FRACTIONATION OF RNA FROM BRAIN SYNAPTOSOMES AND CYTOPLASMIC SUBCELLULAR FRACTIONS

—RNA from rat brain synaptosomes, mitochondria and microsomes was analysed by gel electrophoresis under conditions allowing good resolution in three different molecular weight ranges: 4s-16s, 16s-28s and >28s. Two synaptosome specific RNA bands were found, one with comparatively low molecular weight (8-9 × 10⁴ Daltons) and another very large (s_E > 60s). RNA species with electrophoretic characteristics similar to those reported for liver mitochondrial RNA were found in brain mitochondria. From the electrophoretic data their mean geometric radii were determined.     

COMPARATIVE STUDIES OF HIGHLY BASIC PROTEINS OF OX BRAIN AND RAT BRAIN. MICROHETEROGENEITY OF BASIC ENCEPHALITOGENIC (MYELIN) PROTEIN

(1) The total amount of highly basic proteins in acid extracts of whole ox brain, ox white matter and ox grey matter was determined quantitatively after electrophoresis on 5% polyacrylamide gels at pH 10-6 in the presence of 8 M-urea. (2) Ox white matter gave 13 mg and ox grey matter 2 mg of highly basic proteins per g fresh tissue on treatment with 0-03 n -HCl. The yield of total basic proteins of ox white matter increased to 17-6 mg/g fresh brain on stepwise extraction at pH 3-0, 2-0 and 1-0; the extract at pH 3.0 accounted for 90 per cent of the total basic proteins. (3) The high encephalitogenic activity of the fraction of highly basic proteins extracted at pH 3.0 from ox white matter indicated that these basic proteins were derived from myelin. It is suggested that the amount of basic proteins in a sample of brain extracted under these conditions is proportional to the amount of white matter in the sample. (4) The encephalitogenic (myelin) basic protein fraction was homogeneous with respect to molecular size but could be resolved into at least six components by electrophoresis at high pH. (5) The myelin basic proteins extracted from ox white matter had lower electrophoretic mobilities at high pH than did those of two basic proteins of rat brain apparently derived from myelin.     

AROMATIC AMINOTRANSFERASE ACTIVITY OF RAT BRAIN CYTOPLASMIC AND MITOCHONDRIAL ASPARTATE AMINOTRANSFERASES

Abstract— Mitochondrial and cytoplasmic forms of aspartate aminotransferase were purified from rat brain homogenates and tested for their ability to catalyze transamination of various aromatic amino acids. The mitochondrial enzyme exhibited activity toward tyrosine and phenylalanine with 2-oxoglutar-ate as acceptor, although the specific activities were less than 1% of the corresponding aspartate activity when all substrates were 10 mM. Even less activity was seen with DOPA, 5-hydroxytryptophan and tryptophan. The cytoplasmic aspartate aminotransferase was active toward tryptophan, 5-hydroxytryptophan and DOPA, but these transaminations were favored by pyruvate or oxaloacetate rather than 2-oxoglutarate as keto acid. Based on co-migration of aromatic activities with the respective aspartate aminotransferases during isoelectric focusing and based on equal sensitivities of aromatic transamination and aspartate transamination to inhibition by vinylglycine, it was concluded that all activities resided in the aspartate aminotransferase enzymes. Some doubt exists, however, as to the physiological significance of these alternate activities in view of the requirement that aromatic amino acids must compete with aspartate for transamination by these enzymes.     

MYELIN BASIC PROTEIN MICROHETEROGENEITY IN SUBFRACTIONS OF RAT BRAIN MYELIN

Abstract— Myelin subfractions were prepared from adult rat brain by discontinuous sucrose gradient ultracentrifugation. Gel electrophoretic studies at pH 10.6 in the presence of urea revealed differences in basic protein microheterogeneity among subfractions. With increasing myelin density there was a decrease in the most positively charged components of both large BP and small BP. Since these components are the least modified by deamidation and phosphorylation, it seems likely that the heavier myelin subfractions are enriched in the more modified components of the microheterogeneous population of BP. These observed differences may be related to the regulatory processes controlling biosynthesis, organization, and catabolism of BP in CNS myelin.     

CITRATE OXIDATION IN THE CYTOPLASMIC FRACTION OF RAT BRAIN

Abstract— The cytoplasmic fraction derived from rat brain was shown to possess the ability to oxidize citrate in the presence of NADP, but not in that of NAD. The rate of citrate oxidation is limited by the rate of the aconitase-catalysed isomerization. The dependence of the reaction rate on the protein, citrate and NADP concentrations, and on reaction time, was determined. It was found that 2-oxoglutarate and NADPH, both formed in the citrate oxidation reaction, do not appear in amounts equivalent to the losses of citrate. The possible ways of utilization of the above products in the rat brain cytoplasmic fraction are discussed.
It is suggested that the oxidation of citrate in rat brain cytoplasm may proceed–among other metabolic routes–through its conversion into isocitrate (aconitase) and then 2-oxoglutarate (NADP dehydrogenase) and oxaloacetate (aspartate aminotransferase). In addition it has been shown that hypoxia may markedly effect the activity of the cytoplasmic citrateoxidizing system.     

REGIONAL AND SEXUAL DISTRIBUTION PATTERNS OF CHROMATIN PROTEINS AND OF SOLUBLE CYTOPLASMIC PROTEINS IN THE RAT BRAIN1

Abstract— Electrophoretic patterns of forebrain and hindbrain proteins were compared in normal adult rats of both sexes, as well as in adult female rats following neonatal androgenization. There were significant differences between the forebrains and hindbrains in the relative densities of several chromatin proteins and soluble cytoplasmic protein bands. Minor qualitative differences between forebrains and hindbrains were observed in patterns of chromatin proteins. No qualitative sex difference was detectable in the patterns of chromatin-derived proteins or in soluble cytoplasmic proteins. The relative densities of the brain protein bands were very similar for both sexes, indicating quantitative equivalence as well. The brain protein electrophoretic patterns of adult females following neonatal androgenization were identical qualitatively to those of normal females in respect to both chromatin and cytoplasmic proteins.     

萌发花粉细胞质纤丝网架与细胞质颗粒运动

萌发的丝瓜花粉中存在一个动态细胞质纤丝网架,它具有一定的刚性和柔性,是细胞质一流动的基础,制约着细胞质颗粒的运动。电镜照片显示出,网架纤丝主要由许多直径为5-7nm 的微丝相互平行排列而成,并且在两条微丝的相接处具有定向排列的箭头状物。我们认为在微丝上的某些部位可能存在肌球蛋白分子,它们在细胞质纤丝网架变化及细胞质颗粒的运动中起着动力和调节的作用。     

MICROHETEROGENEITY AND PHOSPHOAMINO ACIDS IN THE CARBOXY-TERMINAL HALF OF MYELIN BASIC PROTEIN

—Three chromatographically distinct peptic peptides (F80-1, F80-2 and F80-3) derived from the C-terminal half of the bovine and guinea-pig myelin basic proteins were characterized. The three peptides of each animal species had the same N-terminal residue (phenylalanine) and essentially the same amino acid composition, but they differed in electrophoretic mobility at alkaline pH. The least basic peptide (F80-3) differed from the others in showing a deficit of C-terminal arginine residues and in containing phosphorus, 0·37 and 0·46 g-atom/mol of bovine and guinea-pig peptides, respectively. Other peptic peptides. derived from the N-terminal half of the basic protein, were essentially phosphorus-frcc. Analyses of partial acid hydrolyzates of peptide F80-3 by high voltage electrophoresis showed the presence of both phosphoserine and phosphothreonine. After incubation with E. coli alkaline phosphatase (EC 3.1.3.1). 34 and 40% of the bovine and guinea-pig F80-3 peptides. respectively, were converted to peptide F80-1. This reaction involved the loss of 2 net negative charges, and its extent corresponded to loss of essentially all of the phosphate originally present in the peptide. This result indicated that the phosphorylated species of peptide F80-3 contained one phosphate group per molecule.     

CYTOPLASMIC EFFECTS ON THE DIFFERENTIATION OF ANTHERS AND OVULES OF COTTON

Reciprocal crosses of Upland cotton with a hybrid derived from (Gossypium anomalum × G. thurberi) × G. hirsutum produced progenies differing significantly in anther development and in the production of supernumerary ovules outside the ovary. Plants with G. anomalum cytoplasm produced fewer anthers than the reciprocal hybrids with G. hirsutum cytoplasm, had a higher percentage of sterile anthers, and were far more likely to form external ovules on an abnormally thickened tip of the staminal tube. The number of locules and ovules within the ovary was not significantly affected by cytoplasm.     

KINETICS OF NUCLEOSIDE INCORPORATION INTO NUCLEAR AND CYTOPLASMIC RNA

The difficulties in sectioning frozen biological objects for electron microscopic investigations are overcome by Steere's freezing-etching method. In order to test this method and to open up a wide field of application, the new freezing-ultramicrotome has been designed. The apparatus consists of the combination of an ultramicrotome with freezing-drying and shadow-casting installations in the same vacuum container. The preliminary results show, on the one hand, the practicability of all preparational steps and, on the other, that it is possible to resolve internal structures of cell organelles and even macromolecular patterns.     

OCCURRENCE AND LOCALIZATION OF BRAIN PHENOLSULPHOTRANSFERASE

—Rat brain contains the enzyme which forms sulphate conjugates of phenols, phenolsulphotransferase (EC 2.8.2.1), but the physiological role of the enzyme is unclear. The enzyme is unevenly distributed in rat brain, with the activity 13 times higher in the hypothalamus than in the cerebellum. Phenolsulphotransferase does not seem to be primarily located in glial cells. Cultured cells (type C6 astrocytoma) derived from rat glia had less than 1 per cent of the phenolsulphotransferase activity of whole rat brain. Sulphate conjugation of neutral compounds may be important in their removal from brain. The pineal and pituitary glands, areas outside the blood-brain barrier had very low phenolsulphotransferase activity. The activity of the enzyme in brain varied widely among different species: rabbit and rat had much higher levels of activity than mouse or frog; the activity in human brain was intermediate. Phenolsulphotransferase also occurred in other organs, including liver, heart, testes, lung, spleen, salivary glands, and intact or decentralized superior cervical ganglion. There was no correlation of enzyme activity with adrenergic or cholinergic innervation, or with the known roles of various tissues in drug metabolism or detoxification. The enzyme activity does not seem to be under neuronal control since ganglionectomy did not affect the phenolsulphotransferase activity of salivary glands. The precise localization of phenolsulphotransferase remains to be established, as well as the physiological importance of sulphate conjugation of phenols in brain and other organs.     

ARGINASES OF MOUSE BRAIN AND LIVER

The arginase present in mouse brain and liver was studied in order to determine if the activity in both tissues was due to the same enzyme. Mouse liver contains only one arginase enzyme whereas mouse brain contains two. One of the arginases in the brain is distinct from the liver enzyme as determined by fractionation on DEAE-cellulose, CM-cellulose and disc gel electrophoresis. The second enzyme from brain tissue has the same properties as the liver enzyme when subjected to these same fractionation techniques. However this arginase can be distinguished from the liver enzyme by its K_m for arginine heat lability and MnCl₂ activation curve. Thus both arginases in brain differ from the liver enzyme. No interconversion of the brain enzymes was detected, and the molecular weight of all the arginases appears to be the same. The observation of multiple distinct brain and liver arginases in mouse brain and liver was confirmed with bovine tissues.