bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation of the Role of Uncharged tRNA in the Regulation of Polypeptide Chain Initiation by Amino Acid Starvation in Cultured Mammalian Cells; a Reappraisal
Authors: AUSTIN Sara A. , PAIN Virginia M. , LEWIS John A. , CLEMENS Micshael J. 
Published: July 2010

The rate of initiation of protein synthesis in mammalian cells in culture is regulated by the availability of essential amino acids. Previous reports have suggested that this effect is mediated by changes in the ratio of uncharged to charged tRNA species in these cells. We have investigated this possibility by examining the formation of 40‐S subunit. Met‐tRNAfribosomal initiation complexes in extracts of Ehrlich ascites tumour cells; this is the stage in initiation previously shown to be regulated in response to lysine starvation of the intact cells.Exposure of cells to a low concentration of cycloheximide makes polypeptide chain elongation the ratelimiting step, allows reformation of polysomes in starved cells and abolishes the effect of lysine deprivation on overall protein synthesis. Treatment with a high concentration of cycloheximide inhibits all protein synthesis and ‘freezes’ the polysomes in the state of aggregation which exists in the cell at that moment. The latter conditions also permit the tRNA pool to become fully charged in both fed and lysine‐starved cells. In cell‐free extracts prepared from cells treated with either low or high concentrations of cyclolieximide 40‐S initiation complex formation nevertheless remains defective after lysine starvation of the cells.Addition of deacylated tRNA, oxidized with periodate to prevent its recharging, to initiating cell‐free extracts inhibits neither 40‐S initiation complex formation nor overall protein synthesisin vitro.Addition of lysine to an extract from lysine‐starved cells, under conditions where this amino acid is charged on to endogenous tRNA, fails to stimulate either 40‐S initiation complex formation or protein synthesisin vitro.These results suggest that amino acid starvation regulates the formation of 40‐S polypeptide chain initiation complexes in mammalian cells by a mechanism which is not influenced by the rate of protein synthesis and does not directly involve uncharged tRNA.
Covalent Bonding of Protein to Polyamine in the Cyst Coat of the Protozoan Colpoda steinii
Authors: TIBBS Jack 
Published: July 2010

High‐voltage electrophoresis and chromatography before and after reaction with 5‐dimethylaminonaphthalene‐1‐sulphonyl chloride have identified putrescine and spermidine in hydrolysates of cyst coat proteins from the protozonn Colpodu steinii.
Amounts present varied with putrescine up to 19.7 and spermidine up to 16.9 residues per 1000 amino acid residues.
The amines were not. in the main. removed by acid or alkaline extraction or by reprecipitation. They were present in hydrolysates of peptides isolated electrophoretically from acid‐degraded coat protein.
Proteolysis of oxidised coat protein produced a soluble core polypeptide to which the major proportion of the amines were attached and which had a simple composition. It was composed almost entirely of glutamie acid or glutamine, glycine. serine and cysteic acid. these residues being present in the approximate ratio of 10:2:1:l.
When coat protein was treated with 5‐dimetliylaminonaphthallene‐1‐sulphonyl chloride mid hydrolysed no fully substituted amities could be detected but putrescine with one group substituted and sperniidine derivatives with one and two groups substituted were present.
Differential Study of Phosphatidylcholine and Sphingomyelin in Human High‐Density Lipoproteins with Lipid‐Specific Fluorescent Probes
Authors: MOLOTKOVSKY Julian G. , MANEVICH Yefim M. , GERASIMOVA Elena N. , MOLOTKOVSKAYA Irina M. , POLESSKY Vladimir A. , BERGELSON Lev D. 
Published: July 2010

Modified phosphatidylcholine and sphingomyelin containing an anthryl end group attached to one of the Fatty acyl chains were used as fluorescent probes in an investigation of the molecular organization of human high‐density lipoproteins (HDL). Monolayer experiments and NMR measurements showed the anthryl‐labeled lipids to mimic closely the corresponding host phospholipids, the fluorophores being located near to the terminal CH3 groups of the fatty acid residues. The above fluorescent phospholipid probes made it possible for the first time to study differentially the behaviour of phosphatidylcholine and sphingomyelin in HDL. The probes were shown to interact in a different way with the apoprotein tryptophans and to be non‐randomly distributed at the surface of the globules. The probable sphingomyelin binding site of apolipoprotein A‐I was defined. Evidence was obtained suggesting the existence in high‐density lipoproteins of two slowly exchanging phospholipid pools: one strongly bound to apoproteins, and the other free or loosely bound.Fluorescence parameters characterizing the fluidity of HDL phospholipids and their interaction with the apoprotein tryptophans were found to correlate with the HDL cholesterol level. The possible significance of the obtained results for a better understanding of the relation of high‐density lipoproteins to coronary heart diseases is discussed.
Attachment of Lipoprotein to the Murein of Escherichia coli
Authors: WENSINK Jan , GILDEN Nanda , WITHOLT Bernard 
Published: July 2010

The rate of incorporation of [3H]diaminopimelate into the lipoprotein attachment sites was compared to the rate of its incorporation into total acid‐insoluble and dodecylsulphate‐insoluble murein. In contrast to earlier findings, we could detect no significant difference in these rates of incorporation, suggesting that the lipoprotein molecules are not pecifically excluded from the murein growth sites.The amount of lipoprotein bound to the murein increased when the cells entered the stationary phase of growth.
Synthesis, Characterisation and Biological Significance of (2′‐5′)Oiigoadenylate Derivatives of NAD+, ADP‐Ribose and Adenosine(5′)Tetraphosph0(5′)Adenosine
Authors: CAYLEY P. Jane , KERR Ian M. 
Published: July 2010

The (2′‐5′)oligoadenylate synthetase [ppp(A2′p)n,A synthetase] from interferon‐treated HeLa cells has been used to synthesize NAD+2′p5′A and NAD′2′p5′A2′p5′A. High‐performance liquid chromatography conditions have been developed for the separation of these products and the structures of the purified nucleotides confirmed by proton nuclear magnetic resonance spectroscopy. Using A5′p45′A and ADP‐ribose as primers the corresponding series of (2′‐5′)oligoadenylate products have been similarly synthesised and purified. All of the analogues whose structure includes pp(A2′p)2A were found to displace ppp(A2′p)3A[32P]pCp in the radiobinding assay for ppp(A2′p)nA (n = 2 to ≥ 4) [M. Knight et al. (1980) Nature (Lond.) 288, 189–192] at the same concentration (nanomolar) as does ppp(A2′p),A. They also have the same stability as ppp(A2′p)nA under protein‐synthesis conditions in Ehrlich ascites tumour cell extracts. A5′p,5′(A2′p)4A and A5′p45′(A2′p)3A inhibit protein synthesis in Ehrlich ascites tumour extracts as effectively as ppp(A2′p)2A. A5′p45′(A2′p)3A activates the ppp(A2′p),A‐dependent nuclease in reticulocyte lysates at the same concentration as ppp(A2′p)3A. The biological activity of these molecules may reflect their conversion to ppp(A2′p)nA.NAD+ is a preferred primer for the ppp(A2′p)nA synthetase in HeLa extracts. Despite this, using thc radiobinding assay in combination with high‐performance liquid chromatographic analysis, no evidence was found for the natural occurrence of NAD+2′p5′A2′p5′A or the corresponding A5′p45′A or ADP‐ribose derivatives in trichloroacetic acid/ether extracts froin interferon‐treated, encephalomyocarditis‐virus‐infected mouse L cells. In contrast ppp(A2′p)2A and ppp(A2′p)3A were detected at levels corresponding to approximately 100 nM in these cells. It appears therefore that none of the derivatives of NAD+ A5′p45′A and ADP‐ribose having two or more additional AMP residues in 2′‐5′ linkage, occurs naturally in these cells at concentrations ≥ 5 nM under conditions in which the ppp(A2′p)nA synthetase is actively synthesising ppp(A2′p)nA.
The Isolation, from Electromotor Neurone Perikarya, of Messenger RNAs Coding for Synaptic Proteins
Authors: SCHMID Dieter , STADLER Herbert , WHITTAKER Visctor P. 
Published: July 2010

Poly(A)‐containing mRNA was isolated from the electric lobe. cerebellum and forebrain of Torpedo marmorata and from cholinergic electromotor perikarya isolated from the electric lobe. All the mRNA preparations were translated by a cell‐free protein‐synthesizing system from rabbit reticulocytes; no brain‐specific factors were required. The highest stimulation rate was found with the perikaryal mRNA suggesting that this purcly neuronal mRNA is a preferred template in the protein‐synthesis system; the molecular basis of this phenomenon remains to be elucidated. The translation products of the perikaryal mRNA were analysed by two‐dimensional gel electrophoresis and compared with the proteins of synaptosomes derived from the electromotor ncrve terminals. The majority of the synaptosonial proteins comigrated with synthesized products. More than 100 synthesized proteins were detected as individual spots in the gel pattern, among them actin, subunits of neurofilamentous proteins and a protein considered to be a specific component of electromotor synaptic vesicles. Identities were confirmed in some cases by immunochemical methods. The results suggest that protein synthesis in the perikaryon of the electromotor neurone is largely directed to the production of proteins needed to maintain synaptic integrity.A comparison of the translation products of mRNA derived from the highly cholinergic electric lobe and a brain region, the cerebellum, which is non‐cholinergic, revealed, as expected, some coinnion translation products and others which appeared to be specific for the brain regions concerned. This approach may lead to the identification of proteins specific for neurones of different transmitter types.
Ovarian and Fat‐Body Vitellogenin Synthesis in Drosophila melanogaster
Authors: ISAAC Peter G. , BOWNES Mary 
Published: July 2010

The ovary and the fat body of Drosophilu melanogaster both synthesise vitellogenins in vivo. The ovary contributes nearly as much vitellogenin to the yolk of an oocyte as does the fat body. Densitometry of fluorographs and gels has been used to compare the amount of the smallest vitellogenin polypeptide, yolk protein 3, synthesised by each tissue. Cell‐free translations indicate that the ovary, in contrast to the fat body, contains a much reduced level of the mRNA for yolk protein 3 compared with the mRNAs for the other vitellogenin polypeptides. However, if tissues are cultured in vitro, the underproduction of this protein by the ovary is not significant. Because young embryos have levels of this polypeptide which are expected if the ovary has a low level of its corresponding mRNA, we argue that the ovary genuinely underproduces this protein in vivo and that the relative levels synthesised by the ovary in vitro are an artefact.Egg chambers of previtellogenic stages can synthesise vitellogenins, but the maximum level of vitellogenin synthesis occurs in egg chambers of the early vitellogenic stages.We conclude that the expression of the vitellogenin genes is subject to different controls at each site of synthesis. The possible cell types responsible for ovarian vitellogenin synthesis are discussed; the follicle epithelial cells are tentatively nominated for this role. We also suggest that a specific repression mechanism for vitellogenin gene expression exists in the ovary.
Enzymic Synthesis of Lignin Precursors
Authors: LÜDERITZ Thomas , SCHATZ Gudrun , GRISEBACH Hans 
Published: July 2010

4‐Coumarate:CoA ligase was purified from cambial sap of spruce (Picea abies). A 1627‐fold purification of the enzyme with ayield of 37% was achieved by a six‐step preocedure including dye‐ligand chromatography. Isozymes of the ligasewere not detected. The enzyme has an Mr of about 63000 and is a single polypeptide chain.Ferulic, 4‐coumaric and caffeic acids are efficient substrates for the ligase. In constrast to some ligase from angiosperms, the ligase from spruce (gymnosperm) does not activate sinapic acid. The substrate specificity of the ligase os consistent with the lignin composition of spruce.
Biosynthesis of Polysaccharides in Acetobacter xylinum
Authors: COUSO Roberto O. , IELPI Luis , GARCIA Rodolfo C. , DANKERT Marcelo A. 
Published: July 2010

The sequential synthesis in vitro of a heptasaccharide diphosphate prenol, containing glucose, mannose, glucuronic acid and rhamnose in the ratio 4:1:1:1 is described. The enzyme preparation consisted of EDTA‐treated Acetobacter xylinum cells and UDP‐glucose, GDO‐mannose, UDP‐glucuronic acid and TDP‐rhamnose were employed as sugar donors.The compounds soluble in chloroform/methanol/water (1:2:0.3) formed from incubations carried out under different conditions in the presence of a variety of combinations of the donors labeled with 14C, 3H or 32P were analysed by DEAE‐cellulose column chromatography, gel filtraton, partial acid hydrolysis, acetolysis, periodate oxidation, etc.The following structure is proposed for the most complex compound characterized: rhamnosyL(1 → 6)‐β‐glucosyl‐(1 → 6)‐α‐glucosyl‐(1 → 4)‐β‐glucuronyl‐(1 → 6)‐β‐mannosyl‐(1 → 3)‐β‐glucosyl‐(1 → 4)‐α‐glucosyl diphosphate prenol.The smaller oligosaccharide diphosphate prenols formed as intermediate steps are also characterized in this or in previous work [Garcia, R. C., Recondo, E. and Dankert, M. A. (1974) Eur. J. Biochem. 43, 93–105; Couso, R. O., Ielpi, L., and Damkert, M. A. (1980) Arch. Biochem. Biophys. 204, 434–443].The role of these compounds in the biosynthesis of a complex exopolysaccharide that this microorganism forms in addition to cellulose is discussed.
Regulation of N‐Acetylglutamate Synthetase in Mouse Liver
Authors: KAWAMOTO Susumu , ISHIDA Hisae , MORI Masataka , TATIBANA Masamiti 
Published: July 2010

N‐Acetyl‐l‐glutamte synthetase catalyzes the synthesis of N‐Acetyl‐l‐glutamte, an allosteric and essential activator of carbamoyl‐phosphate synthetase I in the liver of uretelic animals. The enzyme is activated specifically by arginine.

Mice were fed laboratory chow (protein content, 21%) for 3 h and, after various times, N‐Acetyl‐l‐glutamte synthetase activity in the sonicated mitochondria was measured both in the presence and absence of 1 mM l‐arginine. The activity in the absence of arginine did not significantly change after the feeding, while the activity in the presence of arginine increased with time and reached a maximum (fivefold increase) 9 h after the start of feeding, then decreased gradually. The ratio of the activity with arginine to that without arginine was 1.6 at the start of feeding and 5.5 at 9 h.
Similar posprandial changes were observed with the mice on a protein‐free diet, when these mice had previously been on a protein diet. No such change was seen with a protein‐free diet when the mice previously given a protein‐free diet for a few days.
Cycloheximide injection, which inhibited the total protein synthesis in the mouse liver by 90%, did not inhibit the postprandial increase in arginine activation of the synthetase. Thus, protein synthesis is presumably not required for the change.
The enzymes with a low and a high arginine sensitivity were purified over tenfold from mitochondrial extracts of fased and fed animals respectively. The extent of arginine activation of the enzymes remained unchanged, low or high, throughout the purification. In Sephacryl S‐300 chromatography both types of the enzyme were eluted as a single peak and corresponded to a Mr of 220000–250000. the change in arginine activation may be due to a modification of the enzyme molecule without a gross change in the molecular weight.
The diet‐dependent change in sensitivity of the synthetase to arginine presents a new type of regulation of this enzyme and this regulation may contribute to the prevention of a rapid decrease in heptic acetylglutamate levels after food ingestion.
Low Level Chemiluminescence of Liver Microsomal Fractions Initiated by tert‐Butyl Hydroperoxide
Authors: CADENAS Enrique , SIES Helmut 
Published: July 2010

Low‐level chemiluminescence emitted by rat liver microsomal fractions supplemented with organic hydro‐peroxides (notablytert‐butyl hydroperoxide) is resolved in a time‐course consisting of two phases. Chemiluminescence was accompanied by a loss of cytochrome P‐450 as well as by lipid peroxidation (malondialdehyde accumulation and O2uptake). Cytochrome b5, in contrast, remained intact.With the organic hydroperoxides, lipid peroxidation is initiated without addition of electron donors such as NADPH, so that mainly the propagation reactions are investigated here (phase I1 of chemiluminescence). In fact, addition of NADPH or NADH leads to substantial restriction in chemiluminescence emission and exerts protection on cytochrome P‐450 against degradation. Optimal conditions for light emission were with cytochrome P‐450 in the oxidized state, and inhibitors of this cytochrome, such as SKF‐525A or cyanide, decreased light emission. Conversely, there was increased chemiluminescence in microsomal fractions from rats treated with phenobarbital. a condition with increased levels of cytochrome P‐450.Chemiluminescence was sensitive to a free radical trap, di‐trrt‐butylquinol, and it was enhanced by 1,4‐diazabicyclo[2.2.2]octane, an enhancer of singlet oxygen dimol emission. This and the spectral distribution of emitted light suggest that singlet molecular oxygen is involved in this chemiluminescence system.The present observations establish for cytochrome P‐450, but not for cytochrome b5, its capability to effectively catalyze homolytic scission of hydroperoxides, an activity different from the known monooxygenase and peroxidase activities. This photoemissive reaction system is similar to that previously observed for the system consisting of lipid vesicles supplemented with cytochrome c and hydroperoxides [Cadenas. E., Boveris, A., and Chance, B. (1980)Biochrm. J.188, 577–583], The former system is 70‐fold more effective than the latter, compared on a basis of heme content.
Removal of Wheat‐Germ Agglutinin Increases Protein Synthesis in Wheat‐Germ Extracts
Authors: ABRAHAM Abraham K. , KOLSETH Svein , PIHL Alexander 
Published: July 2010

Affinity chromatography of wheat germ extracts on a chitin column increased the rate and extent of protein synthesis, programmed by rabbit globin mRNA. Addition of purified wheat germ agglutinin to the chitintreated extract reduced the rate of protein synthesis to about the levels seen in the untreated extracts. Experiments where the ratio of messenger to extract and the ratio of supernatant to ribosomes were varied. indicated that addition of wheat germ agglutinin reduced the amount of available ribosomes.Reduced and carboxymethylated wheat germ agglutinin failed to inhibit protein synthesis and was unable to bind to the ribosomes. However, labelled intact agglutinin was found to be bound to ribosomes. The bound agglutinin was not released by acid treatment.The inhibiting effect of wheat germ, agglutinin on protein synthesis could not be counteracted by addition N‐acetyl‐d‐glucosamine or sialic acid, whereas thiols partially diminished the inhibition. The data indicate wheat germ agglutinin binds reversibly to ribosomes, probably through mixed disulfide formation, chitin treatment increases the ability of wheat germ extracts to support protein synthesis. at least in removing the wheat germ agglutinin. The possibility that chitin treatment also removed other inhibitor of protein synthesis cannot be excluded.
DNA Fragment Conformations
Authors: TRAN‐DINH Son , NEUMANN Jean‐Michel , HUYNH‐DINH Tam , GENISSEL Bernadette , IGOLEN Jean , SIMONNOT Guy 
Published: July 2010

The helix‐coil transition, conformations and dynamic structures of d(A‐C‐A‐T‐G‐T) we re investigated as a function of temperature by 1H‐NMR spectroscopy at various frequencies (400, 276, 250 arid 90 MHz). Imino exchangeable protons of the d(A‐C‐A‐T‐G‐T) duplex were observed between 5°C and 25°C at 276 MHz. The results suggest that the fraying process mainly consists of two stages: the extreme dA · cT base pairs open at first; the internal dG · dC and central dA · dT base pairs then open simultaneously at higher temperatures. This conclusion was supported by the midpoint temperature measurements: at1/2 value of 32 2 °C was obtained for the extreme residues; the same t1/2 value of 43± 3 °C was obtained for the internal and central base pairs. The helix and coil proportions and the dissociation constant were determined as a function of temperature from the $=f(t) curves corresponding to the non‐exchangeable protons of the internal and central residues. An enthalpy of 150 kJ/mol was found for the helix‐coil transition. At 400 MHz, all the non‐exchangeable proton resonances were assigned and most of the coupling constants were determined. The H‐1' resonance of the dC residue is particularly broad compared with that of the other H‐' protons at I> 42°C. This was attributed to the fact this proton is located in the proximity of the hydrogen bond between C= 0 of cytidine and H2 of guanine. Several sugar protons, in particular H‐2', exhibited large chemical shift variations (between 20–72°C) which decrease from the central to the extreme residues; this seems to indicate a maximum helical distorsion for the residues. Comparison between the chemical shifts and tempereture‐dependent behaviours associated with d(A‐C‐A‐T‐G‐T) and d(A‐T‐G‐C‐A‐T) protons enabled the H‐1' and thymine H‐6 resonances of d(A‐T‐G‐C‐A‐T) to be assigned. The average sugar conformations of the d(A‐C‐A‐T‐G‐T) residues are very similar for both the helix and coil forms. The large predominance of the S conformation for all d(A‐C‐A‐T‐G‐T) residues and the insignificant chemical shift variation of the guanine 1–8 proton with increasing temperature are consistent with the B‐helical conformation.The initial relaxation time T1 of the base protons was measured as a function of tem;perature at 90 and 250 MHz. At 250 MHz, the T1 values vary slightly with temperature (T), while at 90 MHz most of the curves of log T1=f(T−1) present a break point corresponding to the midpoint temperature for the hielix‐coil transition. The T1 values of the adenine H‐8, H‐2 and thymine H‐6 protons are longer for the extreme than for the internal residues whatever the frequency and temperature. The correlation time for the coil form was determined at high temperatures from the proton TI ratios, T1 (90 Hz)/Tl (250 Hz); tc= 0.2 ns at 65°C. For the helix state, the mobility appears to be different for the purine and pyrimidine bases: the former show a greater base‐sugar flexibility and the latter a stronger anisotropic character.