Allosteric activation of brain hexokinase by magnesium ions and by magnesium-ion–adenosine triphosphate complex

1. The highest blood concentrations of ketone bodies were found at 5 days of age, after which time the concentration fell to reach the adult value by 30 days of age. 2. Both mitochondrial and cytoplasmic hydroxymethylglutaryl-CoA synthase activities were detected, with highest activities being found in the mitochondria at all stages of development. Activity of the mitochondrial enzyme increases rapidly immediately after birth, showing a maximum at 15 days of age, thereafter falling to adult values. The cytoplasmic enzyme, on the other hand, increased steadily in activity after birth to reach a maximum at 40 days of age, after which time activity fell to adult values. 3. Both mitochondrial and cytoplasmic aceto-acetyl-CoA thiolase activities were detected, with the mitochondrial enzyme having considerably higher activities at all stages of development. The developmental patterns for both enzymes were very similar to those for the corresponding hydroxymethylglutaryl-CoA synthases. 4. The activity of heart acetoacetyl-CoA transferase remains constant from late foetal life until the end of the suckling period, after which time there is a gradual threefold increase in activity to reach the adult values. The activity of brain 3-oxo acid CoA-transferase increases steadily after birth, reaching a maximum at 30 days of age, thereafter decreasing to adult values, which are similar to foetal activities. Although at all stages of development the specific activity of the heart enzyme is higher than that of brain, the total enzymic capacity of the brain is higher than that of the heart during the suckling period.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]labelled material into subcellular fractions of 30-day-old rat brain was studied as a function of time, following intracerebral injection of [2-¹⁴C] mevalonic acid. As in the adult and 15-day-old brain, the microsomal fraction was indicated as the site of sterol synthesis. Unlike the 15-day-old animal, the myelin fraction from the 30-day-old rat was the predominately labelled fraction at 2 weeks after injection of the animal. Significant amounts of [¹⁴C]cholesterol were not present until about 4 h after injection. In order to ascertain whether different populations of cholesterol were being labelled, depending on the age of the animal injected, we compared the labelling of myelin and non-myelin components in animals injected at 15 or at 30 days of age, and sacrificed, respectively, from 14 to 29 days or from 1 to 28 days after injection. Our results indicated that there was an apparent shift of labelled sterol from non-myelin to myelin fractions at about 37–44 days of age.     

ACETYLCHOLINE,CHOLINE AND CHOLINE ACETYLTRANSFERASE ACTIVITY IN THE DEVELOPING BRAIN OF NORMAL AND HYPOTHYROID RATS1

Abstract— Acetylcholine, choline and choline acetyltransferase activity were measured in the whole brains of normal and hypothyroid rats during development. At 1 day postpartum, brain acetylcholine was 73 per cent of adult levels. Propylthiouracil-induced hypothyroidism up to age 20 days did not alter brain acetylcholine concentrations, but at 30 days resulted in significantly decreased levels. At day 1, brain choline was 20 per cent higher than adult levels and decreased between days 8 and 10. In hypothyroid rats this phenomenon did not occur until days 15–20. At day 1 postnatally, choline acetyltransferase activity was only 7 per cent of adult levels, then between days 5 and 20 rose to 77 per cent of adult levels. Beginning at day 8, hypothyroidism resulted in significantly decreased enzyme levels. This effect could be reversed at day 17 by concurrent tri-iodothyronine substitution therapy. In hypothyroid rats, maximum brain choline acetyltransferase activity was 30 per cent less than normal adult levels.     

NUCLEAR RIBONUCLEASE ACTIVITIES OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— The activities of alkaline and acidic RNAses were determined in soluble and insoluble fractions from nuclei of brain hemispheres of rats, aged from 1 day to adult. The activities increased rapidly and reached a maximum, at 30 days, of about 10 times (alkaline RNAsel or 5 times (acidic RNAse) that seen at day 1.     

MICROTUBULE PROTEIN DURING CILIOGENESIS IN THE MOUSE OVIDUCT

A colchicine-binding assay and quantitative sodium dodecyl sulfate gel electrophoresis have been used to determine the changes which occur in microtubule protein (tubulin) concentrations in the particulate and soluble fractions of mouse oviduct homogenates during that period of development when centriole formation and cilium formation are at a maximum. When mouse oviducts, at various ages after birth, are homogenized in Tris-sucrose buffer, tubulin concentration is partitioned between the soluble (70%) and particulate (30%) fractions. During the period of most active organelle formation (3–12 days), there is a marked increase in colchicine-binding specific activity, in both the soluble and particulate fractions. Microtubule protein concentration increases from 16 to 24% in the soluble fraction, declining to 14% in the adult. In the particulate fractions, microtubule protein concentration increases from 16 to 27%, leveling off at 16% in the adult. We have concluded from these observations and from electron microscopy that colchicine-binding activity in the particulate fractions is related to the presence of centriole precursors in the pellets of homogenized oviducts from newborn mice. These data further suggest that centriole precursor structures are conveniently packaged aggregates of microtubule protein actively synthesized between 3 and 5 days, and maintained at a maximum during the most active period of organelle assembly.     

CHANGES DURING DEVELOPMENT OF MOUSE BRAIN IN THE ACTIVITIES AND SUBCELLULAR DISTRIBUTIONS OF CREATINE AND ADENYLATE KINASES

Abstract— The activities and electrophoretic patterns of creatine (CK) and adenylate (AK) phosphokinases were determined in the mitochondrial and high speed supernatant fractions of developing and adult mouse brain. The CK activity of the cytosol increases three-fold between 9 and 19 days of age and four-fold by 29 days. Mitochondrial CK activity increases from birth to reach a maximum four-fold that at birth by 19 days of age. The developmental changes in soluble AK activity parallels that of CK. Although the increase in CK activity of the mitochondria exceeds that of the mitochondrial proteins during the period of rapid brain development, mitochondrial AK specific activity changes little. No major changes were found during development in the electrophoretic patterns of proteins with CK or AK activity in the supernatant and mitochondrial fractions. The changes in mitochondrial CK and AK activity parallel the change from a predominantly glycolytic to an aerobic mode of brain metabolism and support the concept that these enzymes play an important role in mitochondrial energy metabolism. Soluble CK and AK activity increase concomitant with neuronal maturation and the associated greatly increased need for rapidly available high energy phosphate.     

钙调蛋白及其调节酶PDE在鸡脑胚胎发育过程中的变化

钙调蛋白(CaM)参与脑中多种细胞过程的调节,推测它与大脑的分化发育有关。本实验研究了鸡脑从胚胎早期(原基)至大脑成熟各发育阶段可溶部分的CaM及其调节酶——环核苷酸磷酸二酯酶(PDE)的含量或活力变化。未经孵育的鸡胚中检测不出CaM,在孵育3—14天的胚脑中CaM含量逐渐增加:总CaM增加了3倍,活性CaM增加了4倍;而且在鸡脑细胞分裂分化最活跃的期间(皮质和髓质形成期间,即孵育8—14天),活性CaM的增加尤为显著(增加近3倍)。提示CaM与脑细胞的分裂分化有关。PDE的活力出现晚于CaM5天,随着胚龄增加,不断上升,提示它与脑细胞的分裂分化无直接关系,可能与大脑的功能活动有关。本实验还研究了鸡脑发育期中可逆性钙结合蛋白的变化。     

THE EFFECT OF NEONATAL THYROIDECTOMY ON THE DEVELOPMENT OF THE ADENOSINE 3'',5''-MONOPHOSPHATE SYSTEM IN THE RAT BRAIN

Abstract— The effect of neonatal thyroidectomy on the cyclic AMP system in the developing rat brain was examined. Administration of ¹³¹I at birth led to a 16 per cent reduction in brain weight and a 70 per cent reduction in body weight by 40 days of age. The level of cyclic AMP in the brain increased 5-fold between birth and 40 days of age and this increase was partially reduced by early thyroidectomy. A similar increase in the activity of adenyl cyclase and phosphodiesterase was observed during development, but thyroidectomy produced no detectable changes in the activity of either enzyme. The activity of the cyclic AMP-dependent protein kinase was already maximal at birth and also was unaffected by thyroidectomy.
Norepinephrine increased levels of cyclic AMP 4- to 5-fold in brain slices prepared from adult rats, but was without effect on slices prepared from newborn or 3-day-old rats. The response to norepinephrine in thyroidectomized rats did not differ from that in control rats at any of the ages examined. Our findings indicate that neonatal hypothyroidism does not deleteriously affect the development of the cyclic AMP system in the rat brain.     

Postnatal ontogeny of uridine kinase in the cerebellum,hypothalamus, and cerebral cortex of the rat

Postnatal developmental patterns of uridine kinase were determined in crude subcellular fractions of the rat cerebellum, hypothalamus and cerebral cortex at ages 3 through 60 days. The highest specific activity and predominant distribution of enzyme was in the 105,000g supernatant of the 3 brain regions. Enzyme activity in hypothalamus and cerebral cortex was maximum at 3 days and decreased with age; in cerebellum it increased through 13 days and decreased thereafter. Thus, the pattern of activity in hypothalamus and cerebral cortex paralleled changes in DNA and RNA synthesis through age 60 days; in cerebellum, it more closely approximated changes in DNA synthesis during early development. Changes inK _m with aging suggest that the brain regions contain more than one form of enzyme. The highest particulate activity was in the microsomal fraction of the cerebellum and hypothalamus at all ages and in the cortex at 35 and 60 days. Relative specific activity for microsomal fractions of the brain regions at 60 days indicate a concentration of the enzyme which may be relevant in the maintenance of RNA activity in adult brain.     

HISTOCHEMISTRY OF RAT BRAIN STEM MONOAMINE OXIDASE DURING MATURATION

—Monoamine oxidase (MAO) activity in the nuclei and tracts of the medulla and pons of the rat from birth to 90 days is reported. Prominent MAO activity was present in the locus coeruleus and nucleus ambiguus at birth. At 5 days a weak reaction localized mainly within the neuropil and glia cells was detected in several other nuclei. By 10 days all nuclei were identified with MAO activity varying from weak to intense, the activity showing further increases at 15 and 20 days. Staining in nerve fibres was negligible at 5 days but increased rapidly to 15 days in some tracts when the characteristic beading pattern was distinct. At 30 days differentiation in intensity of MAO activity between the nuclei diminished and no increase was apparent after 55 days. The results are compared with the distribution of brain stem acetylcholinesterase during maturation and also with regions specific in catecholamine or serotonin content in the adult rat brain stem. This and an earlier study on the cerebrum suggest that MAO is another component of the brain that falls into the caudal-rostral concept of biochemical maturation and that it fits into a group of enzymes exhibiting a similar pattern of increase in activity during development.     

Ontogenetic Development of Histamine H1- Receptor Binding in Rat Brain

Abstract: Histamine H_1- receptors labeled with [³H]mepyramine in rat brain show an age-dependent development. [³H]Mepyramine receptor density and histidine decarboxylase activity in whole rat brain reach adult levels at 25–30 days after birth and they attain 50% of adult level at day 10 and 17, respectively. The apparently later development of histidine decarboxylase activity in whole rat brain is partly accounted for by a biphasic developmental increase of this enzymatic activity in cerebral cortex. For all other brain regions examined, the development of histamine H_1- receptors parallels that of histidine decarboxylase. The increase in [³H]mepyramine binding can be accounted for by an absolute increase in the numbers of the receptor sites, with no change in affinity. Subcellular fractionation studies indicate that histamine H_1- receptors are predominantly associated with synaptosomal fractions derived from both newborn and adult rat.     

BRAIN PROTEINS: QUALITATIVE AND QUANTITATIVE CHANGES, SYNTHESIS AND DEGRADATION DURING FETAL DEVELOPMENT OF THE RABBIT

The composition and metabolism of the proteins of the cerebral pallium of the rabbit during the final one-third of the gestational period were measured. During this period, the brain increased in size almost 10-fold and the migration of neuroblasts to form the cerebral cortex became complete. Concurrent with the marked structural changes, the solubility characteristics and electrophoretic distribution of various brain proteins showed little change. However, at the time of birth and in the adult, significant differences in gel electrophoresis patterns were apparent. The rate of synthesis of protein in brain slices from the fetus of 20 days gestation was 3-fold higher per mg of tissue than in the neonate and about 30-fold higher than in the adult. Activities of acidic and neutral proteases per unit weight were virtually the same and nearly constant throughout the late fetal period. However, during this stage, while rapid growth persists, the total protein synthetic activity of the pallium predominated over the total proteolytic activity, whereas sometime after birth the ratios of these activities reversed consequent to a shutdown of the synthetic process.     

PROTEIN SYNTHESIS IN FRACTIONS FROM ISOLATED BRAIN CELL NUCLEI

Abstract— 1. The incorporation in vivo and in vitro of isotopically labelled leucine into fractions of nuclear proteins from young and adult rat brain was investigated.
2. During post-natal cerebral maturation, the ability of nuclei from brain cells to synthesize proteins decreased. The specific activities of all the fractions of nuclear protein were highest in 3-day-old rats and declined thereafter. Nuclei from adult brain cells exhibited only 10 per cent of the activity found in nuclei from brain cells of 3-day-old rats.
3. The 'residual protein' fraction was most rapidly labelled, peak activity being reached within 30 min after injection. In vitro , the 'residual protein' fraction attained maximum activity within 40 min.
4. The specific activity of the chromatin acidic proteins (HCl-insoluble) was considerably higher than that of the histones both in vivo and in vitro. Histones were the most inert of all the nuclear protein fractions studied.
The possible functional significance of the various protein fractions during the process of cerebral maturation and in the adult brain is discussed.     

Ontogenic pattern of dopamine, acetylcholine, and acetylcholinesterase in the brains of normal and hypothyroid rats.

The influence of neonatal thyroidectomy (Tx) on developmental changes in dopamine (DA), acetylcholine (ACh), and acetylcholinesterase (AChE) was studied in the whole brain of rats. In control animals, brain levels of ACh gradually increased and attained adult values at the 70th day. In contrast, AChE activity showed a rapid increase between the 7th and 30th days. Levels of DA were low during the early postnatal life but markedly increased to reach adult values of 1.47 mug/g at the 30th day, after which no further enhancement was noted. Neonatal Tx interfered with the normal growth of the animals, decreased brain weights, and markedly influenced the developmental pattern of both DA and ACh in the brain. The concentration of DA in 30-day-old hypothyroid rats was 46% of the control values. In contrast, brain ACh levels in Tx rats were consistently above those seen in controls, being significantly higher, by 49 and 64%, at 15 and 30 days, respectively. Activity of AChE in brains of hypothyroid animals was not significantly different from that in controls. Treatment of Tx rats with thyroid hormone virtually restored the levels of DA and ACh to values in control animals.     

DEVELOPMENT OF THE UPTAKE AND STORAGE OF L-[3H]NOREPINEPHRINE IN THE RAT BRAIN

The uptake and storage of L-[³H]norepinephrine at various stages of development was examined in homogenates of rat brain. For the adult animal, active uptake accounted for 80 per cent of the total uptake. At 14 days of gestation, no active uptake was demonstrable At 18 days of gestation, saturable uptake of L-[³H]norepinephrine with a K_m of 3 × 10 ^?7m was first demonstrable; the K_m value did not vary during subsequent development. The V_max. of uptake increased five-fold between 18 days of gestation and 28 days postnatally, at which stage it was the same as the adult value. The development of saturable uptake paralleled but preceded the increase in endogenous norepinephrine. When homogenates were incubated with l -[³H]norepinephrine and subjected to centrifugation on linear sucrose gradients, there was a peak of tritium in the synaptosomal fractions; the magnitude of the peak increased with maturation of the brain. The increase in the peak of tritium paralleled the increase in particulate LDH activity and was distinct from the peak of MAO activity. Desipramine, a compound that blocks the initial uptake of norepinephrine, first exhibited inhibition of uptake at 19 days of gestation; the degree of inhibition did not vary during subsequent development. In contrast, reserpine, a compound which inhibits the intra-neuronal storage of norepinephrine, exhibited a progressive increase of inhibition with maturation of the brain at and subsequent to 19 days of gestation.     

Glycosaminoglycans of brain during development.

The concentration of hyaluronic acid, chondroitin sulfate, and heparan sulfate was measured in rat brain at 2-day intervals from birth to 1 month of age, and in 40-day-old and adult animals. The levels of all three glycosaminoglycans increased after birth to reach a peak at 7 days after which they declined steadily, attaining by 30 days concentrations within 10% of those present in adult brain. The greatest change was seen in hyaluronic acid, which decreased by 50% in 3 days, and declined to adult levels (28% of the peak concentration) by 18 days of age. Only heparan sulfate showed a significant change in metabolic activity during development (a fourfold increase in the relative specific activity of glucosamine), most of which occurred after 1 week of age. In 7-day-old rats almost 90% of the hyaluronic acid in brain is extractable by water alone, as compared to only 15% in adult animals, and this large amount of soluble hyaluronic acid in young rat brain is relatively inactive metabolically. On the basis of our data we propose that the higher amounts of hyaluronic acid found in very young brain may be responsible for the higher water content of brain at these ages, and that the hydrated hyaluronic acid serves as a matrix through which neuronal migration and differentiation may take place during early brain development.     

Ornithine decarboxylase activity in developing rat brain

—Total ornithine decarboxylase (ODC) (EC 4.1.1.17) activity per rat brain was elevated markedly from 14 days after conception to 12 days postnatum. ODC activity in the brainstem was very low and changed little during postnatal development. Activity in the cerebral hemispheres declined from a high level at birth to the low adult level by 8 days postnatum. Conversely activity in the cerebellum increased markedly from 3 days until 11 days postnatum, then suddenly decreased. Hence, the periods of greatest ODC activity paralleled those of maximal cell proliferation in each brain region. During perinatal brain development ODC activity changed considerably; it declined at about one day prior to term, and then increased rapidly to its highest level of activity at 4 h postnatum. Premature birth by caesarian section or lack of maternal care and nutrition did not affect this early postnatal response. The postnatal burst in ODC activity appears to be unique for brain tissue, since this response did not occur in heart, skeletal muscle or liver. Data from studies in which portions of fractions characterized by high or low enzymatic activity, respectively, were mixed or in which the supernatant enzyme fraction was dialysed are not consistent with the presence of direct inhibitors or activators of the enzyme. In addition, administration of cycloheximide to newborn rats abolished the 4-h postnatal burst in ODC activity. Our results suggest that the increase in ODC activity reflects enzyme synthesis de novo.     

Effects of oestradiol, testosterone and medroxyprogesterone on subcellular fraction marker enzyme activities from rat liver and brain

The following enzymes have been studied (subcellular fractions are shown between parentheses): NAG and beta-glucuronidase (lysosomes); SDH (mitochondrial); glucose-6-phosphatase (endoplasmic reticulum); 5'-nucleotidase and (Na+, K+)Mg2+ ATPase (plasma membranes). Alterations on their activities were observed after subcutaneous injection of sex hormones, compared with controls. NAG activity from liver was always significantly decreased in lysosomal and microsomal fractions after the hormonal treatment. In the same conditions, NAG from brain was always increased. beta-Glucuronidase behaves like NAG in brain; in liver it was not modified by testosterone and it was slightly increased in lysosomal fraction after oestradiol treatment. SDH activity was not modified in mitochondrial fractions from liver, but this activity was always significantly increased in brain. Glucose-6-phosphatase activity was always significantly decreased in microsomal fractions from liver. It was increased in brain after oestradiol and testosterone injection, but medroxyprogesterone treatment caused a decreased activity. 5'-Nucleotidase and (Na+, K+)Mg2+ ATPase from brain were significantly increased in microsomal fractions by oestradiol and testosterone. Medroxyprogesterone, however, caused an increase in ATPase, but did not affect 5'-nucleotidase. Both activities in liver were decreased by oestradiol and increased by testosterone, but medroxyprogesterone caused (Na+, K+)Mg2+ ATPase to rise and 5'-nucleotidase to fall.     

CHANGES IN TITER OF THE BRAIN HORMONE DURING DEVELOPMENT OF THE SILKWORM, BOMBYX MORI

Changes in titer of brain hormone (BH) extracted from the brain, head, and thorax and abdomen during the development of Bombyx mori from the 4th larval molting up to 6 days after adult emergence were investigated. The hypothesis is presented that high secretory activity of the brain is not reflected in a corresponding change in its BH titer, while low secretory activity brings about an accumulation of BH in the brain. An interesting finding is that extremely high titers of BH were found in the thorax and abdomen, from 4 days after pupation to adult emergence, of females only. This sexual difference was, however, not found in all of the races examined.     

Perinatal changes in amylase and serum corticosterone levels in rats

In rats amylase activity in the pancreas increased greatly from day 15 of gestation to a maximum on day 21. Then it decreased to less than one-tenth of this maximum value on about day 5 after birth. It increased again about 15 days after birth and reached the adult level about 30 days after birth.No amylase activity was in the parotid gland before birth: it appeared about 12 days after birth and reached the adult level, which was higher than that in the pancreas, about 30 days after birth.The serum corticosterone level was as high as the adult level before birth. Then it decreased to less than one-tenth of the adult level 5 days after birth and increased again from 15 to 25 days after birth to the adult level. The developmental change in the serum corticosterone level seemed to influence amylase activity in the pancreas both before and after birth, and that in the parotid gland only after birth.The serum contained both pancreatic and paratoid type isozymes of amylase until 1 day after birth but only the parotid type from 3 days after birth.