Ernst Boris Chain

Production of a new lysergic acid derivative in submerged culture by a strain of Claviceps paspali Stevens & Hall

1. The production of a new lysergic acid derivative, identified as D-lysergic acid α-hydroxyethylamide, in submerged culture and in yields up to 1 mg/ml. and above, by a strain of Claviceps paspali Stevens & Hall is reported; this substance can be converted in high yields into D-lysergic acid amide. 2. The morphological properties of this strain under different culture conditions are examined and it is shown that it grows in a form resembling natural sclerotia. 3. The biochemical culture conditions for the production of the new lysergic acid derivative in shake flasks and in stirred fermenters, the course of the fermentation, the methods for the isolation and purification of the substance and its transformation products and some of its chemical and physical properties are described.

The Influence of Insulin on Carbohydrate Metabolism in the Isolated Diaphragm Muscle of Normal and Alloxan Diabetic Rats

The fate of uniformly labelled 14C glucose in the isolated diaphragm muscle of normal and alloxan diabetic rats has been studied by a quantitative application of the radio paper-chromatographic technique. No significant differences were observed in the metabolism of glucose by muscle from normal and diabetic rats. About 80% of the glucose metabolized by the muscle in the absence and presence of insulin has been accounted for. The extra glucose disappearing from the incubation medium in the presence of insulin was found to be incorporated in oligo- and polysaccharides, the percentage of glucose converted into the other main metabolites (i.e. lactic acid, hexose-phosphate esters and CO2) was shown to be unchanged or decreased by insulin. Insulin markedly accelerated the synthesis of oligo- and polysaccharides. The amount of free glucose in the tissues, which reached a maximum after 15 min of incubation, was uninfluenced by insulin. When the muscle was first incubated with 14C glucose and then in the absence of substrate, there was a sharp decrease in the radioactivity of all the intermediates with the exception of the hexose-phosphate esters which were found to accumulate. Under anaerobic conditions the total glucose metabolism was very reduced, and the greater part of the glucose disappearing from the medium was recovered as free glucose in the tissues. Very little radioactive lactic acid was formed under anaerobic conditions, except when the muscle was first incubated with 14C glucose aerobically and then anaerobically; under these conditions there was a considerable accumulation of radioactive lactic acid. Current theories on the mode of action of insulin have been discussed in reference to the findings reported in the present work.

Fate of uniformly labelled 14C glucose in brain slices

The fate of uniformly labelled 14C glucose in rat-brain slices has been followed by a quantitative application of the radio paper-chromatography technique. After 60 min incubation with brain tissue approximately 60% of the glucose disappearing from the medium was accounted for as lactic acid, about 20% as CO2 and most of the remainder as free amino-acids. Of the total glucose metabolized approximately 9% was converted into glutamic acid, 1.5% alanine, 3% γ-amino-butyric acid and 2.4% aspartic acid. Glutamic acid was the first of the amino-acids to be formed from glucose, and was detectable after 3 min incubation of brain tissue with 14C glucose. Insulin had no effect on glucose metabolism in brain slices. Under anaerobic conditions the total glucose metabolized by brain slices was only about 10% of that found under aerobic conditions; of the total glucose disappearing from the medium anaerobically about 80% was accounted for as lactic acid and the remainder as free unchanged glucose in the tissue cells.

Metabolism of hexoses in rat cerebral cortex slices.

Abstract— 1 The metabolism of two 14C‐labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro. 2 The metabolism of [U‐14C]mannose was essentially identical to that of glucose; oxygen consumption and CO3 production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration. 3 With [U‐14C]fructose, maximal oxygen consumption and CO3 production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3‐14C]pyruvate into CO2 or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO2 from [U‐14C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent. 4 By comparison with [1‐14C]glucose, incorporation of radioactivity from [1‐14C]‐glucosamine into lactate, CO2, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine‐1‐phosphate, glucosamine‐6‐phosphate, and an unidentified metabolite, all accumulated.

The fate of glucose in different parts of the rabbit brain

The fate of uniformly 14C labelled glucose has been followed by a quantitative application of the radio paper-chromatographic technique in different parts of the rabbit brain-hypo-thalamus medialis anterior, hypothalamus medialis posterior, hypothalamus lateralis anterior, cerebral cortex, cerebellar cortex—and in the optic chiasma. Qualitatively the metabolic pattern of glucose was similar in all the different parts of the brain which were studied. The glucose disappearing from the medium after 60 min of incubation was accounted for as lactic acid, CO2, alanine, aspartic acid, glutamic acid, γ-aminobutyric acid and glutamine. However, in the cerebral cortex significantly greater quantities of alanine, aspartic acid, glutamic acid and glutamine were found than in any of the other tissues. The hypothalamus formed more γ-aminobutyric acid from glucose than the cerebral and cerebellar cortex. The inclusion of potassium in the medium augmented the oxygen uptake and the production of radioactive glutamine in all the different parts of the brain and the production of radioactive CO2 in the cerebral cortex. On the other hand, the presence of potassium diminished the production of radioactive CO2 in the hypothalamus but had no effect on CO2 production in the cerebellar cortex.

The Metabolism of Glutamic Acid in Rat Retina

Investigation of the metabolism of L-glutamic acid in slices of adult rat retina under aerobic conditions using the technique of quantitative automatic paper radio-chromatography is described, L-glutamic acid was converted into aspartate, γ-aminobutyrate, glutamine and carbon dioxide. In the presence of added glucose, the formation of glutamine and γ aminobutyrate from glutamate was markedly increased, whereas the formation of glutamine was not appreciably affected by the addition of pyruvate to the medium. The addition of either glucose or pyruvate caused a marked decrease in the production of aspartate from glutamate. The production of carbon dioxide from glutamate, or from glutamate in the presence of glucose or pyruvate, appears to be quantitatively the same.

Interrelationship of Glucose, Glutamate and Aspartate Metabolism in Rat Cerebral Cortical Slices

The relative specific activities of the respiratory CO2 deriving from the metabolism of [14C]glutamate, and [14C]aspartate in the presence and absence of non-radioactive glucose, and of [14C]glucose in the presence and absence of non-radioactive glutamate and aspartate in slices of rat brain cortex were determined. The values for glutamate and aspartate in the absence of glucose were 0·4 and 0·3 respectively, showing that 30 to 40% of the respiratory CO2 derived from these amino-acids. Aspartate, in contra-distinction to glutamate, does not increase the rate of oxygen of brain tissue above that due to endogenous substrates; yet the determination of the specific activity of the respiratory CO2 shows that this amino-acid too is readily oxidized by this tissue, replacing about a third of the endogenous sources of respiratory CO2. The addition of non-radioactive glucose as a co-substrate considerably stimulated the rate of oxygen uptake by brain tissue both in the presence of glutamate as well as aspartate, yet did not affect the specific activity of the respiratory CO2, indicating that glucose stimulated the oxidation of the amino-acids to the same extent as the oxidation of glucose itself or other non-amino acid substrates was increased. The addition of non-radioactive glutamate or aspartate to [14C]glucose resulted in a considerable decrease of the relative specific activity of the respiratory CO2, without affecting the rate of respiration, indicating that the oxidation of amino-acids replaced that of glucose, i.e. in this sense the amino-acids were preferentially oxidized. These results indicate that in the pattern of the oxidative processes of brain cortical tissue the oxidation of amino-acids plays an important part.

The metabolism of glucose and pyruvate in rat retina

The metabolism of [U-14C]glucose and [3-14C]pyruvate in the adult rat retina is described. In vitro under aerobic conditions, in either phosphate or bicarbonate medium, glucose was converted into lactate, carbon dioxide, glutamate, γ-aminobutyrate, aspartate, glutamine and alanine. Under anaerobic conditions, total glucose metabolized was reduced to 60 to 70% of that under aerobic conditions, lactic acid being the only metabolic product detected. Under aerobic conditions [3-14C]pyruvate was converted by the retina into the same metabolites as was glucose. The quantitative data for oxygen uptake and 14CO2 formation were similar to those obtained with glucose as substrate; lactate production was lower and amino acid formation higher.

The Metabolism of Glutamate and Aspartate in Rat Cerebral Cortical Slices

The fate of [14C]glutamate and [14C]aspartate in rat cerebral cortical slices was followed quantitatively by means of the previously described automatic scanning technique of paper radio-chromatograms, in the presence and absence of non-radiocative glucose, and in the case of [14C]glutamate, also of the following non-radioactive tricarboxylic cycle acids: pyruvate, citrate, α-oxoglutarate, malate, oxalo-acetate and succinate. Both amino-acids were readily oxidized to CO2, glutamate increasing, aspartate not affecting the rate of endogenous oxygen uptake of the tissue. In addition the following metabolites accumulated: (a) From glutamate: aspartate, glutamine and γ-aminobutyrate, in order of decreasmg amounts. (b) From aspartate: glutamate, malate and citrate, in order of decreasmg amounts, with traces of glutamine, γ aminobutyrate and lactate. The presence of non-radioactive glucose as co-substrate caused the following effects. 1. On glutamate-.The total production of 14CO2aswell as the oxygen uptake increased. This indicated that glucose oxidation had not replaced glutamate oxidation, but actually caused its stimulation. The conversion into glutamine was increased by a factor of 3, an effect similar to that caused by K+ ions. The conversion into aspartate was decreased by a factor of 4. The accumulation of intracellular glutamate was much enhanced. 2. On aspartate: both 14CO2 production and oxygen uptake were stimulated, indicating that, as in the case of glutamate, glucose oxidation not only had not replaced the oxidation of amino-acid, but stimulated it. The production of glutamate, glutamine, γ-aminobutyrate and lactate was considerably increased while that of malate and citrate was unaffected. The following effects on glutamate metabolism were noted in the presence of non-radioactive tricarboxylic cycle acids: None of the acids affected 14CO2 production except α-oxoglutarate which caused a decrease of over half. This decrease was shown to be due to a dilution of [14C]glutamate through equilibration with non-radioactive α-oxoglutarate; the latter acquired about half of the radioactivity of the [14C]glutarnate added during the incubation. Pyruvate, like glucose, decreased the conversion to aspartate and increased glutamine formation, the latter, however, to a much lower degree than glucose. It also enhanced the accumulation of intracellular glutamate. Citrate, succinate, malate and oxaloacetate, particularly the latter two co-substrates, repressed the formation of [14C]glutamine, but had no other effect on the pattern of distiibution of the metabolites formed from glutamate and on the permeability of this amino-acid into the cell.

Studies on Ferroverdin, a Green Iron-Containing Pigment Produced by a Streptomyces Wak. Species

The submerged culture production of ferroverdin, an iron-containing green pigment of a novel type, by a new species of Streptomyces Wak. is reported. The morphological properties of this micro-organism are described, and the optimum culture conditions for pigmentation in shake flasks and in stirred fermenters are determined. The Preparation and identification of reductive and alkali degradation products of ferroverdin are reported; on the basis of their structures it is suggested that ferroverdin is the ferrous complex of the p-vinylphenylester of 3-nitroso-4-hydroxybenzoic acid.