leucyl-tRNA synthetase. At the non-permissive temperature of 40 degrees C cytosolic protein synthesis is rapidly inhibited. The protein synthesis which continues at 40 degrees C appears to be mitochondrial, since: (a) whole-cell protein synthesis at the permissive temperature of 34 degrees C is not inhibied by tevenel, the sulfamoyl analogue of chloramphenicol and a specific inhibitor of mitochondrial protein synthesis; however, whole-cell protein synthesis at 40 degrees C is inhibited by tevenel, (b) Protein synthesis by isolated mitochondria from tsH1 cells is not significantly inhibited at 40 degrees C. (c) At 40 degrees C [14C]leucine is incorporated predominantly into the mitochondrial fraction of tsH1 cells. (d) The incorporation of [14C]leucine at 40 degrees C into mitochondrial proteins of tsH1 cells is inh-bited by tevenel but not by cycloheximide. These results suggest that the mitochondria of tsH1 cells contain a leucyl-tRNA synthetase which is different from the cytosolic enzyme. The inhibition of cytosolic, but not of mitochondrial protein synthesis in tsH1 cells at 40 degrees C allows the selective labelling of mitochondrial translation products in the absence of inhibitors. The mitochondrial translation products labelled in tsH1 cells at 40 degrees C and at 34 degrees C in the presence of cycloheximide have been compared by sodium dodecylsulphate-polyacrylamide gel electrophoresis. Both conditions of labelling give similar profiles. The mitochondrial translation products are resolved into two components, one with an apparent molecular weight range from 40,000 to 20,000 and a second with an apparent molecular weight range from 20,000 to 10,000.     

Suppression of ribosomal gene expression in oocytes and nurse cells of a polychaete as a result of polyamine synthesis inhibition

The role of the polyamines in ribosomal gene expression was evaluated in the polychaete Ophryotrocha labronica by analyzing the effects of polyamine synthesis inhibition on RNA synthesis during oogenesis, a period characterized by intense nucleolar activity. At various stages of oogenesis adult polychaete females were blocked in their polyamine synthesis by the addition of 10 mM DL-alpha-difluoromethylornithine (DFMO) to the sea water in which they were cultivated. To monitor RNA synthesis during DFMO treatment the animals were pulse-labeled with [5-3H]uridine and processed for autoradiography. Light and electron microscope autoradiographs demonstrate that DFMO treatment suppresses incorporation of label into nucleolar RNA (rRNA) both in the oocytes and their associated nurse cells. The ultrastructural appearance of both cell types reveals interference with nucleolar and ribosomal activity; the endoplasmic reticulum is deprived of ribosomes, and the production of protein granules (vitellogenesis) is reduced. The high specificity of DFMO for polyamine synthesis and the fact that the effects of DFMO were counteracted by addition of a low concentration (10 microM) of putrescine shows that the observed interference with ribosomal gene expression is indeed due to polyamine deficiency.     

Genetics of a low-density lipoprotein allotype in a cattle.

The paper describes a cattle serum antigen (LdlA1) located on a low-density lipoprotein and detected by single radial diffusion. The specificity is inherited in a simple Mendelian manner and the gene controlling its synthesis is inherited independently from the one controlling the synthesis of the alpha 2 macroglobulin McA1 antigen.     

Genetics of a low-density lipoprotein allotype in a cattle*

The paper describes a cattle serum antigen (LdlA1) located on a low-density lipoprotein and detected by single radial diffusion. The specificity is inherited in a simple Mendelian manner and the gene controlling its synthesis is inherited independently from the one controlling the synthesis of the α₂ macroglobulin McA1 antigen.     

Self-replication of chemical systems based on recognition within a double or a triple helix: a realistic hypothesis.

A scenario is proposed by which non-enzymatic self-replication of short RNA molecules could occur. The hypothesis is illustrated for the self-replication of an oligopyrimidine (Y) strand. The successful replication of Y requires a series of plausible steps. The first, experimentally feasible, step involves the template-directed polynucleotide synthesis, based on Watson-Crick base pairing, of an oligopurine (R) strand using Y as the template, and chemically activated mononucleotides as the building blocks. This step will result in the formation of an oligopyrimidine.oligopurine (YR) double helix. The second step requires the use of the double helix as the template for the synthesis of a second oligopyrimidine (Y') strand from activated pyrimidine monomers. This synthesis could be facilitated by the binding of the monopyrimidines in the major groove of the YR double helix, via Hoogsteen-type base pairing with the R strand, establishing in that sense triple helix recognition. This step, if successful, should result in the formation of a new strand, Y', that runs parallel to the oligopurine strand. Y' differs from Y in that all 3'-5' phosphodiester linkages in Y are replaced by 5'-3' linkages in Y'. The resulting triple helix (YRY') is in dynamic equilibrium with YR and free Y'. In subsequent steps, unassociated Y' directs the synthesis of the complementary oligopurine (R') strand forming a new double helix Y'R' that may direct the synthesis of an oligopyrimidine strand, Y, that is expected to be identical to the first strand that started the whole sequence. An attempt is made to generalize the above hypothesis to mixed oligonucleotides containing all four bases and identify the limitations of this hypothesis.     

Regulation of synthesis of alkaline phosphatase by deoxyribonucleic acid synthesis in a constitutive mutant of Bacillus subtilis

Hiraga, Sota (Osaka University, Osaka, Japan). Regulation of synthesis of alkaline phosphatase by deoxyribonucleic acid synthesis in a constitutive mutant of Bacillus subtilis. J. Bacteriol. 91:2192-2199. 1966.-It was found that synthesis of alkaline phosphatase (APase) correlated with deoxyribonucleic acid (DNA) synthesis in a partially constitutive mutant of Bacillus subtilis. When cultures of the mutant were made to undergo synchronous growth by germination of spores in an excess-phosphate medium, synthesis of APase was repressed at the beginning of DNA synthesis. If the initiation of DNA synthesis was inhibited by thymine starvation, the repression of APase was not observed. When DNA synthesis, previously initiated, was inhibited by thymine or uracil starvation, or by addition of mitomycin C, the repression was partially released at a later stage. In contrast, this correlation between repression and DNA synthesis was not observed in a repressible strain.     

Characterization of a novel 5' subgenomic RNA3a derived from RNA3 of Brome mosaic bromovirus

The synthesis of 3' subgenomic RNA4 (sgRNA4) by initiation from an internal sg promoter in the RNA3 segment was first described for Brome mosaic bromovirus (BMV), a model tripartite positive-sense RNA virus (W. A. Miller, T. W. Dreher, and T. C. Hall, Nature 313:68-70, 1985). In this work, we describe a novel 5' sgRNA of BMV (sgRNA3a) that we propose arises by premature internal termination and that encapsidates in BMV virions. Cloning and sequencing revealed that, unlike any other BMV RNA segment, sgRNA3a carries a 3' oligo(A) tail, in which respect it resembles cellular mRNAs. Indeed, both the accumulation of sgRNA3a in polysomes and the synthesis of movement protein 3a in in vitro systems suggest active functions of sgRNA3a during protein synthesis. Moreover, when copied in the BMV replicase in vitro reaction, the minus-strand RNA3 template generated the sgRNA3a product, likely by premature termination at the minus-strand oligo(U) tract. Deletion of the oligo(A) tract in BMV RNA3 inhibited synthesis of sgRNA3a during infection. We propose a model in which the synthesis of RNA3 is terminated prematurely near the sg promoter. The discovery of 5' sgRNA3a sheds new light on strategies viruses can use to separate replication from the translation functions of their genomic RNAs.     

Insulin stimulation of muscle protein synthesis in obese Zucker rats is not via a rapamycin-sensitive pathway

The obese Zucker rat is resistant to insulin for glucose disposal, but it is unknown whether this insulin resistance is accompanied by alterations of insulin-mediated muscle protein synthesis. We examined rates of muscle protein synthesis either with or without insulin in lean and obese Zucker rats with the use of a bilateral hindlimb preparation. Additional experiments examined insulin's effect on protein synthesis with or without rapamycin, an inhibitor of protein synthesis. Protein synthesis in red and white gastrocnemius was stimulated by insulin compared with control (no insulin) in obese (n = 10, P<0.05) but not in lean (n = 10, P>0.05) Zucker rats. In white gastrocnemius, rapamycin significantly reduced rates of protein synthesis compared with control in lean (n = 6) and obese (n = 6) rats; however, in red gastrocnemius, the attenuating effect of rapamycin occurred only in obese rats. The addition of insulin to rapamycin resulted in rates of synthesis that were similar to those for rapamycin alone for lean rats and to those for insulin alone (augmented) for obese rats in both tissues. Our results demonstrate that insulin enhances protein synthesis in muscle that is otherwise characterized as insulin resistant. Furthermore, rapamycin inhibits protein synthesis in muscle of obese Zucker rats; however, stimulation of protein synthesis by insulin is not via a rapamycin-sensitive pathway.     

Glucocorticoid induction of growth hormone synthesis in a strain of rat pituitary cells

Conditions were determined for measuring growth hormone synthesis by a clonal strain of rat pituitary cells grown in suspension culture. Incubation of the cells with [3H]leucine in either continuous labeling or pulse-chase experiments showed that secretion of newly synthesized growth hormone commences only after a lag of about 15 min. The pulse-chase experiments also demonstrated that there is no detectable degradation by the cells of growth hormone. Thus growth hormone synthesis could be measured, in the absence of complications arising either from secretion or degradation of growth hormone, by incubating the cells with [3H]leucine for 10 min. Exposure of cells grown under the usual culture conditions to dexamethasone (a synthetic glucocorticoid) led to an average stimulation of specific growth hormone synthesis (growth hormone synthesis/total cytoplasmic protein synthesis) of only 2.6-fold. However, two other growth conditions were found in which dexamethasone routinely yielded a 5- to 15-fold stimulation of specific growth hormone synthesis. One of these conditions, involving substitution of 10% fetal calf serum for the normal serum supplement, was employed in subsequent experiments. A stimulation of specific growth hormone synthesis could be observed at 10(-9) M dexamethasone, and the maximum stimulation was observed at dexamethasone concentrations of about 10(-8) to 10(-7) M. There was a lag of about 6 h before a stimulation by dexamethasone of specific growth hormone synthesis was detected. Thereafter, the stimulation increased in a nearly linear fashion until maximum stimulation was reached at about 48 h.     

Control of the Corpora Allata During a Reproductive Cycle in a Viviparous Cockroach

SYNOPSIS. This paper describes factors which regulate the corporaallata (CA) during the reproductive cycle in the viviparouscockroach Diploptera punctata. Experiments, designed to alterthe rate of juvenile hormone (JH) synthesis by the CA, are performedon whole animals and their effects are demonstrated by an invitro radiochemical assay for JH synthesis by the CA. The CAis both inhibited and stimulated. Axonal pathways from the brainto the CA are required for inhibition of the CA in virgin femalesbut not in young pregnant females. Humorally transmitted inhibitionof the CA is caused in vivo by JH (in high titers) and ecdysteroidhormone. JH in low titers also stimulates the CA. Since thesehormones do not affect the CA in vitro we suggest that theyexert their effect via the central nervous system. The ovary,a possible source of ecdysteroids in the hemolymph, is necessaryboth for maintenance of JH synthesis and for inhibition in therate of JH synthesis at the end of egg growth.     

Localization of DNA repair synthesis by human cell extracts to a short region at the site of a lesion

Double-stranded bacteriophage M13 DNA molecules were constructed containing a single specifically placed 2-(acetylamino)fluorene adduct or a single 4'-hydroxymethyl-4,5',8-trimethylpsoralen monoadduct. These circular DNA molecules were used to analyze in vitro DNA repair synthesis by cell extracts from normal human lymphoid cell lines. Both types of lesions stimulate DNA repair synthesis at the site of the adduct. DNA repair synthesis induced by the 2-(acetyl-amino)fluorene adduct took place in the damaged strand and was confined to a region within a 31-base pair restriction fragment around the adduct.     

Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesis.

Multiple signalling pathways are involved in the mechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase-3 (GSK-3) and an allosteric inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a, to determine the relative contributions of inactivation of GSK-3 and dephosphorylation of phosphorylase a as alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes. GSK-3 inhibitors (SB-216763 and Li+) caused a greater activation of glycogen synthase than insulin (90% vs. 40%) but a smaller stimulation of glycogen synthesis (30% vs. 150%). The contribution of GSK-3 inactivation to insulin stimulation of glycogen synthesis was estimated to be less than 20%. Dephosphorylation of phosphorylase a with CP-91149 caused activation of glycogen synthase and translocation of the protein from a soluble to a particulate fraction and mimicked the stimulation of glycogen synthesis by insulin. The stimulation of glycogen synthesis by phosphorylase inactivation cannot be explained by either inhibition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translocation of synthase. Titrations with the phosphorylase inactivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phosphorylase a. We conclude that a signalling pathway involving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Selective inactivation of phosphorylase can mimic insulin stimulation of hepatic glycogen synthesis.     

Chemical synthesis of cellulose and cello-oligomers using a hydrolysis enzyme as a catalyst

Regio- and stereo-selective synthesis of polysaccharides and oligosaccharides has been achieved by using glycosyl fluorides as substrates for cellulases. This methodology has successfully been applied to the first synthesis of cellulose via a non-biosynthetic pathway as well as to a selective preparation of cello-oligosaccharides and unnatural oligosaccharides. Using the enzymatic polymerization, it is possible to control the relative direction (parallel or anti-parallel) of each glucan chain in the synthetic cellulose in vitro. Based on these results, a new concept of ‘allos-selectivity’ in polymer synthesis has been proposed.     

Effect of Competence Induction on Macromolecular Synthesis in a Group H Streptococcus

The effects of competence induction by competence factor (CF) on macromolecular synthesis in group H streptococcus strain Wicky were investigated. CF preparations (culture filtrates from competent group H streptococcus strain Challis) were either heated or partially purified to remove a bacteriocin. These preparations did not inhibit growth, although they induced high levels of competence in strain Wicky. The action of the CF preparations did not affect the overall rates of deoxyribonucleic acid and protein synthesis, but caused a reduction in the rates of ribonucleic acid (RNA) and peptidoglycan synthesis. When competence induction by CF was prevented, no alterations in RNA or peptidoglycan synthesis were observed, indicating that these changes are in fact related to the development of competence.     

Stimulation of protein synthesis by a 34 KD DNA-binding protein from human placenta

A 34 KD DNA-binding protein fraction from human placenta stimulated endogenous protein synthesis in rabbit reticulocyte and wheat-germ cell-free systems. Though the synthesis of several proteins were stimulated by the 34 KD protein, a dose-dependent increase of two polypeptides of molecular weights 42,000 and 51,000 were distinctly observed in reticulocyte lysates. The synthesis of the major protein (beta-globin) was not affected by the 34 KD protein. In both hemin supplemented and unsupplemented lysates, the ability of 34 KD protein to stimulate the synthesis of high molecular weight (HMW) proteins was drastically reduced by Mg++ and not by dsRNA.     

A convenient synthetic method of a 5,7-diarylcyclopenteno-[1,2-b]pyridine-6-carboxylate: a key intermediate for potent endothelin receptor antagonists

A convenient method for the synthesis of the title intermediate 4 was described. The key steps of this synthesis involved: (1) regioselective addition reaction of arylzinc reagent to quinolic anhydride in 42% isolated yield, (2) conversion of a ketoacid to an enone, which was achieved in 65% yield by intramolecular Knoevenagel reaction of beta-ketoester generated by condensation of an acid imidazolide with an ester enolate, followed by dehydration assisted with silica gel, and (3) stereoselective reduction of an allyl alcohol in 75% yield with zinc under acidic conditions. This synthesis enabled us to provide hundreds of grams of without chromatographic purification.     

Existence of malfunctioning pro alpha2(I) collagen genes in a patient with a pro alpha 2(I)-chain-defective variant of Ehlers-Danlos syndrome

Collagen synthesis was examined in skin fibroblasts from a patient with a variant of Ehlers-Danlos syndrome. The relative rate of collagen synthesis to total protein synthesis in the patient's fibroblasts was always one-half of that in fibroblasts from normal controls. Total collagen synthesis, as assessed by quantification of total hydroxyproline, was also significantly lower than that of controls, indicating that the rate of collagen synthesis by the patient's fibroblasts was decreased compared with that by normal fibroblasts. Analysis of procollagen and collagen components showed the absence of the pro alpha 2(I) chain and its derivatives. Dot-blot and Northern-blot analyses showed the patient's fibroblasts to contain less than 10% of the mRNAs for pro alpha 2(I) found in control fibroblasts. In spite of these results, Southern blot analysis of genomic DNA indicated the presence of the same number of genes for the pro alpha 2(I) collagen chain in the patient's fibroblasts as in control fibroblasts, suggesting malfunctioning pro alpha 2(I) collagen genes as the cause for failure of the patient's fibroblasts to synthesize pro alpha 2(I) collagen chains.     

Control of RNA synthesis in Escherichia coli after a shift to higher temperature.

Parameters of RNA synthesis were measured after a temperature upshift in a pair of Escherichia coli B/r strains that are isogenic except for having relA and relA+ loci, to examine the cause for a reported anomaly in the correlation between guanosine tetraphosphate (ppGpp) and stable RNA (rRNA, tRNA) synthesis under such conditions. Two main results were: (i) the specific stable RNA gene activity (stable RNA per total RNA synthesis) correlated in the conventionally expected fashion with the level of ppGpp but was obscured by a nonspecific increase in the RNA chain elongation rate due to the higher temperature; (ii) the temperature upshift caused a transient reduction in the RNA polymerase activity (transcribing per total enzyme) that accounts for the previously observed oscillating RNA synthesis rate after a temperature shift.