Effetto dell'Irraggiamento X sulla Biosintesi degli Amminoacidi in Piante di Orzo

Abstract

The effects of irradiation on aminoacids biosynthesis in barley. — The effect of radiations on the biosynthesis of aminoacids in barley has been studied. Seeds have been irradiated with X-tays of 7.5 and seedlings with 7.5, 15 and 30 Kr. After 6–7 days of growth the control and irradiated seedlings, and the seedlings from control and irradiated seeds, have been supplied with 10–100 μC of C¹⁴O₂ in a closed chamber for 44–42 hours. The material was extracted with hot ethanol (85%–80% and 40%). Total radioactivity and the radioactivity of the basic fraction, eluted with HCl from Dowex 50, X-8′ (200–400 mesh, H^?) have been measured. It was observed that radiations increase the radioactivity of the aminoacid fraction, both in leaves and roots. A correlation between the decrease of fresh weight and increase of aminoacids radioactivity was shown. A possible explanation might be visualized in terms of the inhibition of growth and protein synthesis by radiations.     

Hypotheses on the establishment of a genetic code and transfer of information from proteinoids to nucleic acids

An hypothesis is proposed in which the specificity of interaction between an aminoacid and a nucleotide sequence of a tRNA would be enhanced by a ternary association with a specific proteinoid. These strict relations would have led to the present genetic code that we know. It is also proposed that the origin of the enzymatic activity of the primitive proteinoids would have arisen from the presence of different substrates during polymerisation, which would have favored specific sequences of aminoacids by forming more stable complexes with them, corresponding to the lowest free enthalpy. The information included in the aminoacid sequences of the proteinoids would have been transferred to messenger type RNA, according to a mechanism reverse of that for the present process for protein synthesis, and then to DNA.     

Effect of different oxygen pressure levels on the transport of alpha-aminoisobutyric acid in myocardial cell cultures

The activity of aminoacid transport, as measured by alpha-aminoisobutyrate uptake, has been studied in confluent myocardial cell cultures exposed to different oxygen tensions. The results obtained indicate that the rate of cellular uptake and accumulation of the inert aminoacid increase with time as the fraction of oxygen is reduced. When alpha-aminoisobutyrate was added in presence of all other aminoacids of the medium, the effect of oxygen was also evident, suggesting a mechanism which overcomes the competitive action of the other aminoacids assigned to the same transport system of alpha-aminoisobutyrate (A system). The modulation of aminoacid transport activity may represent one of the possible mechanisms by which environmental oxygen affect the rate of cellular protein synthesis.     

"In vivo" (35S)methionine interaction with rat liver tRNA

As part of a study to characterize the methionine role in tumorigenesis, we report that methionine sulfur interacts with rat liver tRNA "in vivo" (35S) radioactivity remained associated to the nucleic acid after a number of treatments, including tRNA deacylation. Similar data were obtained after administration of (methyl-3H) methionine, while no comparable tRNA labelling was detected when the aminoacid labelled in the aliphatic chain was given. Hplc analysis of (35S) tRNA enzymic hydrolysate showed two unidentified UV-absorbing radioactive peaks. NMR spectra of these two peaks did not reveal any thiomethyl group.     

Partial purification and study of gamma-glutamyl transpeptidase from sheep cerebral capillaries]

Gamma-glutamyl transpeptidase was purified 53 times from Sheep brain cortex capillaries. On gel filtration it appears homogeneous with a M.W. = 350 000. The enzyme is likely a glycoprotein, the properties of which are close to hog kidney gamma-glutamyl transpeptidase ; gamma-glutamyl aminoacid formation is assayed electrophoretically. The results obtained using several aminoacids are in favour of the existence of different units, specific of each group of aminoacids ; together with the data from structural analogs, they support the hypothesis that gamma-glutamyl transpeptidase participates in aminoacid transport accross blood brain barrier.     

Free amino acids in rat blood serum during A-hypovitaminosis]

A pool of free aminoacids of the blood serum has been studied in growing rats with a decreased retinol content in the liver tissue (to 5 g/g). The study shows a reliable drop in the concentrations of all essential aminoacids, except for phenylalanine. It is determined that the total amount of essential aminoacids decreases by 31%. At the same time concentrations of replaceable aminoacids increase, namely: glutamic acid and arginine--by 15.7%, ornithine and histidine--by 24%-45%, though proline concentration decreases abruptly by 31%. A disturbance in ultrastructural organization of microvilli in the apical membrane cells absorptive epithelium of the small intestine has been found. The results of the study confirm changes in a free aminoacid pool of the blood serum in growing rats and these changes occur first of all due to the disturbance of the absorption processes in the small intestine.     

The imbalance of brain large-chain aminoacid availability in amyotrophic lateral sclerosis patients treated with high doses of branched-chain aminoacids

Following the report of an increased mortality among patients with amyotrophic lateral sclerosis given high daily doses of branched-chain aminoacids, we assessed the plasma concentrations of large neutral aminoacids and glutamic acid and the large neutral aminoacid brain influx in 24 amyotrophic lateral sclerosis patients receiving placebo or branched-chain aminoacids ( -leucine 12 g, -isoleucine 6 g, -valine 6 g daily), in 15 untreated amyotrophic lateral sclerosis patients and in 15 healthy volunteers. The branched-chain aminoacid plasma concentrations increased three- to six-fold in the treated group compared to the patients receiving placebo or no treatment and to the healthy controls. Plasma glutamic acid concentrations in healthy volunteers were 51.59±7.53 nmol/ml while in the amyotrophic lateral sclerosis patients receiving no treatment, placebo or branched-chain aminoacids were 92.33±12.15 nmol/ml, 91.21±15.86 nmol/ml and 95.08±17.96 nmol/ml respectively. The glutamic acid concentration was significantly higher (P<0.01) in amyotrophic lateral sclerosis patients than in healthy individuals. Plasma phenylalanine and tyrosine were lower in the amyotrophic lateral sclerosis patients than in healthy controls, regardless of treatment, whereas tryptophan levels were not significantly different. The branched-chain aminoacid brain influx of the treated group was 110-140% of that measured in the patients receiving placebo and in the healthy controls. The aromatic aminoacid brain influx was lower in the treated group than in the placebo group or healthy controls. An impairment of brain large neutral aminoacid availability might possible contribute to enhancing the progression of symptoms in patients with amyotrophic lateral sclerosis.     

Two forms of methionyl-transfer RNA synthetase from Mycobacterium smegmatis

Two methionyl-transfer RNA synthetases (A and B forms) have been isolated from $\text{Mycobacterium smegmatis}$. The homogeneous preparations of the enzymes showed 1500 fold increase in specific activity in aminoacylation of methionine specific tRNA. The A and B forms differed in their specificity of aminoacylation of tRNA_m^Met and tRNA_f^Met; enzyme B exhibited much higher specificity for tRNA_f^Met. The molecular activities of A and B enzymes for aminoacid and tRNA were identical. The turnover number for aminoacid was 27 fold greater than that for tRNA, while the Km values for tRNA were lower by a factor of 10⁶ as compared to the aminoacid. Both the enzymes catalysed ATP-pyrophosphate exchange reaction to the same extent.     

The sequence of the gene for cytochrome c oxidase subunit I, a frameshift containing gene for cytochrome c oxidase subunit II and seven unassigned reading frames in Trypanosoma brucei mitochrondrial maxi-circle DNA.

A 9.2 kb segment of the maxi-circle of Trypanosoma brucei mitochondrial DNA contains the genes for cytochrome c oxidase subunits I and II (coxI and coxII) and seven Unassigned Reading Frames ("URFs"). The genes for coxI and coxII display considerable homology at the aminoacid level (38 and 25%, respectively) to the corresponding genes in fungal and mammalian mtDNA, the only striking point of divergence being an unusually high cysteine content (about 4.5%). The reading frame coding for cytochrome c oxidase subunit II is discontinuous: the C-terminal portion of about 40 aminoacids, is present in the DNA-sequence in a -1 reading frame with respect to the N-terminal moiety. URF5, 8 and 10, show a low but distinct homology (about 20%) to mammalian mitochondrial URF-1, 4 and 5, respectively. In URF5, the first AUG is found at codon 145, whereas extensive homology to mammalian URF-1 sequences occurs upstream of this position. The possibility exists that UUG can serve as an initiator codon. URF7 and URF9 have a highly unusual aminoacid composition and do not possess AUG or UUG initiator codons. These URFs probably do not have a protein-coding function. The segment does not contain conventional tRNA genes.     

Changes in rate of RNA synthesis and ribosomal gene number during oogenesis of Drosophila melanogaster.

A new method for separating Drosophila egg chambers into different developmental classes (Jacobs-Lorena and Crippa, 1977) made it possible to study changes in the rate of ribosomal RNA (rRNA), 5S RNA, and tRNA synthesis and the changes in ribosomal gene number during oogenesis. Synthesis of RNA was measured by [³H]uridine incorporation in vivo and subsequent analysis on sucrose gradients or gel electrophoresis. Specific radioactivity of nucleotide pools has also been determined. Ribosomal gene number has been measured by hybridization of egg chamber DNA to rRNA of high specific radioactivity. Our findings led us to conclude that in Drosophila melanogaster: (i) rRNA, 5S RNA, and tRNA are synthesized in all stages of oogenesis. (ii) In every stage, rRNA is the main RNA species synthesized. (iii) The rate of rRNA, 5S RNA, and tRNA synthesis increases greatly during oogenesis and is paralleled by a similar increase in ribosomal gene number resulting from the polyploidization of the nurse cell nuclei.     

A new water soluble Zn-salophen derivative as a receptor for alpha-aminoacids: unexpected chiral discrimination

A new water soluble zinc-salophen complex with appended D-glucose moieties was synthesized and characterized. Its binding properties toward six aminoacids were investigated by UV-vis spectrophotometric titrations. Association constants showed to be unfavorably affected by increasing steric hindrance of the side chain. Quite surprisingly, different association constants were measured for the L and D enantiomers. These data suggest that aminoacids are bound via two interactions, zinc-carboxylate coordination and two hydrogen bonds between the ammonium group of the aminoacid and two oxygen atoms of one D-glucose moiety. Such conclusion is supported by the result of semiempirical (PM3) calculations. Notably, the K(L)/K(D) value of 9.6 observed for the association of phenylalanine rivals with the highest values found for the chiral recognition of aminoacids in water.     

Fractionation of Rat Liver tRNA by Reversed-Phase High Performance Liquid Chromatography: Isolation of ISO-tRNAsPro

Abstract

This paper illustrates the fractionation of cytoplasmic transfer ribonucleic acid from rat liver by reversed-phase high performance liquid chromatography using a gradient of acetonitrile/ammonium acetate. The procedure is fast, highly reproducible, and gives an excellent resolution of the numerous tRNA population: about 50 peaks with area peak percentages ranging from 0.001 to 5 can be monitored. Uncharged tRNA preparations exhibited a chromatographic profile different from aminoacylated tRNA, thus suggesting a possible strategy to distinguish between aminoacylated and nonacylated tRNA species. Moreover, a first approach to map the HPLC peaks was attempted by chromatographing preparations of tRNA which had been aminoacylated with individual ³H-labeled aminoacids. Here is reported the case of tRNA^Pro, which gave three well separated radioactive peaks, most likely corresponding to tRNA^Pro isoacceptor species.     

Synthesis of 125I-labeled N-3-(4-hydroxyphenyl)propionyl aminoacyl transfer ribonucleic acids.

A method for the isolation and labeling to high specific radioactivity of individual isoaccepting tRNAs is described. After blocking reactive minor bases by acetylation and iodination of the crude tRNA, a single family of isoacceptors was aminoacylated. Individual isoacceptors were separated by chromatography on RPC-5 and then acylated with the 3-(4-hydroxyphenyl)propionyl ester of N-hydroxysuccinimide. The product was purified by chromatography on BD-cellulose and RPC-5. This derivatized tRNA was then iodinated with 125I- and Chloramine-T to give a product containing between 5 X 10(7) and 3 X 10(8) dpm/microgram. The suitability of such labeled tRNAs for hybridization to homologous DNA in solution and cytological preparations of chromosomes is discussed with particular reference to Drosophila melanogaster.     

The Partitioning of Photosynthetically Assimilated 14C into Amino Acids in Wheat: 2. Distribution in Amino Acids of Wheat Grain Fractions

Changes in the distribution of ¹⁴C between free and bound aminoacids in wheat grains (Triticum aestivum L. cv. Arkas) at 10and 20 d post-anthesis are described. After ¹⁴CO₂, labellingof the flag leaf, ¹⁴C was initially more rapidly transferredto the grains of 20 d post-anthesis plants than for 10 d post-anthesisplants. However, after a 460 min chase period in the light theamount of ¹⁴C in the grains of the younger and older plantswere similar. In the younger, more rapidly growing grains, agreater proportion of the ¹⁴C was incorporated into structuraltissue and starch. ¹⁴C accumulation in the grains continuedduring the dark in the younger grains but not in the older grains. Although the overall ¹⁴C distribution between the free aminoacid and protein pools of the grain was similar for both treatments,the distribution within the albumin, prolamin and globulin fractionsand between the individual non-bound amino acids differed. Ofthe protein fractions, the albumins were initially the mostheavily labelled but after 460 min chase the prolamins containedmore ¹⁴C. The majority of the ¹⁴C in the albumin and globulinfractions after 280 min chase was in hydrolysable, non-aminoacid compounds. In both tissues, the free amino acid pools lostradioactivity in the dark but the solid residues and proteinscontinued to function as ¹⁴C sinks. Daily fluctuations in the radioactivity in free and bound alanineare consistent with the role of free alanine as a diurnal metabolicnitrogen pool. Wheat, Triticum aestivum¹⁴CO₂, amino acids, proteins, carbon metabolism     

Anticonvulsive activity of antiepileptic drugs and quantum-chemical modelling of their interaction with GABA(A) receptor

The results of experimental analysis of the clinical activity of the antiepileptic drugs' (Phenobarbital, Carbamazepine, Valproic acid, Lamotrigine, Topiramate, Felbamate) widely used in clinic, that was carried out using the standard convulsion test with bicuculline in vivo were compared with characteristics of these drugs' interaction with the key aminoacids of GABA(A) receptor calculated by quantum chemical method (program HyperChem7, semi-empirical method AM1 technique). The correlation between the activity of the drugs in the experiment in vivo and energy of system's interaction of the drugs with aminoacid residue Thr201-Thr202-Gly203- Ala204-Tyr205-Pro206 was found out.     

Cloning and expression of the coding regions of the heat shock proteins HSP10 and HSP16 from Piscirickettsia salmonis

The genes encoding the heat shock proteins HSP10 and HSP16 of the salmon pathogen Piscirickettsia salmonis have been isolated and sequenced. The HSP10 coding sequence is located in an open reading frame of 291 base pairs encoding 96 aminoacids. The HSP16 coding region was isolated as a 471 base pair fragment encoding a protein of 156 aminoacids. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from other prokaryotic organisms. Both proteins were expressed in E. coli as fusion proteins with thioredoxin and purified by chromatography on Nicolumn. A rabbit serum against P. salmonis total proteins reacts with the recombinant HSP10 and HSP16 proteins. Similar reactivity was determined by ELISA using serum from salmon infected with P. salmonis. The possibility of formulating a vaccine containing these two proteins is discussed.     

Activity of gamma-glutamyl transferase of sheep cerebral cortex with respect to amino acids and glutathione]

gamma-glutamyl Transferase fron Sheep brain cortex capillaries was studied from the point of view of transport of aminoacids across blood brain barrier. Excess substrate inhibition was competitive and observed both with donor (glutathione) and various acceptors (methionine, alanine, tryptophan) but not with arginine. Excess glutathione inhibition of transfer reaction is concomitant with an increase of total reaction (transfer + hydrolysis + autotranspeptidation). With regard to aminoacids, the greater the K'm the stronger the inhibition. This inhibition is the result of formation of a dead complex. Lineweaver-Burk plots 1/v versus 1/[acceptor] give straight lines meeting at the same point, whereas 1/v verus 1/[donor] plots are roughly parallel for high aminoacid concentrations and become secant for the low ones. Replots of slopes vs. 1/[acceptor] are not linear: the lower the aminoacid affinity the more pronounced the slope replot curvature. Thus kinetic patterns are consistent with a branched ping-pong mechanism including a ternary complex (Enzyme-acceptor-H2O) at high or low relative concentration, which balances the two branches. The estimated value of kinetic parameters does not support the hypothesis of major implication of the enzyme in brain uptake of aminoacids.     

Molecular structure and function of the bacteriocin gene and bacteriocin protein of plasmid Clo DF13.

In this paper we present the complete nucleotide sequence of the bacteriocin gene of plasmid Clo DF13. According to the predicted aminoacid sequence the bacteriocin, cloacin DF13, consists of 561 aminoacids and has a molecular weight of 59,293 D. To obtain insight into the structure and function of specific parts of the cloacin molecule, we constructed a hydration profile and we predicted the secondary structure of the protein. According to our predictions, the N-terminus of cloacin DF13 (corresponding to the first 150-180 aminoacids) is relatively hydrophobic and is rich in glycine residues. The data obtained support previous findings that the N-terminal part of cloacin DF13 is involved in translocation of this protein across the cell membrane. The C-terminal part of the cloacin protein is rich in positively charged aminoacids; this might reflect the RNase activity located within this domain. A comparison of the bacteriocin genes and corresponding proteins of Clo DF13 and Col E1 did not reveal any homology at the level of either the nucleotide or the aminoacid sequence. The codon usage of both genes, however, exhibits striking similarities. The sequence data obtained during this study enabled us to present the nucleotide sequence of the entire cloacin operon. The structure of this operon and the regulation of expression of the genes, located within this operon, is discussed.     

Blood amino acids in chronic HBsAg positive liver disease

Typical changes in blood aminoacid concentrations have been described in patients with severe liver disease. In this study we measured the serum amino acid levels, by Beckman Aminoacid Analyzer, in 11 healthy subjects and 24 HBsAg-positive patients with biopsy-proven liver disease (4 CPH, 10 CAH, 10 cirrhosis). A significant decrease in total aminoacids was observed in CAH and cirrhosis groups (-24% and -22% respectively). The three branched chain aminoacids (BCAA = val + leu + isoleu) were reduced by 24% (P less than 0.002) and 37% (P less than 0.001) in the CAH and cirrhosis groups respectively. Tyrosine was the only of the aromatic aminoacids (AAA) to increase in cirrhotics (+ 34%, P less than 0.02). The molar ratio BCAA/AAA was 3.6 in controls, 3.8 in CPH, 3.1 in CAH (P less than 0.025) and 1.9 in cirrhosis (P less than 0.001). A linear correlation was found between molar ratio BCAA/AAA and serum albumin in all patients (P less than 0.001). These results document the presence of specific quantitative changes in serum aminoacids of HBsAg positive patients, which appear related to severity of liver disease and comparable to the alterations described in non viral chronic liver disease.