1. A method is given for the quantitative determination of free tryptophan or tryptophan in the intact protein by treating with ninhydrin in a mixture of formic acid and hydrochloric acid (reagent b), for 10min at 100 degrees C. Glycyltryptophan was used as a standard for the determination of tryptophan in the intact protein. The extinction at 390nm was linear in the range 0.05-0.5mumol for free tryptophan (in7120) and 0.05-0.30mumol for glycyltryptophan (in15400). 2. Free tryptophan in the presence of protein may be determined by treating with ninhydrin in a mixture of acetic acid and 0.6m-phosphoric acid (reagent a) for 10min at 100 degrees C, the extinction being linear for tryptophan in the range 0.05-0.9mumol. N-Terminal tryptophan peptides also give the typical yellow product on treatment with reagent a. 3. Tryptophan content of several pure intact proteins when treated with the above method gave values in good agreement with those reported by others. A mean tryptophan content of 11.25 (s.e.m. +/-0.08) mumol/100mg of protein was found in rat brain during development from 1 to 82 days after birth.

1. Measurements were made of the rate of incorporation of (14)C from uniformly (14)C-labelled glucose into individual amino acids of rat brain and liver. 2. At 2.5 min. after intravenous injection of uniformly (14)C-labelled glucose, about 30% of the total radioactivity in the brain was present in the five amino acids studied. At 30 min. after subcutaneous injection the distribution of (14)C in amino acids was: in brain, alanine 2%, gamma-aminobutyrate 4%, aspartate 9%, glutamine 9% and glutamate 37% (total 69%); in liver, alanine 3%, aspartate 2.6%, glutamine 5.3% and glutamate 5.2% (total 18%). About 1% of the total radioactivity was in serine and glycine. 3. In both organs the specific radioactivity of alanine was initially higher than that of the other amino acids examined. The specific radioactivity of gamma-aminobutyrate in the brain was about the same as or higher than that of glutamate. 4. Amino acids of the rat brain were separated into ;free' and ;bound' fractions from brain dispersions in saline (or sucrose) media. Definite differences in the specific activities of the ;bound' and ;free' forms were not apparent.

Abstract— Thiamine deficiency produced by administration of pyrithiamine to rats maintained on a thiamine‐deficient diet resulted in a marked disturbance in amino acid and glucose levels of the brain. In the two pyrithiamine‐treated groups of rats (Expt. A and Expt. B) there was a significant decrease in the levels of glutamate (23%, 9%) and aspartate (42%, 57%), and an increase in the levels of glycine (26%, 27%) in the brain, irrespective of whether the animals showed signs of paralysis (Expt. A) or not (Expt. B). as a result of thiamine deficiency. A significant decrease in the levels of γ‐aminobutyrate (22%) and serine (28%) in the brain was also observed in those pyrithiamine‐treated rats which showed signs of paralysis (Expt. A). Threonine content increased by 57% in Expt. A and 40% in Expt. B in the brain of pyrithiamine‐treated rats, but these changes were not statistically significant. The utilization of [U‐14C]glucose into amino acids decreased and accumulation of glucose and [U‐14C]glucose increased significantly in the brain after injection of [U‐14C]glucose to pyrithiamine‐treated rats which showed abnormal neurological symptoms (Expt. A). The decrease in 14C‐content of amino acids was due to decreased conversion of [U‐14C]glucose into alanine, glutamate, glutamine, aspartate and γ‐aminobutyrate. The flux of [14C]glutamate into glutamine and γ‐aminobutyrate also decreased significantly only in the brain of animals paralysed on treatment with pyrithiamine. The decrease in the labelling of, amino acids was attributed to a decrease in the activities of pyruvate dehydrogenase and α‐oxoglutarate dehydrogenase in the brain of pyrithiamine‐treated rats. The measurement of specific radioactivity of glucose, glucose‐6‐phosphate and lactate also indicated a decrease in the activities of glycolytic enzymes in the brain of pyrithiamine‐treated animals in Expt. A only. It was suggested that an alteration in the rate of oxidation in vivo of pyruvate in the brain of thiamine‐deficient rats is controlled by the glycolytic enzymes, probably at the hexokinase level. The lack of neurotoxic effect and absence of significant decrease in the metabolism of [U‐14C]glucose in the brain of pyrithiamine‐treated animals in Expt. B were probably due to the fact that animals in Expt. B were older and weighed more than those in Expt. A, both at the start and the termination of the experiments.

Ki-67 over expression was associated with adverse pathological features in cases of upper tract urothelial carcinoma. It was also an independent predictor of recurrence-free survival in patients with high grade upper tract urothelial carcinoma.

A simple and rapid method based on the NADH-linked reduction of a tetrazolium dye was described for the determination of pyruvate dehydrogenase activity in rat brain homogenates. The method (method 3) gave a value of 36.06 +/- 1.24 nmol of pyruvate utilised/min/mg of whole brain protein. This value was higher than that obtained by measurement of the rate of decarboxylation of [1-14C]pyruvate (15.10 +/- 0.88 nmol/min/mg of protein; method 1) and was comparable with the rate of transfer of acetyl groups to an arylamine (39.04 +/- 1.32 nmol/min/mg of protein; method 2). A critique of the values reported by others by different methods was given. The pyruvate dehydrogenase activity in the mitochondria isolated from rat brain was in the "active" (nonphosphorylated) form. A deficiency of thiamine in rats was produced by treatment with pyrithiamine, an antagonist of thiamine. This treatment resulted in abnormal neurological signs, such as ataxia and convulsions. The measurement of the total activity of pyruvate dehydrogenase in the brain by all three methods showed no significant change in the enzymic activity in thiamine-deficient rats after treatment with pyrithiamine. The activities of the enzyme in the brains of pair-fed animals were similar to those in the controls.

No abstract

WHEN [14C]glucose is administered in oiuo, part is oxidized to CO,, part is stored as glycogen and part is used for the synthesis of amino acids and other compounds in the tissues (WINZLER et al., 1952;PROCHOROVA, 1954; ALLWEIS and MAGNES, 1958a,b; GEIGER, 1958;BUSCH et al., 1960). ROBERTS, FLEXNER and FLEXNER (1959) reported a relatively high rate of conversion of glucose carbon into protein in the liver and brain in the mouse. They found that the distribution of I4C in the different amino acids of the proteins was different in the two organs : there was a greater incorporation of I4C into alanine than into glutamic acid in the liver, whereas in brain tissue the converse was true.The object of the present work was to study the conversion of glucose-carbon into protein in the rat brain. Orientating experiments were first carried out to find out to what extent carbon from [14C]glucose is incorporated in the free amino acids and proteins of other organs besides the brain. The incorporation was studied for a period of up to 12 hr and the distribution of I4C was examined in the dicarboxylic, neutral and basic amino acid fractions. METHODSMale or female littermates (100-150 g) of a Wistar albino strain of rat were used. D-[UJ~C]-Glucose (410 pclmg) was obtained from the Radiochemical Centre, Amersham, England. The isotopic purity determined by the suppliers by dilution with glucose and conversion to the pentaacetate was 100 per cent 14C: analysis by paper chromatography gave 99 per cent purity both in n-butanolethanol-water and in phenol-water. Each animal received 0.2 ml of a solution of D-[u-' *C]glUCOSe (5 pc) containing 2 mg of added carrier glucose by subcutaneous injection. The animals were decapitated with a guillotine at intervals between 0 3 and 12 hr after injection of radioactive glucose. The brains and other organs were quickly removed, blotted on filter paper, frozen in liquid nitrogen and then kept at -20" until analysed. Blood was taken immediately after decapitation in a 25 ml beaker coated with Na-oxalate and 1 ml was pipetted with a similarly coated pipette into 5 ml of 10 per cent (w/v) trichloroacetic acid (TCA).Protein fractions. Brain, liver, kidney, spleen, heart, blood and skeletal muscle (gastrocnemius) were taken for analysis. The organs were weighed on a torsion balance, defrosted in 5 ml of ice-cold 10% TCA in a MSE-homogenizer tube (Measuring & Scientific Equipment Ltd., London), and then homogenized for 1 min at 0". The sediment obtained by centrifugation for 15 min at 1000 g was resuspended in 5 ml of TCA at 0" and the treatment was repeated with 4 consecutive 5 ml portions of TCA. The combined TCA-extracts were kept for analysis of acid-soluble amino acids.The residue after TCA extraction was treated with organic solvents to remove lipids as described by VRBA, FOLBERGROV~ and KANTBREK (1957). The tissue lipids were extracted consecutively with acetone, ethanol-ethyl ether (3 : I, v/v), chloroform-methanol (2 : 1 , v/v) and ether. The combined lipid extracts were evaporated to dryness ...

No abstract