GLUTATHIONE PEROXIDASE LEVELS IN BRAIN

Glutathione peroxidase activity in brains of various animals was examined. Enzyme activity was low, approximately 10 nmol of glutathione oxidized min^?1 mg protein^?1 or less. This result suggests that brain tissues contain insufficient glutathione peroxidase activity to provide protection from peroxidative damage and that an alternative mechanism may be present.     

SIALOGLYCOPROTEINS AND SEVERAL GLYCOSIDASES IN DEVELOPING RAT BRAIN

Abstract— The amount of sialoglycoproteins expressed as μmol of sialic acid per g of lipid-free residue remained fairly constant in developing rat brain. However, the activity of various enzymes which may be involved in glycoprotein metabolism varied in an inconstant fashion during the period of development. The specific activity of a neuraminidase increased, N -acetyl-β-glucosaminidase remained relatively constant, while the specific activities of α-mannosidase and α-fucosidase decreased.     

PHOSPHOFRUCTOKINASE AND FUMARATE HYDRATASE IN DEVELOPING RAT BRAIN

The developmental patterns of phosphofructokinase, fumarate hydratase and lactate dehydrogenase were determined and compared using homogenates of rat brain. Phosphofructokinase activity, expressed in terms of tissue wet wt., was relatively constant from 5 days before birth to 8 days postnatal; a 110 per cent increase in activity occurred between 12 and 21 days of age, when adult levels were achieved. The degree of inhibition of phosphofructokinase by 1-0 mM-ATP changed little during development; inhibition by 2-5 mM-citrate was about 50 per cent in both newborn and adult brain. Phosphofructokinase development more closely resembled that of lactate dehydrogenase than that of fumarate hydratase.     

HISTAMINE AND MAST CELLS IN DEVELOPING RAT BRAIN

The number and distribution of mast cells in rat brain were determined at different postnatal ages. The number of brain mast cells was found to change during ontogenic development following the same pattern as brain histamine (HA) levels. The calculated HA content of brain mast cells was close to the HA content of the crude nuclear fraction at every age studied. Since most of the brain HA in the newborn sediments with the crude nuclear fraction, these results suggest that the developmental pattern of brain HA reflects changes in the number of brain mast cells, that is, in the size of the mast cell HA pool. The HA content of the supernatant of the crude nuclear fraction corrected for mast cell HA contamination, on the other hand, follows a developmental pattern similar to that of other known neurotransmitters.     

SYNTHESIS OF GLYCOPROTEINS AND GANGLIO-SIDES IN DEVELOPING RAT BRAIN

Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [³H]fucose and [¹⁴C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -³H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[³H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [¹⁴C]fucose and N -[³H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [¹⁴C]fucose relative to that of N- [³H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt.     

ADENOSYLMETHIONINE DECARBOXYLASE IN DEVELOPING RAT BRAIN

Adenosylmethionine decarboxylase from rat brain has been found to be similar to the same enzyme isolated from other rat tissues in regard to kinetic parameters, pH optimum, putrescine requirement, and subcellular location. Evidence is presented that pyridoxal phosphate is not the functional cofactor in enzymatic decarboxylation by the rat brain preparation. The capacity for spermidine synthesis in developing rat brain was determined by measurement of the activity of adenosylmethionine decarboxylase. The activity increased dramatically after 10 days of postnatal age. This increase occurred after the period of maximum nucleic acid synthesis, an observation which suggests that spermidine may have a role in the functional development of the brain.     

OXIDIZED AND REDUCED GLUTATHIONE IN THE RAT BRAIN UNDER NORMOXIC AND HYPOXIC CONDITIONS

In order to test the proposition that hypoxia leads to a change in the concentration ratio of reduced (GSH) and oxidized (GSSG) glutathione in the brain, enzymatic, fluorometric assays were worked out for measuring GSH and GSSG. In lightly anaesthetized and immobilized rats. GSH concentrations in the cerebral cortex and the cerebellum were close to 2 μmol.g^-1 while a slightly lower concentration (approx 1.4μmol.g^-1) was found in the brain stem. In order to avoid artefactual oxidation of GSH during sample preparation for GSSG determination the tissue was extracted with trichloroacetic acid, following alkylation of SH groups with N-ethylmaleimide. With these precautions GSSG concentrations were approx 0.7% of the corresponding GSH concentrations. However. the results indicated that the true GSSG concentrations may be even lower. During hypoxia there was neither a decrease in GSH nor an increase in GSSG concentrations in cortical tissue or cisternal CSF.     

TRIPHOSPHOINOSITIDE PHOSPHODIESTERASE IN DEVELOPING BRAIN OF THE RAT AND IN SUBCELLULAR FRACTIONS OF BRAIN

Abstract— An assay system for the measurement of triphosphoinositide phosphodiesterase in homogenates of rat brain is described. With triphosphoinositide (TPI) as substrate, and in the presence of 0·1 m -KCI and saturating amounts of diethyl ether, the activity of phosphodiesterase in myelinated brain was 400–500 μmoles of TPI hydrolysed per g wet wt. per hr. One quarter of the adult level of the enzyme was present in rat brain one day after birth, with the remainder being added prior to and during the early stages of myelination. On subfractionation of brain homogenates, substantial activity of the enzyme was located in the soluble portion and in the paniculate fractions enriched in myelin and synaptosomes. The enzyme associated with the particulate fractions could not be detached from the membranes by any of several methods employed. There was a rough correlation between distribution of phosphodiesterase and that of 5'-nucleotidase, an enzyme associated with plasma membrane in a number of tissues. Some implications of the results are discussed.     

THYMIDINE METABOLISM AND DEOXYRIBONUCLEIC ACID SYNTHESIS IN THE DEVELOPING RAT BRAIN

—In growing rat brain, the specific activity of DNA at 12 h after the subcutaneous injection of [³H]thymidine underwent a sharp rise during the first 6 days of life, dropping just as precipitously by 15 days, thereafter continuing to decrease with increasing age. When [³H]thymidine was given to 6-day-old rats, a considerable amount was taken up immediately into the brain. Thymidine taken up into the acid-soluble fraction was readily phosphorylated to its nucleotides, thymidine mono-, di-, and triphosphate (TMP, TDP and TTP) within only 30 min following injection. The highest specific activity was found in TTP. The incorporation of of [3H]thymidine into DNA took place over a longer period of time after injection.     

MYELIN SUBFRACTIONS IN DEVELOPING RAT BRAIN: CHARACTERIZATION AND SULPHATIDE METABOLISM

Centrifugation of isolated myelin on discontinuous sucrose gradients resulted in a separation into three bands and a pellet. The three bands were morphologically identical to myelin, whereas the pellet consisted primarily of vesicular membranes. These four fractions differed from one another in their lipid-to-protein ratios and in molar ratios of cholesterol:phospholipid:galactolipid. All of the fractions contained proteins typical of myelin, although the proportions of the proteins varied, with the pellet being the lowest in basic protein and proteolipid protein. High activity of 2′,3′-cyclic nucleotidase and low activity of cerebroside sulphotransferase further distinguished these fractions from the microsomal fraction. Distribution of radioactive sulphatide in the subfractions at 15 min after intracranial injection of radioactive sulphate indicated that newly-labelled sulphatide first appeared in the lipid-poor fractions, followed by the lipid-rich fractions; results of pulse-chase experiments also suggested this relationship. Several days or weeks after the injection of radioactive sulphate, most of the radioactive sulphatide was in the lipid-rich fractions.     

THE ACCUMULATION OF ARACHIDONATE AND DOCOSAHEXAENOATE IN THE DEVELOPING RAT BRAIN

—Fatty acids typical of grey matter lipids (C20:4 and C22:6) and of myelin lipids (C20:1 and C24:1) were estimated in developing rat brains. The polyenoic fatty acids (C20:4 and C22:6) are synthesized from the essential fatty acids (C18:2 and C18:3). The results showed that more than 50 per cent of the adult content of the brain polyenoic acids were laid down by day 15. In contrast, the fatty acids characteristic of myelin lipids did not appear in significant quantities until after this time. These findings distinguish biochemically the different periods of brain development associated firstly with cell division (formation of neurons and glial cells) and secondly with myelination. It is of special interest that the period of cell proliferation is accompanied by the appearance in brain lipids of long-chain polyenoic acids derived from the essential fatty acids.     

SYNTHESIS OF RNA IN DEVELOPING RAT BRAIN IN VITRO

—Incorporation of [8-¹⁴C]adenine into a rapidly-labelled fraction of RNA derived from the nucleus, and into a cytoplasmic RNA of high molecular weight was studied in brain slices from new born rats. The kinetic behaviour of the two fractions of RNA was compatible with a precursor-product relationship between them. The change in the specific activity of adenine and the reduction of radioactivity in prelabelled RNA of brain slices in the presence of actinomycin D, suggest that the observed degradation of nuclear RNA is not due to random changes, but is limited to a relatively small fraction, presumably messenger RNA.     

CHANGES IN POLYSOMES OF THE DEVELOPING RAT BRAIN

Abstract— Rat brain polysomes were prepared from a deoxycholate-treated postmito-chondrial supernatant in the presence of 2% bentonite and 1 mg/ml of yeast RNA to prevent partial degradation during preparation.

• 1 The polysomal preparations had an absorption maximum at 260 mμ and an absorption minimum at 235 mμ. The ratio of absorption maximum to minimum and the RNA to protein ratio were 1·58 and 1·06 respectively in 6-day-old rat brain polysomes. The sedimentation patterns showed six distinct peaks with sedimentation coefficients of 235S, 185S, 173S, 135S, 100S and 80S, indicating that these preparations have the characteristics of pure heavy polysomes.

• 2 The rate of [¹⁴C]phenylalanine incorporation into brain polysomal protein was maximal at approximately 10 days of age and decreased thereafter. A similar progressive reduction with increasing age was found in the stimulation of phenylalanine incorporation by the addition of 60 μg/tube of polyuridylic acid. However, the incorporation of phenylalanine into young rat brain polysomes was usually greater even with the addition of polyuridylic acid than in the older animals.

• 3 The comparative studies on sucrose density gradient centrifugation of polysomes between young and adult rat brains showed a considerable decrease of heavy polysomes in the older animals.

• 4 The effect of various factors on the stability of brain polysomes from both ages has been studied. The rates of RNA, protein and acid-soluble phosphorus release from polysomes of the adult rat brains were usually greater in the presence of high salt concentration, ethylenediaminetetra-acetic acid and urea than those from the corresponding preparations of younger animals. On the basis of evidence obtained from the above results it suggested that the adult brain polysomes were more unstable than those of younger animals.

• 5 The amount of polysomal RNA linearly increased up to the first 20 days after birth and then levelled off. The ratio of G + C/A + U of polysomal RNA was less in the young rat brains, falling to 1·30 as compared to 1·50 in older animals. The differences were statistically significant at less than a 1% level of confidence.

• 6 Polysomal preparations also contained RNase, phosphomonoesterase, phospho-diesterase and 5′-nucleotidase activities which cannot be washed off. The specific activities of these enzymes were generally higher in young rat brains than those in the adult.

     

NEURONAL AND NEUROPIL FRACTIONS FROM DEVELOPING RAT BRAIN

Abstract— A method is described for the preparation of enriched fractions containing isolated neuronal and glial cells from brains derived from 1 to 20-day-old rats. The method is based on mechanical disaggregation in a medium containing Ficoll-PVP followed by centrifugation on a single-stage two-step gradient at 13,000 g for 30min. The neuronal and neuropil (glial) fractions are approx 70–80% pure in cellular terms.
The cells showed well-preserved cytoplasmic and nuclear morphology at the light and electron microscope level and between 70 and 80% excluded trypan blue. Despite changes in the total cell population with age due to glial proliferation, the proportionate recovery of cells in the separated fractions was fairly constant: based on DNA determination, 23 and 29% of all neurons and 15 and 17% of glia were recovered in the purified fractions from Day 1 and Day 20 animals respectively.
Changes in neuronal cell size with age were reflected in a 2.5-fold increase in protein recovered in the neuronal fraction per mg DNA. Protein and RNA levels/mg DNA in the neuropil fraction reached a maximum at Day 10. It is concluded that the method produces a defined and reliable purification of cells in the separated fractions throughout the studied age range and therefore provides a sound basis for studies on the distribution of biochemical systems between cell types during post-natal development.     

OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA OF DEVELOPING RAT BRAIN

Abstract—

• 1 Oxygen uptake, ADP/O ratios and respiratory control ratios (RCR) were studied by oxygen electrode techniques in mitochondria prepared from developing rat brain.
• 2 Oxygen consumption, ADP/O ratios and RCR based on mitochondrial protein concentration increased with maturation. Of the substrates employed, succinate supported oxygen uptake best and malate poorest.
• 3 The addition of exogenous NAD to the mitochondrial preparation had no effect on rate of oxygen uptake.
• 4 Lack of change in ADP/O ratio in the presence of glucose, a tricarboxylic acid cycle intermediate (α-oxoglutarate), and ATP leads us to believe that there is no significant hexokinase activity in this preparation.

     

THE SYNTHESIS OF MYELIN IN DEVELOPING RAT BRAIN

Abstract— —The synthesis of myelin proteins has been studied in the grey and white matter slices of developing rat brain by measuring the incorporation of [³H]lysine and [¹⁴C]arginine into polypeptide. The incorporation was sensitive to cycloheximide and puromycin at 1 mM concentration. Developing rat optic nerve slices, free of retinal ganglion cells, were able to synthesize myelin basic and proteolipid proteins, but rat retinal preparation failed to synthesize myelin basic protein. Rabbit retinae were able to synthesize myelin basic and proteolipid proteins. Significant activity of the myelin marker enzyme 2',3'-cyclic nucleotide-2'-phosphodiesterase has been found in the rabbit retina but not in rat retina. The results presented in this communication suggest that myelin proteins in the rat CNS are synthesized by the oligodendroglial cells and that neurons probably do not participate.     

日本沼虾含硒谷胱甘肽过氧化物酶全长克隆及表达分析

为探讨日本沼虾(Macrobrachium nipponense)的免疫机制及含硒谷胱甘肽过氧化物酶(Se-GPx)在甲壳动物解毒和免疫应激反应中的作用,本文采用RACE法克隆了日本沼虾Se-GPx cDNA全长,其cDNA全长为908 bp,5′-UTR的长度为91 bp,3′-UTR长为256 bp,包括1个保守的硒代半胱氨酸插入序列(SECIS)和1个polyA尾,开放阅读框长度为561 bp,编码由186个氨基酸组成的多肽,其中,第39个氨基酸为TGA编码的硒代半胱氨酸。同源性和相似性分析显示日本沼虾的Se-GPx在甲壳动物中与罗氏沼虾相似度最高,在脊椎动物中与GPx1和GPx2家族的相似度比较高。日本沼虾Se-GPx基因在血细胞、肝胰腺、肌肉、卵巢、表皮以及大颚器官中都有表达,尤其在血细胞中表达量相对较高。用嗜水气单胞菌(Aeromonas hydrophila)刺激后3h和6h,血细胞Se-GPx基因表达量显著升高。结果表明,日本沼虾Se-GPx作为一种重要的抗氧化酶,在维护组织正常功能方面及免疫应激反应中发挥重要作用。     

TAURINE IN DEVELOPING RAT BRAIN: CHANGES IN BLOOD-BRAIN BARRIER

Abstract— The fate of [³⁵S]taurine injected intraperitoneally or intracranially has been compared in rats throughout neonatal development. The amount of [³⁵S]taurine present in the whole rat pup 24 h after intraperitoneal injection increases during development to a maximum 15 days after birth, and declines thereafter, whereas the amount of [³⁵S]taurine reaching the brain 24 h after intraperitoneal injection was greatest in the first 5 days after birth. The amount of [³⁵S]taurine remaining in the brain 24 h after intracranial injection does not vary throughout the period of neonatal development. These results suggest that the 'blood-brain' barrier is more accessible to taurine in the first few days after birth than later in neonatal development, and that factors other than simple exchange are involved.     

CHANGES OF THYMIDINE KINASE IN THE DEVELOPING RAT BRAIN

Abstract— Thymidine kinase (ATP: thymidine-5'-phosphotransferase EC 2.7.1.21) of the supernatant fraction from 6-day-old rat brain possessed a pH optimum of 8.0 and required the presence of 5mM-ATP and 2.5 mM-MgCl₂ for maximum activity. The activity was completely inhibited by addition of 1.8 mM-TTP. The enzyme activity was lost if the same supernatant fraction was refrozen and thawed. K_m was 2.8 × 10⁻⁶ M for [6-³H]thymidine.
Following subcellular fractionation of rat brain, the greatest proportion and highest specific activity of thymidine kinase was found in the supernatant fraction. Thymidine kinase activities reached a maximum at 6 days of age and then dropped sharply during maturation. Comparative studies of thymidine kinase activities of cerebrum, cerebellum and the remainder of the brain during growth indicated that the activity in the cerebellum was usually higher than those in the cerebrum and the remainder, and the biggest differences obtained at 6 days after birth corresponded with the peak in cerebellar activity.