Studies on evolutionary and selective properties of hypercycles using a Monte Carlo method

Summary The most relevant properties of hypercycles were previously studied mainly from a theoretical point of view. We have developed a Monte Carlo method simulating hypercyclic organization to obtain information about the dynamics of this prebiotic organization. Nucleation, growth, and selective properties have been tested and the results obtained are in good agreement with those of the theoretical predictions. The influence of hypercyclic organization of the error threshold has also been studied. As a consequence of the emergence of a hypercycle, the value of this threshold decreases. The amount of this decrease depends on the population size. Moreover, for some interval of quality factor values, either the hypercycle organization or an error catastrophe can be produced, depending on the initial conditions. The influence of these phenomena on both the dynamic behavior and evolutionary advantages of the hypercycle, as well as their decisive roles on genome size, are discussed.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

A study of free radicals in paraffin embedded and deparaffinized human heart muscle tissue using electron spin resonance (ESR)

Summary Free radicals (spectroscopic splitting factor; g factor=2.003–2.005) were investigated in formol-fixed, paraffin embedded heart-muscle tissue sections using electron spin resonance (ESR) spectra. Changes in signal amplitude, g factor and line width were registered during deparaffinization, chloroform-methanol extraction, vapour treatment and bromination. An attempt was made to identify the source of the ESR signals by a correlation between the signal amplitude and number of fluorescent and/or Sudan-black-positive granules counted in the tissue sections. An increase in signal amplitude, g value and line narrowing were characteristic of the ascorbyl radical after deparafinization in air. Vapour treatment revelated that the broader signal has lower g factor, a characteristic that is tentatively assigned to oxidized lipids. The bromination resistant minor fraction of free radical centres with small g factor might be associated with the pigment content of the samples.     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioates from adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate)

The chiral and achiral phosphorothioate analogues of 2',5'-oligoadenylates (2-5A) have been enzymatically synthesized from the Sp and Rp isomers of adenosine 5'-O-(2-thiotriphosphate) [(Sp)-ATP beta S and (Rp)-ATP beta S, respectively] and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) by 2-5A synthetase from L929 cells and lysed rabbit reticulocytes. These 2',5'-phosphorothioate analogues were separated, purified, and structurally characterized. While ATP gamma S and (Sp)-ATP beta S were as efficient substrates for the 2-5A synthetase as was ATP, (Rp)-ATP beta S was more than 50-fold less efficient a substrate. The beta- and gamma-phosphorothioates were more resistant to enzymatic hydrolysis than was authentic 2-5A. Compared to 2-5A, there were marked differences in the biological activities of the 2',5'-phosphorothioates as determined by (i) binding to 2-5A-dependent endoribonuclease (RNase L), (ii) activation of RNase L to hydrolyze RNA, and (iii) inhibition of protein synthesis in intact L929 cells. These studies extend previous reports on the elucidation of the stereochemical requirements of 2-5A synthetase and RNase L [Karikó, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (first of three papers in this issue); Karikó, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (second of three papers in this issue)] with the phosphorothioate analogues of 2-5A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity of Mg2+-ATPase and cytochrome c-Oxidase in microsporocytes and anther wall during microsporogenesis inLarix europaea D.C.

Studies have been made on the activity of two mitochondrial enzymes, Mg²⁺ ATPase (E.C.3.6.1.3.) and cytochrome c-oxidase (E.C.I.9.3.2.) in microsporocytes and somatic cells of anther in larch. The material for study were homogeneous fractions of microsporocytes from 15 stages of meiosis and the attendant anther somatic cells. The results have demonstrated that cells undergoing meiosis exhibit considerable mitochondrial metabolic activity. It is characterized by considerable variations in the activity level of both enzymes studied. The level and dynamics of variations of Mg²⁺-ATPase and cytochrome c-oxidase activity in microsporocytes are clearly different from those in the anther somatic cells. The cytochrome c-oxidase activity in microsporocytes throughout microsporogenesis is higher compared with that in the anther wall cells, whereas the Mg²⁺-ATPase activity in microsporocytes averagesca. one half that in the anther somatic cells The dynamics of activity variations of the enzymes under study suggests enhanced mitochondrial metabolism in the period of middle diplotene and young dyad. This result supports the thesis following from our earlier studies that the middle diplotene and young dyad constitute specific metabolic switches in microsporogenesis in larch.     

Biochemical indicators of water stress in sunflower seedlings

The free proline and chlorophyll contents, and the chlorophyllase, peroxidase and nitrate-reductase activities were determined in sunflower seedlings grown under controlled conditions and submitted to water stress induced by 14 % polyethyleneglycolj (M_r = 4000) or isotonic NaCl solution. Combined free proline content and peroxidase activity may be used for detection of the factor inducing water stress.     

Evaluation of the effectiveness factor along immobilized enzyme fixed-bed reactors: Design of a reactor with naringinase covalently immobilized into glycophase-coated porous glass

A design equation is presented for packed-bed reactors containing immobilized enzymes in spherical porous particles with internal diffusion effects and obeying reversible one-intermediate Michaelis-Menten kinetics. The equation is also able to explain irreversible and competitive product inhibition kinetics. It allows the axial substrate profiles to be calculated and the dependence of the effectiveness factor along the reactor length to be continuously evaluated. The design equation was applied to explain the behavior of naringinase immobilized in Glycophase-coated porous glass operating in a packed-bed reactor and hydrolyzing both p-nitrophenyl-alpha-L-rhamnoside and naringin. The theoretically predicted results were found to fit well with experimentally measured values.     

Application of extended Kalman filter to identification of enzymatic deactivation

A recursive estimation scheme, the Extended Kalman Filter (EKF) technique, was applied to study enzymatic deactivation in the enzymatic hydrolysis of pretreated cellulose using a model previously developed by the authors. When no deactivation model was assumed, the results showed no variation with time for all the model parameters except for the maximum rate of cellobiose-to-glucose conversion (r'(m)).The r'(m) variation occurred in two zones with a grace period. A new model of enzymatic hydrolysis of pretreated cellulose deactivation was proposed and validated showing better behavior than the old deactivation model. This approach allows one to study enzyme deactivation without additional experiments and within operational conditions.     

Partial purification and characterization of an aldohexose 1-P phosphatase from pig skeletal muscle

An enzyme with a molecular weight of 54,000 which possesses phosphatase activity acting on glucose 1-P, galactose 1-P and mannose 1-P has been partially purified and characterized from pig skeletal muscle. The enzyme is free of phosphoglucomutase and galactokinase activities, and it possesses a neutral optimum pH. Pi acts as an inhibitor; glucose, galactose and mannose do not produce any effect. Divalent cations are required for activity, Mg2+ being the most effective activator. Micromolar levels of fluoride and millimolar levels of chloride act as inhibitors; however, vanadate does not produce any effect. The enzyme may have an important role when galactose accumulates in tissues; for example, in galactosemic patients and in young animals ingesting high-galactose diets.     

Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparinum

During the investigation of alternative methods for the large scale preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast to the chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashion, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chondroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of delta-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30 degrees C and at pH 6.0-7.5. Only 15% of the activity was observed at 37 degrees C, indicating that the enzyme is very sensitive to thermal denaturation. It is strongly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.     

Glutathione status and sensitivity to GSH-reacting compounds of Escherichia coli strains deficient in glutathione metabolism and/or catalase activity

The intracellular concentrations of total glutathione, GSSG and protein · S-SG, the total excreted glutathione concentration, and the susceptibility towards GSH-reacting compounds were assayed in strains of Escherichia coli deficient in biosynthesis and/or reduction of glutathione. A deficiency in glutathione reductase displaced the glutathione status towards the oxidized forms. This displacement was more clearly appreciated in strains additionally deficient in glutathione biosynthesis. A deficiency in catalase activity also produced an increase in the oxidation of glutathione. The most severe changes were observed in the concentrations of protein-glutathione mixed disulfides and in the amount of glutathione excreted to the medium. Increased sensitivities towards compounds known to interact with cellular GSH were observed in glutathione reductase deficient strains, although these effects were enhanced in strains additionally deficient in GSH biosynthesis