Postnatal Changes in Cathepsin D in Rat Neural Tissue

Cathepsin D, an aspartyl endopeptidase, was analyzed in cortex from forebrain and cerebellum, spinal cord, and optic and sciatic nerves, and in the liver of rats from 1 to 120 days of age. Cathepsin D was quantitated in tissue extracts by measurement of enzyme specific activity on a substrate of [methyl-¹⁴C]-methylated hemoglobin and by radioimmunoassay. Immunocytochemistry was used to ascertain the identity of the mixed cell types that contributed to the cathepsin D detected. As quantitated by radioimmunoassay, immunoreactive cathepsin D varied between 0.2 and 1 ng/μg of total protein. Maximum activity occurred at approximately the 15th postnatal day; the least amount of immunoreactive cathepsin D was found at 30 or 60 days of age. A subsequent increase of varying magnitude occurred at postnatal day 120. There was good correspondence between immunoreactive enzyme and enzyme specific activity, which ranged from 1 to 4 ng/μg of total protein, and the activities determined by the two methods provided similar, but not identical, developmental profiles. Cathepsin D was demonstrated by immunocytochemistry to be present in most neurons, in all choroid plexus epithelium, and in certain oligodendrocytes from the first postnatal day. Cathepsin D was present in oligodendrocytes in cord lateral funiculi and optic nerve by the first postnatal day, and by the sixth postnatal day many oligodendrocytes were abundantly stained. In contrast, oligodendrocytes in the corpus callosum and in the cerebellar white matter did not contain demonstrable cathepsin D until postnatal days 10 and 15, respectively. These results indicate a role for cathepsin D during the postnatal development of rat CNS and suggest that this proteinase may be involved in the steps of myelination.     

Intracellular distribution of creatine kinase isoenzymes in the brains and hearts of rats at different stages of postnatal development]

The total activity and range of the creatine kinase (CK) isozymes have been studied in the homogenate and subcellular fractions (nuclei, mitochondria, cytoplasm) of the rat brain and heart during postnatal ontogenesis. The total activity of CK in the brain and heart of newborn rats was found to be 4 and 2 times less, resp., than in those of adults. The age patterns were established in the activity of cytoplasmic (CK-1, CK-2 and CK-3) and mitochondrial (CK-4) isozymes. During the whole postnatal development the rat brain contains only one cytoplasmic isozyme, CK-1. In the heart of newborn rats, as compared with adults, the content of CK-1 and CK-2 is much higher and that of CK-3 lower. On the 12-15th day of life the range of the CK isozymes approaches that characteristic of adult animals. The activity of CK-4 was found in the brain on the 5-7th day of life and in the heart on 12-15th day. In the range of the CK isozymes in the adult brain the content of mitochondrial CK amounts to 19.3% and in the heart to 16.5%. The data obtained complement the literary ones suggesting the low level of energy-forming processes in the brain and heart cells at the early stages of the rat postnatal development.     

Neuronal Membrane D2-Protein During Rat Brain Ontogeny

Abstract The D2-protein exists as a sialylated form in fetal and in perinatal rat brain, and as a desialylated form in adult rat brain. By crossed Immunoelectrophoresis the concentrations and amounts of these forms were investigated during ontogeny of both forebrain and cerebellum. The concentration of sialylated D2-protein reached two peaks during ontogeny. The first peak occurred in forebrain around embryonic day 13, and in cerebellum just after birth. In both brain areas it coincided with the periods of major neuronal migration. The second peak occurred in forebrain around postnatal day 6 and in cerebellum around postnatal day 20, during the initial period of synaptogenesis in both brain areas. Moreover, the desialylated form of D2-protein was found only in postnatal rats and it increased to a slight maximum at postnatal day 25 in forebrain, and postnatal day 35 in cerebellum. The findings are discussed in relation to the possible role of D2-protein as an adhesion molecule.     

Membrane-bound neuraminidase in the developing mouse brain

The specific activity of membrane-bound neuraminidase towards exogenously added gangliosides in the developing mouse brain was investigated. While whole brain was examined in fetuses, studies were carried out comparatively in cortex and cerebellum of postnatal stages. Considerable differences were found among brain areas. There was a rapid increase in forebrain neuraminidase activity before birth. This high level of activity was maintained throughout the first postnatal week, followed by a slow leveling-off to adult values on the 30th day. In the cerebellum a rapid increase in neuraminidase activity occurred between the 10th and 16th postnatal day. After having reached the maximum, enzyme activity declined rapidly, with adult values being reached on the 21st day. Neuraminidase activity in the adult cerebellum exceeds that in the cortex by 65.9 %. The results are discussed in reference to developmental changes in ganglioside metabolism and the possible involvement of neuraminidase in its regulation is pointed out.     

Changes of malic enzyme activity in the developing rat brain are due to both the increase of mitochondrial protein content and the increase of specific activity.

1. The pattern of NADP-linked malic enzyme activity estimated in the whole brain homogenate did not parallel that found in liver of developing rat. 2. Studies on intracellular distribution of malic enzyme in brain showed that the mitochondrial enzyme increased about three-fold between 10th and 40th day of life. Thereafter, a slow gradual increase to the adult level was observed. 3. The extramitochondrial malic enzyme from brain, like the liver enzyme, increased at the time of weaning, although to a lesser extent. At day 5 the brain malic enzyme was equally distributed between mitochondria and cytosol. 4. During the postnatal development, the contribution of the mitochondrial malic enzyme in the total activity was increasing, reaching the value approx. 80% at day 150 after birth. 5. The increase with age of the malic enzyme specific activity was observed in both synaptosomal and non-synaptosomal mitochondria, the changes in the last fraction being more pronounced. 6. The activity of citrate synthase developed markedly between 10-40 postnatal days, increasing about five-fold, while the specific activity of the enzyme did change neither in the synaptosomal nor in non-synaptosomal mitochondria at this period. 7. We conclude that the changes in malic enzyme activity in the developing rat brain are mainly due both to the increase of mitochondrial protein content and to the increase of specific activity of the mitochondrial malic enzyme.     

Studies on cystathionase activity in rat liver and brain during development. Effects of hormones and amino acids in vivo

High activity of cystathionase was present in rat liver but only low amounts of activity in rat brain during development. Triamcinolone had no effect on liver cystathionase activity in foetuses but increased the enzyme activity significantly in postnatal rats. l-Thyroxine decreased liver cystathionase activity significantly in newborn rats; administration of pyridoxal 5'-phosphate did not prevent this effect. l-Methionine significantly increased liver cystathionase activity in newborn rats.     

Developmental changes in alcohol-dehydrogenase activity in rat and guinea-pig liver

1. Alcohol-dehydrogenase activity is first detectable in the rat foetus on about the eighteenth day of gestation, after which time it increases to about 25% of the adult activity at birth. Adult activity is reached at about 18 days after birth. The ethanol-oxidizing capacity of liver slices from rats correlates well with the increase of the enzyme activity in vitro. 2. In the guinea pig there is a steady linear increase from about 17 days before term to 5 days after birth. Adult activity is reached between the sixth and eighth postnatal day. 3. Some kinetic properties of liver alcohol dehydrogenase are very similar in newborn and adult rats. 4. Administration of ethanol to pregnant rats during the latter half of gestation had no effect on alcohol-dehydrogenase activity in the liver of the newborn offspring. Intraperitoneal injections of ethanol to newborn and young rats had no effect on the alcohol-dehydrogenase activity of the livers. 5. Intraperitoneal injections of hydrocortisone and triamcinolone to newborn and adult non-adrenalectomized rats had no significant effect on the increase of the alcohol-dehydrogenase activity as studied up to 4 days after the injection.     

Biochemical and immunohistochemical study on physiological activity and distribution of hepatic cathepsin D

Cathepsin D (EC 3.4.23.5) is a lysosomal endopeptidase physiologically present at very low concentration in different tissues. The aim of the study was to estimate the physiological activity and distribution of cathepsin D in the liver. Four groups of ten-week-old male Wistar rats were raised without xenobiotics and sacrificed on day 4, 42, 47 and 84 of the experiment, and their livers were taken for immunohistochemical and biochemical investigation. Immunostaining for cathepsin D was evaluated by light microscope. Activity of the free and bound fractions of hepatic cathepsin D was measured spectrophotometrically. Immunohistochemical staining for cathepsin D was positive in Browicz-Kupffer cells in some but not in all rat liver specimens of each experimental group. The staining pattern was cytoplasmic and granular. Occasionally the positive stained endothelial cells were also found. No activity of cathepsin D in hepatocytes was detected. The positive immunostaining was found in livers with high enzyme activity in the biochemical investigation. No significant differences in activity of the free and bound fractions of cathepsin D among the different age groups were noted. However, the higher, age-dependent activity (p>0.05) of the free fraction was observed in the youngest and the two-middle groups of rats that were sacrificed on day 42 and 47 than in the oldest one. The bound fraction did not reveal such changes. It could be concluded that there were no differences in the activity of hepatic free and bound fractions of cathepsin D in male Wistar rats of various reproductive age. The rat Browicz-Kupffer cells revealed the highest activity of cathepsin D.     

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.     

Developmental aspects of uridine diphosphate galactose 4-epimerase in rat intestine

Kinetic and developmental characteristics of rat intestinal UDP-galactose 4-epimerase activity have been examined. The enzyme in the adult rat had a V_max. value 2–3 times higher than that of the newborn animal, but the K_m values for the enzyme in the newborn and adult rat were the same (0.17mm). No differences in epimerase activity were found along the length of the jejuno-ileum of adult animals, but higher activity was detected in the lower portion of the villi and crypts. The specific activity of the enzyme in the newborn rat began to rise at about 17 days of age, reaching a peak at 29 days of age, and then became constant at adult values. Total epimerase activity in the newborn rat liver was 2–5 times higher than the total activity in the intestine, and total epimerase activity in the adult intestine was 3–4 times higher than the total activity in the liver. Cortisone injection did not enhance the increase of epimerase normally seen during development, but caused a decrease in activity of this enzyme in the jejunum in rats up to 17 days of age. After 17 days, cortisone treatment had no effect on epimerase activity.     

Regulation of NAD- and NADP-linked isocitrate dehydrogenase by hydrocortisone in the brain and liver of male rats of various ages

The activity and hormonal regulation of NAD- and NADP-linked isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42, respectively) in the brain and liver of rats of various ages were investigated. The activity of NAD-linked isocitrate dehydrogenase of the brain was greater than cytoplasmic or mitochondrial NADP-linked isocitrate dehydrogenase. In contrast, the cytoplasmic NADP-isocitrate dehydrogenase of the liver predominates over both NAD- and mitochondrial NADP-isocitrate dehydrogenases at the three ages studied. The activity of NAD-isocitrate dehydrogenase increased in the brain (139%) and liver (17%) of rats upt o 33 weeks of age and decreased (57 and 39%, respectively) in old rats (85-week-old). The activity of cytoplasmic NADP-isocitrate dehydrogenase was maximum in immature (6-week-old) rat brain and decreased as the age of the rats increased; whereas, in liver, the activity of this enzyme was found to be maximum in adult rats (33-week-old). Brain mitochondrial NADP-isocitrate dehydrogenase activity increased (64%) in adult rats, but in liver it decreased (45 and 33% in 33- and 85-week-old rats, respectively). In both tissues, adrenalectomy and hydrocortisone treatment showed differential age-dependent response. Hydrocortisone-mediated induction of the level of enzymes was inhibited by actinomycin D.     

Thyroxine-induced changes in the glycosylation pattern and in brain and serum levels of rat alpha-fetoprotein

We have studied the effect of thyroid disfunction during the postnatal period, on the serum and brain levels of rat alpha-fetoprotein (AFP) and albumin. Hypothyroidism was induced by treatment of pregnant rats and their newborn pups with 2-mercapto-1-methylimidazole(methimazole). Hyperthyroidism was provoked in newborns by daily injections of thyroxine (0.25 micrograms/g body wt) from the 3rd postnatal day weaning. Impaired growth, lower brain size, altered behaviour and morphological features observed were according to an altered thyroid status. Hypothyroid rats showed a significantly reduction in serum AFP concentration (78% of control values at 8 days of age) and a slight increase in that of albumin. level could be appreciated. Thyroxine supplementation (0.2 micrograms/rat/day) corrected most of these alterations. Hyperthyroidism induced a drastic fall in both serum and brain AFP levels (about 48% of the corresponding control values). Albumin concentration in serum was augmented significantly from the 12th postnatal day, but its brain levels did not change significantly. In hyperthyroid rats, a significant reduction (37% relative to controls) in the concanavalin A-non reactive microform of AFP, was observed. This alteration of the glycosylation pattern of AFP could be due to the inhibition by thyroxine of the activity of the hepatic enzyme GlcNAc-transferase III.     

ONTOGENETIC DEVELOPMENT OF A PHOSPHODIESTERASE ACTIVATOR AND THE MULTIPLE FORMS OF CYCLIC AMP PHOSPHODIESTERASE OF RAT BRAIN

Abstract— The activity of cyclic AMP phosphodiesterase of rat cerebral homogenates increased several-fold between 1 and 60 days of age. Enzyme activity in the cerebellum, on the other hand, did not increase during this period. A kinetic analysis of the phosphodiesterase activity revealed evidence for multiple forms of the enzyme and indicated that the postnatal increase in phosphodiesterase activity of rat cerebrum was due almost exclusively to the high K_m enzyme. In cerebellum, the ratio of the high and low K_m enzyme remained fairly constant during ontogenetic development. Physical separation of the phosphodiesterases contained in 100,000 g soluble supernatant fractions of sonicated brain homogenates by polyacrylamide disc gel electrophoresis confirmed the presence of multiple enzyme forms. In adult rats we found six distinct peaks of phosphodiesterase activity (designated I to VI according to the order in which they were eluted from the column) in cerebellum and 4 forms of the enzyme (Peaks I through IV) in cerebrum. Brains of newborn rats had a different pattern and ratio of phosphodiesterase activities. For example, Peak I phosphodiesterase was undetectable in cerebrum or cerebellum of newborn rats. Moreover, in the cerebellum of newborn rats Peak II was the dominant peak whereas in the cerebellum of adult rats Peak III was the largest peak. A comparison of the multiple forms of phosphodiesterase from the cerebrum of newborn and adult animals suggested that the postnatal increase in phosphodiesterase activity previously seen in crude homogenates was due largely to an increase in a high K, Peak II phosphodiesterase. The ratios of activities of the other peaks and their sensitivities to an activator of phosphodiesterase were similar in newborn and adult rats. An endogenous heat-stable activator of phosphodiesterase was found in cerebrum, cerebellum and brain stem. In newborn rats, the cerebellum contained several-fold less activity of this activator than did cerebrum or brain stem. However, the activity of this activator increased with age in the cerebellum and would appear to have decreased postnatally in cerebrum and brain stem. These results suggest that some multiple forms of phosphodiesterase can develop independently and that changes in activities of these phosphodiesterases may occur by increases in the quantity of enzyme or by changes in the quantity of an endogenous activator of phosphodiesterase.     

Regulation of succinyl-CoA:3-oxoacid CoA-transferase in developing rat brain: responsiveness associated with prenatal but not postnatal hyperketonemia

Activities of ketone body-metabolizing enzymes in rat brain rise 3- to 5-fold during the suckling period, then fall more than 50% after weaning. Our purpose was to determine the mechanism of the developmental changes in activity of 3-oxoacid CoA-transferase in rat brain and to study its regulation by dietary modification. Purified rat brain 3-oxoacid CoA-transferase was used to generate specific antibody. Immunotitrations of the enzyme from brains of 4-, 24-, and 90-day-old rats indicated that changes in 3-oxoacid CoA-transferase activity during development are due to changes in content of the enzyme protein. Pulse-labeling studies showed that changes in enzyme specific activity reflected changes in its relative rate of synthesis, which increased 2.5-fold between the nineteenth day of gestation and the third postnatal day, remained at this high level until the twelfth postnatal day, and declined thereafter, returning by Day 38 to the level observed in utero. The enzyme is apparently degraded very slowly during early postnatal life. Fetal hyperketonemia induced by feeding pregnant rats a high-fat diet was associated with an increase in the relative rate of synthesis of 3-oxoacid CoA-transferase in brains of 19-day-old fetuses and newborn rats and with an increase in the specific activity of the enzyme at birth. To examine the role of postnatal hyperketonemia in the development of the enzyme in brains of suckling rats, neonates received intragastric cannulas and were fed, for up to 13 days, a modified milk formula low in fat. Postnatal hyperketonemia was abolished but cerebral 3-oxoacid CoA-transferase specific activity on Days 10 and 17 was not significantly affected. Thus, the physiological hyperketonemia caused by the high fat content of rat milk is not required for the normal development of 3-oxoacid CoA-transferase in rat brain.     

Postnatal development of β-galactosidase activity in the small intestine of the rat. Effect of adrenalectomy and diet

1. β-Galactosidase activity was studied in homogenates of the proximal and distal thirds of the small intestine from adult and infant rats. o-Nitrophenyl β-d-galactoside served as the substrate. 2. Activity in suckling rats is highest in the distal part of the small intestine. 3. The pH optimum was 3·5 in the distal third of the small intestine in rats aged 5 and 15 days, whereas in the proximal third the maximum was not clearly defined. 4. Activity was higher in both thirds in newborn than in adult rats, expressed per wet wt. or per wt. of protein. In the proximal third activity continually decreases with age, whereas in the distal part there is a rise up to day 15 and then a sudden decrease. Total β-galactosidase activity changes very little in the proximal third during postnatal development; the greatest changes occur in the distal third. 5. Adrenalectomy performed on day 15 postnatally slows down the decrease in β-galactosidase activity, particularly in the distal part. 6. Feeding a lactose diet to infant rats from day 14 postnatally in the presence of the mother rat also slows down the decrease in β-galactosidase activity and this is not found with a diet containing glucose and galactose instead of lactose.     

MAO, COMT, and GABA-T Activities in Primary Astroglial Cultures

Cultures from cerebral hemispheres of newborn rats contain the enzymes monoamine oxidase (MAO), catechol-O-methyltransferase (COMT), and gamma-aminobutyric acid alpha-ketoglutarate transaminase (GABA-T). The COMT activity was higher in the cultures than in adult rat cerebral hemispheres. The MAO activity was comparable in the cultures and in the rat cerebral hemispheres. The activities of both these enzymes increased with age in the cultures and in the rat brain hemispheres. In the culture the activities were further potentiated by removal of fetal calf serum and addition of 0.1 mM dibutyryl-cyclic AMP (dB-cAMP). GABA-T activity was, however, lower in the cultures than in the adult rat brain hemispheres. The activity increased in brain during postnatal maturation. No changes in the enzyme activity were observed in the cultures, either during growth or after removal of fetal calf serum and addition of dB-cAMP.     

Appearance and accumulation of the brain specific S 100 protein in the developing nervous systems of rat, rabbit and guinea pig.

The appearance and accumulation of S 100 protein during postnatal development of rats and rabbit, and during pre- and postnatal development of guinea pig, was studied quantitatively by immunoelectrophoresis. High amounts of S 100 were found in newborn guinea pig brain. In rabbit and rat central nervous systems the content of S 100 rose linearily between the 1st and the 3rd and 4th weeks of postnatal life, respectively. From this later period to the adult animal there was a small increase in the amount of S 100 in rabbit and rat brain. The results of this study are compared with other studies on S 100 accumulation during postnatal development of rat, rabbit and guinea pig, using immunofluorescence and immunoelectron microscopic techniques.     

Identification, Development, and Regional Distribution of Ribonucleotide Reductase in Adult Rat Brain

The development and regional distribution of ribonucleotide reductase (EC 1.17.4.1) were determined in rat brain. Ribonucleotide reductase was partially purified by ammonium sulfate fractionation (20-40% saturation). Enzyme activity was measured by a specific radiochemical assay. This method involved the reduction of [¹⁴C]cytidine diphosphate (CDP) to [¹⁴C]deoxy-cytidine diphosphate with subsequent hydrolysis and separation of the product ([¹⁴C]deoxycytidine) from substrate ([¹⁴C]cytidine) by Dowex-1-borate ion-exchange chro-matography. The specific activity of ribonucleotide reductase in whole brain of newborn rats was 3.78 ± 0.55 units (pmol/h)/mg protein (SEM; n = 6) and declined to 0.17 ± 0.01 units/mg protein (n = 7) at 10-12 weeks of age, with a further decline to 0.11 ± 0.01 units/mg protein (n = 3) at 1 year. Ribonucleotide reductase activity in rat liver decreased from 4.58 ± 0.62 units/mg protein (n = 3) in newborn animals to 0.06 ± 0.01 units/mg protein (n = 7) at 10-12 weeks and was present at trace levels at 6 months of age. The decline in specific activity with age was not due to a change in the K_m for CDP. The K_m for CDP in brain of newborn and adult rats was 80-90 μM. In 10- to 12-week-old rats, the specific activity of ribonucleotide reductase was similar in the various regions of the brain tested except for the brainstem, which had 50% lower specific activity than the whole brain. These results indicate that ribonucleotide reductase activity is present and widely distributed in adult rat brain.     

PHOSPHOCHOLINE DIGLYCERIDE TRANSFERASE ACTIVITY DURING DEVELOPMENT OF THE CHICKEN BRAIN

Abstract— The activity of chicken brain phosphocholine diglyceride transferase was followed during pre- and postnatal development. The specific activity of this enzyme increases from the 10th day of embryonic life, reaches a maximum at hatching and decreases thereafter. Total brain activity increases in parallel with the increase of brain lecithins. The apparent K _m of the enzyme for CDP choline is 1.5 × 10^-4 m before the 10th day of embryonic life, 2.5 × 10^-5 m between the 13th day of the embryo and the 10th day after hatching, and finally 1.3 × 10^-4 m after the 38th day of postnatal life. These data suggest the existence of isoenzymes, one of which appears at the beginning of myelination.     

Expression of GABA and glycine receptors by messenger RNAs from the developing rat cerebral cortex

The ontogenesis of mRNAs coding for GABA and glycine receptors in the cerebral cortex of the rat was examined by extracting poly(A)+ mRNA from the brains of embryonic, postnatal or adult rats and injecting it into Xenopus oocytes. The ability of a messenger to express functional receptors was then assayed by measuring the membrane currents elicited by the agonists. The size of the GABA-induced current increased progressively with age, being undetectable in oocytes injected with mRNA from embryonic day 15 and reaching a maximum in oocytes injected with mRNA from postnatal day 30. In contrast, the glycine-induced response was negligible in oocytes injected with mRNA from the cerebral hemispheres of embryos 15 days old; it increased sharply to a maximum with newborn animals and then decreased with age to become very small with mRNA from adult cortex. GABA and glycine receptors induced by mRNA from the cerebral cortex of all ages are associated with chloride channels.