A PROCEDURE FOR THE MEASUREMENT OF HEMICHOLINIUM-3 IN BRAIN TISSUE

Abstract— The natural fluorescence of hemicholinium-3 (HC-3) was used to provide a method for determining its concentration in brain tissue following intraventricular administration of the compound to rats and rabbits. When hemicholinium-3 was perfused through the cerebral ventricles of the rabbit, the highest concentration was found in the pons-medulla oblongata. The effect of a number of compounds on the uptake of hemicholinium-3 in the brain was studied in rats. Choline and tetramethylammonium significantly reduced the uptake of HC-3 from the cerebral ventricles but tetraethylammonium, physostigmine and atropine had no effect. The results are discussed in relation to the known effects of hemicholinium-3 in the central nervous system.     

LARGE SCALE PREPARATION OF A CRUDE MEMBRANE FRACTION FROM OX BRAIN

Abstract— Two procedures are described for preparing from ox brain grey matter a subcellular fraction enriched in neuronal membranes. In the first, an angle rotor was used to fractionate the crude mitochondrial and nuclear fraction and yielded 1·3–1·4 g of membrane protein from 200 g of tissue. The second procedure employed essentially the same principle except that a zonal rotor was used to fractionate in the final stage. The two methods gave comparable results for yields of protein, Na-K-Mg AT Pase and succinate dehydrogenase.     

THE SUBCELLULAR DISTRIBUTION OF CARBONIC ANHYDRASE IN HOMOGENATES OF PERFUSED RAT BRAIN

Abstract— The distribution of carbonic anhydrase was examined in subcellular fractions of perfused rat brain and compared with those of markers for cytosol (lactic dehydrogenase), mitochondrial matrix (glutamic dehydrogenase), and mitochondrial membranes (succinic dehydrogenase). About half of the total carbonic anhydrase was found in particulate fractions, with the greatest part of this in the crude mitochondrial fraction. This fraction was separated into its components on a discontinuous sucrose gradient either as such or after isotonic mechanical disruption with a French pressure cell, and the resultant fractions were characterized by electron microscopy and by assay of marker enzymes.
Carbonic anhydrase was solubilized by mechanical disruption, but not to the same extent as lactic dehydrogenase. The highest specific activity for carbonic anhydrase was found in the myelin fraction of the gradient. A mitochondrial locus for carbonic anhydrase is unlikely, but the presence of the enzyme in synaptosomes remains in question.
Addition of soluble carbonic anhydrase did not significantly increase the activity of particulate fractions. Treatment of particulate fractions with detergent was necessary to reveal latent activity; this procedure resulted in a more than ten-fold increase in the measurable carbonic anhydrase activity of myelin fragments.     

TECHNICAL NOTE: THE PREPARATION OF MOLLUSCAN TISSUE FOR PRODUCTION ESTIMATES

Oven-drying at 60°C under vacuum, and presumably also dryingat higher temperatures, leads to relatively high losses of volatile,highly-calorific components, with a resultant underestimateof dry weight, lipid content and calorific content. Freeze-dryingis to be generally recommended. Fresh material should be usedwherever possible for the estimation of tissue parameters. Freezingor formalin-fixation may be used for estimations of wet weights,but only freezing if dry weights or calorific contents are alsorequired. (Received 26 November 1981;     

A SENSITIVE FLUOROMETRIC METHOD FOR THE ESTIMATION OF 3,4-DIHYDROXYPHENYLETHYLENE GLYCOL SULPHATE IN BRAIN TISSUE

Abstract— A simple fluorometric method for the estimation of endogenous 3,4-dihydroxyphenylethylene glycol sulphate (DOPEG-SO₄) in brain tissue is described. DOPEG-SO₄ was extracted from brain tissue with 0.4 n -perchloric acid and purified by column chromatography on QAE-Sephadex A-25, Cl⁻ form. The isolated DOPEG-SO₄ was hydrolysed by incubation with a purified preparation of sulphatase from Helix pomatia. The free DOPEG thus formed was further purified on a column of aluminium oxide and determined fluorometrically after conversion to a fluorescent compound by condensation with ethylenediamine.
The sensitivity of this method was about 15 ng per perchloric acid extract applied on the column (3 ml) and the reproducibility was excellent, with a variation coefficient of 1.6% (n = 5). The concentration of DOPEG-SO₄ in whole brains of rats, determined by the present method, was 79.3 ± 4.7 ng/g (mean ± S.D., n = 10) in terms of free DOPEG. No appreciable DOPEG-SO, was detected in the brains of mice and guinea-pigs.     

PYRUVATE METABOLISM BY HOMOGENATES OF HUMAN BRAIN: EFFECTS OF PHENYLPYRUVATE AND IMPLICATIONS FOR THE ETIOLOGY OF THE MENTAL RETARDATION IN PHENYLKETONURIA

Abstract— The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by homogenates of human brain was investigated. In the presence of 5 mM pyruvate as substrate homogenates of human cerebral cortex fixed about 1 μmol of H¹⁴CO₃^-_- per g of tissue in 30 min. Phenylpyruvate at a concentration of 5 raw inhibited the fixation of H¹⁴ CO₃^-_- by homogenates of human brain by approximately 50 per cent, whereas 5 mM phenylalanine had no effect. The inhibition of pyruvate carboxylation by phenylpyruvate was dependent upon the concentration of the inhibitor. The activity of pyruvate carboxylase (EC 6.4.1.1) in human cerebral cortex was 02–0.4 units, with a K_m for pyruvate of about 0.2 mM. Homogenates of human cerebral cortex decarboxylated [1-¹⁴C]pyruvate to ¹⁴CO₂ at a rate of about 5 μmol per g of tissue per 15 min, with a 20–50 per cent reduction in the presence of 5 mM phenylpyruvate; phenylalanine at the same concentration had no effect. The possible toxic effect of phenylpyruvate on the metabolism of pyruvate in the brains of untreated phenylketonuric patients is discussed.     

TRANSAMINATION OF AMINO ACIDS IN HOMOGENATES OF RAT BRAIN

Abstract— The aminotransferase activity of homogenates of brains from adult and neonatal rats has been investigated. Aminotransferase activity was demonstrated wtih 15 of 22 amino acids incubated with seven keto acids. The basic amino acids exhibited little or no activity.

• 1 The greatest activity was obtained when glutamate or aspartate was incubated with α-ketoglutarate or oxaloacetate. Significant activity was also observed when the neutral aliphatic and aromatic amino acids were incubated with these two keto acids.
• 2 Activity with pyruvate was obtained principally upon incubation with glutamate and alanine. Most of the other amino acids that underwent transamination with α-ketoglutarate also did so with pyruvate, although at a lower rate.
• 3 When phenylpyruvate was added to the medium, glutamate, phenylalanine and tyrosine transaminated most actively.
• 4 Incubations with 11 amino acids and glyoxylic acid demonstrated aminotransferase activity, with glutamate and ornithine being the most active substrates.
• 5 α-Ketoisocaproate and α-ketoisovalerate accepted amino groups primarily from the branched-chain amino acids. Except for glutamate, activity with other amino acids was low or not detectable.
• 6 A comparison of aminotransferase activity in the newborn brain with that in the adult brain showed that the greatest change in activity occurred for glutamate with pyruvate or for alanine with α-ketoglutarate, these activities increasing about 10-fold from birth to adulthood; during this time activities with most other amino acids increased two- to threefold. Amino transfers from the branched-chain amino acids showed no increase with maturation, and some reactions, such as that with methionine and a number of keto acids, decreased from birth to adulthood.
• 7 Our results correspond in general to previous studies of aminotransferase activity in brain and in liver. However, our study also indicates a possible second aminotransferase acting on the branched-chain amino acids, the presence of aminotransferase activity for methionine and asparagine, and relatively high aminotransferase activity for glutamine or ornithine when incubated with glyoxylic acid rather than other keto acids. Moreover, phenylpyruvate and glyoxylate are active in amino transfers and may serve as substrates for a number of aminotransferases.

     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]-labeIled material into subcellular fractions of 15-day-old rat brain was studied as a function of time after intracerebral injection of [2-¹⁴C]mevalonic acid. As previously shown for adult brain, the data indicated the microsomal fraction to be the site of sterol biosynthesis. The synaptosomal fraction exhibited a marked early uptake of [¹⁴C]-nonsaponifiable material. Total radioactivity in both myelin and myelin-like fractions remained low in comparison to that in the other subcellular fractions at all time periods examined. At 2 h after injection, labelled digitonin-precipitable material was demonstrable in all subcellular fractions. Examination of the [¹⁴C]-labelled nonsaponifiable material by thin-layer chromatography indicated the rapid appearance of labelled 4-desmethyl sterol in all subcellular fractions, with the most rapid appearance in the myelin fraction, followed in decreasing order by microsomal, synaptosomal, and mitochondrial fractions. Examination of [¹⁴C] digitonin-precipitable material from each fraction by the dibromide method demonstrated that although 4-desmethyl sterol appeared quickly, the formation of cholesterol was slow in all fractions, an effect that had been reported earlier for adult brain.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of ¹⁴C into several subcellular fractions of adult rat brain was studied as a function of time, following intracerebral injection of [2-¹⁴C]mevalonic acid. As expected from previous studies, the microsomal fraction was indicated as the site of sterol biosynthesis. The myelin fraction showed a marked and early uptake of ^I4C-labelled, digitonin-precipitable material. This was assumed to be a non-enzymic uptake of sterol intermediates. The mitochondrial fraction exhibited a rapid uptake of ¹⁴C-labelled, nonsaponifiable material, but a very slow accumulation of ¹⁴C-labelled, digitonin-precipitable product. Examination of the nonsaponifiable ¹⁴C-fractions by TLC showed a rapid appearance of labelled 4-desmethyl sterols in the microsomal fraction. The myelin fraction selectively retained 4,4'-dimethyl sterol but seemed to release this with time, possibly to be further metabolized by the microsomes. Examination of [¹⁴C]digitonin-precipitable material by the dibromide method showed that although labelled 4-desmethyl sterol appeared quite early, cholesterol itself was formed slowly in all fractions.     

ALGAL CELL DISRUPTION USING A PYREX TISSUE GRINDER AND THE B. BRAUN HOMOGENIZER1

A motor-driven Pyrex tissue grinder (PTG) and the B. Braun cell homogenizer (BH) were used to disrupt cells of Selenastrum capricornutum Printz for chlorophyll extraction. Cell disruption efficiency depended upon the number of cells filtered. Within the range of 2 × 10⁶ to 8–9 × 10⁶ algal cells per 2.4 cm filter, the PTG allowed more complete extractions. Beyond that number of cells, the BH was more efficient. The algal mass corresponding to 8–9 × 10⁶ cells was 0.14–0.17 mg dry weight.     

小鼠脑中32种元素含量正常参考值的测定

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TISSUE CULTURE IN THE PRODUCTION OF NOVEL DISEASE-RESISTANT CROP PLANTS

1. Plant tissue cultures form the basis of a number of techniques which have been developed to effect genetic changes in plants. Progress is being made in the application of these techniques in breeding new, disease-resistant cultivars. 2. It is possible to induce and select for mutants among populations of cultured plant cells. Novel disease-resistant plants of a small number of species have been regenerated from cells selected in culture for their resistance to toxins produced by pathogens, both with and without prior exposure to mutagens. It is not known whether such procedures are widely applicable, and the nature of the genetic changes involved has not yet been determined. 3. The tissues of plant species which are propagated vegetatively are normally genetic mosaics with regard to many characteristics, including resistance to disease. Thus, some of the plants regenerated from cultured cells of such species are more resistant to pathogens than the parent plants. Novel plants produced in this way are already being used in some breeding programmes. 4. Many attempts have been made to modify the genomes of cultured plant cells by means of exogenous nucleic acids. The evidence for integration and replication of this genetic material is equivocal. The technique, therefore, offers no immediate prospects for the development of novel disease-resistant plants, but may be important in the long term as methods are perfected for using plasmids and other agents as carriers of useful genes. 5. Steady advances are being made in producing somatic hybrids of crop plants by fusion of isolated protoplasts. In the long term it may be possible to use protoplast fusion to transfer desirable disease-resistance traits between related species which cannot be hybridized by conventional breeding methods. 6. The culture of excised embryos may be used to grow interspecific and inter-generic hybrid plants in cases where incompatibility occurs after normal fertilization. The technique is already being used by breeders in the production of disease-resistant hybrids of crop species. 7. It is concluded that tissue culture has a limited but useful role to play in the development of novel disease-resistant crop plants.