Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450 – (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG – was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained.Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH–cytochrome P450–reductase in L--dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction.CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450–reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.     

The isometric force that induces maximal surface muscle deoxygenation

To determine the external force that induces maximal deoxygenation of brachioradialis muscle 32 trained male subjects maintained isometric contractions using the elbow flexor muscles up to the limit time (isotonic part of the isometric contraction, IIC) and beyond that time for 120 s (anisotonic part of the isometric contraction). During IIC each subject maintained relative forces of either 25% and 70% maximal voluntary contraction (MVC), 50% and 100% MVC, or 40% and 60% MVC. Muscle oxygenation was assessed using a near infrared spectroscope, and expressed as a percentage of the reference value (ΔO_2rest) which was the difference between the minimal oxygenation obtained after 6 min of ischaemia at rest and the maximal reoxygenation following the release of the tourniquet. During IIC at 25% MVC, muscle oxygenation decreased to 17 (SEM 3)% ΔO_2rest, then it levelled off [25 (SEM 1)% ΔO_2rest]. After the point at which target force could not be maintained, reoxygenation was very weak. During IIC at 40%, 50%, 60%, and 70% MVC, the lowest muscle oxygenation values were obtained after 15–20 s of contraction and corresponded to −18 (SEM 6), −59 (SEM 12) −31 (SEM 6), and −29 (SEM 6)% ΔO_2rest, respectively. For the contraction at 100% MVC, the lowest oxygenation [−19 (SEM 9)% ΔO_2rest] was obtained while force was decreasing (69% MVC). During the anisotonic part of the isometric contractions, the greatest reoxygenation rate was obtained after 50% MVC IIC (P < 0.001). Our results showed that during isometric elbow flexions between 25% and 100% MVC, there was no linear relationship between external force and muscle oxygenation, and that the maximal deoxygenation of the brachioradialis muscle was obtained at 50% MVC. Accepted: 16 February 1998     

Anaphylactic Reaction to Penicillin O

Although normally the sympathetic nerves aid vascular dilatation during effort, in certain diseases of the vascular system they have a reverse effect. Abolition of sympathetic vasoconstrictive impulses by sympathectomy is the most effective treatment in some chronic peripheral vascular conditions. The authors have used electrocoagulation for a number of years and found it quick, effective and more likely to prevent regeneration of the affected nerves.Improvement was obtained by sympathectomy in arteriosclerotic vascular insufficiency, thromboangiitis obliterans, Raynaud''s disease, reflex sympathetic dystrophy following thrombophlebitis or trauma, scleroderma, spinal sympathetic dystrophy and acquired megacolon. A case of causalgia was aggravated by the operation.Abstention from the use of tobacco appears to be sufficient for control of symptoms in many cases.Since vasospasm is manifested in many conditions long before a thrombotic catastrophe occurs, not only relief of symptoms but prevention of irreversible changes may be achieved by early operation.     

Catechol oxidase from green olives: Properties and partial purification

Catechol oxidase was extracted from an acetone powder prepared from green olive. The enzyme was purified 240-fold by ammonium sulphate fractionation followed by ion exchange chromatography and gel filtration. The enzyme was characterized by substrate specificity and response to inhibitors. Between 7 and 9 bands having catechol oxidase activity could be detected by gel electrophoresis and electrofocusing. The purified enzyme had an estimated MW of 42 000. The enzyme was strongly inhibited by diethyldithiocarbamate. Inhibition by chloride was strongly dependent on pH. The enzyme did not oxidise monophenols.     

Respiration,ATP level,and sugar accumulation in potato tubers during storage at 4°

A kinetic study was made of the relationship between respiration rate, sugar content and ATP levels, in fresh and aged potato tubers stored at 4°. The ATP content in tubers rose rapidly immediately after the chilling stress, while respiration rate decreased below the initial rate and sugar accumulation was not detected. After 4 days of storage, the ATP level declined and the sugars started to accumulate. The typical increase in respiration rate that usually follows chilling stress, appeared only in fresh tubers (at about the 6th day of storage). In dinitrophenol-treated tubers, the ATP level remained below the initial level and sugar accumulation was blocked completely. The evidence presented suggests that ATP elevation is not generated by the respiration burst.     

Qualitative dynamics of three species predator-prey systems

Summary A qualitative analysis of some two and three species predator-prey models is achieved by application of the method of averaging in conjunction with a Lyapunov function constructed from the appropriate Volterra-Lotka model. We calculate the limit cycle solution for a two-species model with a Holling type functional response of the predator to its prey by means of a time-scaled transformation. The existence of a bifurcation of steady states for a community of three species is discussed and the periodic solution around one of the unstable steady states is calculated to the lowest approximation. Several comments are made regarding the behavior of these systems under changes of some control parameters.This work was supported in parts by USERDA, Contract number E(11-1)-3001.     

Molecular and functional anomalies in two new mutant glucose-phosphate-isomerase variants with enzyme deficiency and chronic hemolysis

Summary Two new deficient glucose-phosphate-isomerase (GPI) variants have been described in patients suffering from severe chronic hemolytic anemias. The patients' parents were consanguineous, such that the patients were true homozygotes for the mutated GPI genes. In both cases the main cause of the defect in enzyme activity was molecular instability of the mutated GPI molecules, their catalytic activity being nearly normal.GPI Paris was characterized by a slow electrophoretic migration and, above all, a drastically altered affinity for the substrates glucose-6-phosphate (decreased) and fructose-6-phosphate (increased). GPI Enfants malades exhibited a slightly reduced electrophoretic mobility, an abnormal curve of the activity in function of pH, and an abnormal ratio of maximal velocity in the backward direction (fructose-6-phosphate»glucose-6-phosphate) to that in the forward direction (glucose-6-phosphate»fructose-6-phosphate).No clear relation could be proved between the kinetic abnormalities of the mutant GPI variants on the one hand and the metabolic changes of the GPI-deficient red cells and the severity of hemolysis on the other.Finally we emphasized the possible role of the impairment of hexosemonophosphate pathway in the reduction of viability of the GPI-deficient red cells.