Jesús Sebastián

Analysis of the structure of international scientific cooperation networks through bibliometric indicators

International scientific cooperation of Latin American countries amongst themselves, with the USA and with the European Union in the period 1991–1995 was studied. The analysis deepens in the differences per subject area and the influence of the regional axis involved. Collaboration patterns differ according to the scientific size of the latin American countries, the thematic areas and whether a bilateral collaboration or a participation in a multilateral network takes place. Some special characteristics of multi-regional cooperation networks are presented.

Oxaloacetate metabolic crossroads in liver. Enzyme compartmentation and regulation of gluconeogenesis

Summary1.The subcellular distribution of the enzymes involved in oxaloacetate metabolism and certain related enzymes in the liver of normal fed and starved rats has been studied. A simplified fractionation procedure was developed in the course of this work. Pyruvate carboxylase and citrate synthase were found to precipitate exclusively associated to mitochondria.2.The precise location of the mitochondrial enzymes was studied in submitochondrial fractions. The existence of two distinct oxaloacetate metabolic crossroads in rat liver has been clearly established. One is located in the mitochondrial matrix and involves the oxaloacetate forming pyruvate carboxylase, the citrate synthase and the mitochondrial malate dehydrogenase and glutamic oxaloacetic transaminase. The second one is located in the cytosol and involves the phosphoenolpyruvate carboxykinase and the cytosolic malate dehydrogenase and glutamic oxaloacetic transaminase.3.The results of similar fractionation studies applied to rabbit liver indicate that in this animal phosphoenolpyruvate carboxykinase is located in the mitochondrial matrix. The other enzymes tested showed the same localization as in the rat. 4. The search in rat liver for a cytosolic activity from 3C to 4C gluconeogenic precursors has been negative. This result stresses the importance of the regulatory problems associated with the exit of oxaloacetate out of the mitochondria in gluconeogenesis. 5. The adaptive behaviour of the enzymes of the mitochondrial and cytoplasmic oxaloacetate crossroads has been studied in rat liver. Only phosphoenolpyruvate carboxykinase shows a marked increase in specific activity when the animals are fasted. 6. The kinetic properties of rat liver citrate synthase have been studied. NADPH inhibition is competitive against oxaloacetate while ATP inhibition is competitive against acetyl-CoA. 7. The Michaelis constants of the mitochondrial malate dehydrogenase and the kinetic constants of the mitochondrial glutamic oxaloacetic transaminase have been measured. 8. A model of the possible regulation of the metabolic flow at the oxaloacetate crossroads in gluconeogenesis in rat liver is proposed.

Identification and properties of an inducible mannokinase from Streptomyces violaceoruber

1. 1. Crude extracts from cells of several species of Streptomyces grown on different sources of carbon and energy, showed a constitutive ATP-dependent phosphorylating activity on glucose. Specifically inducible kinase activities for mannose and fructose were found in cells grown on each of these sugars. 2. 2. The activity on glucose corresponded to a typical bacterial glucokinase (ATP:d-glucose 6-phosphotransferase, EC 2.7.1.2). The activities on mannose and fructose corresponded to two enzymes of a novel pattern. 3. 3. The activity on mannose was due to a mannokinase (ATP:hexose 6-phosphotransferase, EC 2.7.1.7), which has been purified about 100-fold from extracts of Streptomyces violaceoruber. The Km of this enzyme for mannose was 0.05 mM. Glucose was also phosphorylated by this enzyme although with low affinity (Km = 4 mM). Fructose was neither a substrate nor an inhibitor. 4. 4. The inducible activity on fructose corresponded to a highly specific fructokinase (ATP:d-fructose 6-phosphotransferase, EC 2.7.1.4), which is described in the accompanying report.

Identification and properties of an inducible and highly specific fructokinase from Streptomyces violaceoruber

1. 1. An inducible fructokinase (ATP:fructose 6-phosphotransferase, EC 2.7.1.4) found in Streptomyces is described. The enzyme has been purified 400-fold from extracts of Streptomyces violaceoruber. 2. 2. This fructokinase phosphorylated fructose as the apparently unique sugar substrate, with a Km of 0.5 mM under conditions of MgATP saturation. The Km for MgATP was 0.2 mM. MgADP inhibited the enzyme competitively with MgATP (Ki, 0.2 mM). The other product, fructose 6-phosphate, did not inhibit the enzyme appreciably. 3. 3. The enzyme showed complex kinetic properties: apparent activation by free Mg2+, a sigmoid saturation curve for MgATP, and a double loop saturation curve for fructose at low MgATP concentrations.

Purification and properties of three proteases from the larvae of the brine shrimp Artemia salina.

Three proteases, termed A, B and C, have been characterized and partially purified from Artemia salina larvae. Enzyme A is active on benzyloxycarbonyl L-leucin p-nitrophenyl ester (Km = 53 micron, determined at pH 8 and 37 degrees C) but not on alpha-N-benzoyl-DL-arginine p-nitroanilide and is strongly inhibited by micron concentrations of phenylmethylsulfonylfluoride. Enzymes B and C are active on alpha-N-benzoyl-L-arginine p-nitroanilide (Km = 20 and 11 micron, respectively) but not on benzyloxycarbonyl-L-leucine p-nitrophenyl ester and B, but not C, is inhibited by mM concentrations of phenylmethylsulfonyfluoride. Enzymes A, B and C are optimally active at alkaline pH values, do not require either metal ions or -SH groups for their catalytic activity and have molecular weights of 38 000, 33 000 and 34 000, respectively. After heating for 5 min at pH 7.5 and in the presence of 0.7 M KCl half inactivation of proteases A, B and C was attained at 60, 52 and 45 degrees C, respectively.

Characterization and levels of the RNA polymerases during the embryogenesis of Artemia salina

Dormant embryos at the gastrula stage of Artemia salina contain three DNA-dependent RNA polymerases: I, II, and III. The enzymes are solubilized from whole embryos and they are separated by chromatography on DEAE Sephadex. The ratio of activities with native and denatured DNA at the optimal salt concentrations is 3.5 for RNA polymerase I, 0.1 for RNA polymerase II and 1.0 for RNA polymerase III.Mn(i2+) is more efficient than Mg(2+) for the three enzymes. RNA polymerase II is 50% inhibited by 5 ng/ml of alpha-amanitin while RNA polymerases I and III are 10% and 30% inhibited by 1 mg/ml. During the embryonic development there is am independent variation of the levels of the RNA polymerases. RNA polymerase I increases its specific activity 4-5-fold, RNA polymerase III increases 2-fold, and RNA polymerase II less than 2-fold. The increase in RNA polymerase activity may represent a mechanism to control the rate of synthesis of RNA during the embryogenesis of A. salina.

Satellite DNA in the crustacean Artemia

We have isolated a satellite fraction from the Artemia genome by both restriction endonuclease digestion and equilibrium density centrifugation in CsCl gradients containing ligand dye Hoechst 33258. Satellite DNA was arranged in long stretches (approx. 23 kb) of tandem repeats of a basic unit of 113 bp. The basic unit has been sequenced, showing a G + C content very close to that of total DNA. Different amounts of satellite were present in several populations of Artemia, whereas it was absent from others.

Restriction mapping of the rRNA genes from Artemia larvae

Abstract A restriction endonuclease analysis of the genes coding for the ribosomal RNA from Artemia larvae has shown that these genes consist of a repeat unit of 16.2 kilobase pairs (10.7 Mdaltons) and that the repeat unit seems to be homogeneous in size.

Proteolytic origin of a modified form of RNA polymerase I from Artemia salina larvae.

Abstract Extracts from Artemia salina larvae contain a form of RNA polymerase (Ia) not present in the embryos. The appearance of this RNA polymerase during the larval development is correlated with a decrease in the levels of RNA polymerase I. The modification of RNA polymerase I to Ia was obtained in vitro by incubation of enzyme I with either a larvae extract or protease B, one of the multiple proteases induced during the larval development. The results indicate that the modified form of RNA polymerase present in the larvae is produced in vitro by proteolysis of enzyme I during the extraction and solubilization of the RNA polymerases.

Adenylate cyclase activity in permeabilized cells from Dictyostelium discoideum.

Abstract Aggregation competent cells of Dictyostelium discoideum have been permeabilized using the method previously developed for mammalian cells by Aragon et al . (Proc. Natl. Acad. Sci. USA (1980) 77, 6324). The permeabilization allows the “in situ” assay of adenylate cyclase. The enzyme activity is proportional with the amount of cells and the time of incubation. The enzyme exhibits non Michaelian kinetics towards its substrate ATP as recently reported for the enzyme assayed in crude extracts. Permeabilized cells contain lower levels of phosphodies-terase activity as compared with cellular homogenates. The similar properties of the adenylate cyclase from cell homogenates and permeabilized cells, as well as the low phosphodiesterase activity, makes the permeabilization method of great interest to study the regulation of adenylate cyclase during the differentiation of Didiscoideum .