Identification and partial characterization of rabbit brain deoxyuridine 5′-triphosphatase

Adult rabbit brain contains the enzymatic machinery to convert deoxyuridine to deoxyuridine triphosphate (dUTP). Although dUTP as dUMP can be readily incorporated into DNA in place of thymidine monophosphate, we detected no (3H)dUMP in newly synthesized (3H)DNA in adult rabbit brain after the intraventricular injection of (3H)deoxyuridine. Only (3H)thymidine was detected. The probable explanation for the lack of incorporation of uracil into adult rabbit brain DNA is the presence of a specific, high affinity dUTPase which converts dUTP to dUMP and PP. After homogenization and ammonium sulfate fractionation of adult rabbit brain (35 to 75% saturation), a high affinity, specific dUTPase was detected in the dialyzed enzyme preparation. The Km and Vmax of the dUTPase were 0.2 microM and 36 pmol/mg protein/min, respectively. No high affinity dUTPase activity was detectable in liver. In brain, another enzyme hydrolyzed dUTP and dTTP (NTPase( to their respective diphosphates. NTPase, unlike dUTPase, was not sensitive to heating at 65 degrees C for five minutes. Thus, brain, like other tissues, contains a high affinity, specific dUTPase presumably to "sanitize" the cells of dUTP and, thus, protect the integrity of newly synthesized DNA.     

Identification, Development, and Regional Distribution of Thymidylate Synthetase in Adult Rabbit Brain

Abstract: The development and regional distribution of thymidylate synthetase (TS) (EC 2.1.1.45) in rabbit brain were determined. After optimization of the assay for brain, TS activity in brain was measured by a nonspecific (³H₂O release) and specific method. The specific method involved the conversion of [6-³H]deoxyuridine monophosphate (dUMP) to [³H]thymidine phosphate and the subsequent identification of [³H]thymidine. The specific activity of the enzyme in whole brain of newborn rabbits declined from 10.35 ± 1.17 units/mg protein to 0.71 ± 0.09 units/mg protein at 10–12 weeks of age. Two-year-old rabbits had 0.81 ± 0.04 units/mg protein. The decline in specific activity with age was not due to an inhibitor of TS activity or a change in the K_m for dUMP. The K_m for dUMP of the unpurified enzyme in the brains of both 10-day-old and young adult rabbits was 0.8 μ m . In young adult rabbits (3 months) the specific activity of TS was similar in the various regions of the brain tested except for the cerebellum, which had 40% higher specific activity than the whole brain. The results show that TS is widely distributed in adult rabbit brain, and, although the activity declines with age, it stabilizes at adult levels at 3 months of age.     

Kinetics of Neutral Amino Acid Transport Through the Blood-Brain Barrier of the Newborn Rabbit

Abstract: Since protein synthesis in the developing brain may, under certain conditions, be limited by amino acid availability, the present studies were undertaken to characterize the kinetics of large neutral amino acid transport through the blood-brain barrier (BBB) of the newborn rabbit. The K_m, V_max, and K_D of the transport of eight amino acids were determined by a nonlinear regression analysis of data obtained with the carotid injection technique. Compared with kinetic parameters observed for the adult rat, the K_m, V_max, and K_D of amino acid transport were all two- to threefold higher in the newborn. Albumin was found to bind tryptophan actively in vitro , but had no inhibitory effect on tryptophan transport through the newborn BBB. Glutamine was transported through the BBB of the newborn at rates severalfold higher than are seen in the adult rat. However, glutamine transport was not inhibited by high concentrations of N -methylaminoisobutyric acid (NMAIB), a model amino acid that is specific for the alanine-preferring or A-system present in peripheral tissues. In conclusion, these studies show that the BBB neutral amino acid transport system of the newborn rabbit has a lower affinity and higher capacity than does the BBB of the adult rat. Under conditions of high plasma amino acids, the increased capacity of the newborn transport system allows for a higher rate of amino acid transport into brain than would occur via the lower capacity system present in the adult rat brain.     

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.     

Glutamic Acid Decarboxylase in Sea Lamprey (Petromyzon marinus): Characterization, Localization, and Developmental Changes

Abstract: We have carried out assays for glutamic acid decarboxylase (GAD) in homogenates of brain and spinal cord from larval and adult sea lamprey ( Petromyzon marinus ). The enzyme had similar characteristics in both stages. Optimal pH was 6.8; optimal temperature was 27–30° C; K _m at 27°C was 5 mM. GAD activity was distributed uniformly along the length of the spinal cord. Specific activities for the larval cord and brain were 26 and 63 nm CO₂/mg protein/h. respectively. The specific activities for the adult cord and brain were 29 and 236 nm CO₂/mg protein/h, respectively. Thus, the activity of cord homogenates did not change significantly between larval and adult stages, but that of the brain increased about fourfold.     

Inhibition of Aldehyde Dehydrogenases in Rat Brain and Liver by Disulfiram and Coprine

Abstract: Rats were treated with either coprine or disulfiram and the inhibition of aldehyde dehydrogenase (ALDH) in liver and brain mitochondria was measured with acetaldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL), and succinate semialdehyde at different concentrations. The inhibition pattern was similar for both inhibitors, but the degree of inhibition was lower with disulfiram. The ALDH activity both in the liver and the brain was inhibited at low concentrations of acetaldehyde and DOPAL, but not with succinate semialdehyde. The high- K _m enzyme activities with acetaldehyde were not inhibited in liver and brain. The activity at high concentration of DOPAL was inhibited in the liver, but only slightly affected in the brain, suggesting the presence of a brain enzyme with an intermediate K _m value for DOPAL. In contrast with the results observed in viva, it was found that the high- K _m activities with acetaldehyde and DOPAL in brain mitochondrial preparations were more sensitive to the inhibitors in vitro than the low- K _m activities. Kinetic studies on ALDH preparations from brain and liver mitochondria suggested that acetaldehyde and DOPAL are metabolized by the same low- K _m ALDH.     

No effect of maternal niacin defficiency on niacin metabolism in newborn brain

The effects of maternal niacin and tryptophan deficiency on: (1) total niacin levels and (2) niacinnamide entry into brain, blood, and liver of newborn rabbits were studied. The deficient maternal diet produced a decreased concentration of the oxidized niacinamide-containing vitamers in the liver (73% of controls;P<0.05) but not in the brain of newborn rabbits. In both deficient and control newborn rabbits, the entry of [¹⁴C]niacinamide into brain and liver was saturable with an increasing [¹⁴C]niacinamide concentration in plasma. Also, the formation of [¹⁴C] NAD in brain and liver and [¹⁴C]niacinamide mononucleotide in blood was saturable. In vitro, the affinity of the saturable accumulation system for [¹⁴C]niacinamide in both newborn and adult rabbit brain slices was comparable (0.9 M). The avid saturable, uptake system for niacinamide in rabbit brain contributes to total niacin homeostasis in brain.     

Pyruvate Carboxylase Activity in Primary Cultures of Astrocytes and Neurons

Abstract: The activity of the pyruvate carboxylase was determined in brains of newborn and adult mice as well as primary cultures of astrocytes, of cerebral cortex neurons, and of cerebellar granule cells. The activity was found to be 0.25 ± 0.14, 1.24 ± 0.07, and 1.75 ± 0.13 nmol · min⁻¹· mg⁻¹ protein in, respectively, neonatal brain, adult brain, and astrocytes. Neither of the two types of neurons showed any detectable enzyme activity (i.e., < 0.05 nmol · min⁻¹· mg⁻¹). It is therefore concluded that pyruvate carboxylase is an astrocytic enzyme.     

Embryonic Serum Factors Required for Transdifferentiation of Chick Embryo Neuroretinal Cells in Culture

The accumulation of δ crystallin (chick lens marker) in cultures of 9 day chick embryo neuroretinal cells is strongly promoted by chick embryo extract (CEE) or foetal calf serum (FCS), but much less so by adult sera (horse, chicken and newborn bovine serum). The "transdifferentiation-promoting" (TP) activity of FCS is absent from dialysed FCS but is largely recovered in the initial dialysis medium (F_DM). Similarly, the initial dialysis medium from CEE (E_DM) shows strong TP activity, whereas that from chicken or from horse serum does not. We conclude that the proposed TP factor(s) is (are) of relatively low molecular weight. By contrast, horse serum contains macromolecular factor(s) able to inhibit the TP activity of E_DM or F_DM. Rapid loss of neuronal cells (including those expressing choline acetyltransferase activity) is also observed in media based on F_DM, though whether this effect is mediated by the proposed TP factor(s) has not been determined. The TP activity is not directly related to growth rate or cell density, since cultures in F_DM alone grow poorly yet still accumulate δ crystallin.     

Polypeptide Elongation Factors of the Developing Chick Brain

Abstract: The polypeptide elongation factors (EF-1_L, EF-1_H, and EF-2) of the developing chick brain were separated and purified by means of a combination of gel chromatographic methods. The molecular weight of EF-1_H of the chick brain ranged from 5 to 10 × 10⁵, and was different from that of the chick liver (about 7 × 10⁵). The molecular weight of other purified factors was about 5 × 10⁴ for EF-1_L. and 9.4 × 10⁴ for EF-2. High activities of polyphenylalanine (poly-Phe) synthesis per mg protein in the developing chick brain were observed between the 3rd embryonic week and the 1st post-hatch week and declined afterwards. On the other hand, the levels of both EF-1 and EF-2 per mg protein in the brain were observed to be high in an early embryonic stage, gradually declining afterwards to the adult level. The brain EF-1_L was a major component of EF-1 in an early embryonic stage, while EF-1_H became recognizable in the 3rd embryonic week. Moreover, the EF-1_H activities were found to be more than double with regard to the binding reaction and to be more than 10-fold as active in respect to poly-Phe synthesis in comparison with the activities of EF-1_L. It is proposed that the brain EF-1_H could be due to aggregates consisting of EF-1_L, a stimulatory factor, and other components.     

Reaction of Muscimol with 4-Aminobutyrate Aminotransferase

Abstract: The reaction of muscimol as amino donor substrate for GABA transaminase (GABA-T) has been studied using enzyme purified from rabbit brain. Enzyme activity was assayed by measuring the glutamate produced using glutamate dehydrogenase. Kinetic parameters determined at 37°C were for GABA, K _m (app) = 1.92 ± 0.24 m M , specific activity = 7.33 ± 0.27 μmol/min/mg ( k _cat= 13.7s⁻¹), and for muscimol, K _m (app) = 1.27 ± 0.15 m M , specific activity = 0.101 ± 0.009 μmol/min/mg ( k _cat= 0.19s⁻¹). Addition of muscimol to the enzyme caused the spectral changes associated with conversion of the pyridoxaldimine form to the pyridoxamine form, and the first-order rate constant for the reaction showed a dependence on muscimol concentration that followed saturation kinetics, with a K = 1.1 ±0.18 m M and k _max= 0.065 ± 0.004 s⁻¹ (19°C). The rate of spectral change observed on addition of muscimol to ornithine transaminase was extremely slow—at least an order of magnitude slower than that seen with GABA-T.     

IN VITRO PROTEIN SYNTHESIS ON FREE POLYRIBOSOMES ISOLATED FROM THE DEVELOPING MOUSE BRAIN

Abstract— Free polyribosomes were isolated at three ages (newborn, 2 weeks and 8 weeks) from the developing mouse brain. All three preparations were found to be highly polyribosomal in nature and were essentially identical with respect to their chemical composition and sedimentation properties. An estimate of the sedimentation coefficients of the first seven members of these polysome preparations yielded S °_20,w values of 76, 114, 146, 174, 196, 217 and 236. All three preparations were found to be very active when employed in in vitro protein synthesizing systems. An age-dependent response to the concentration of K⁺ was observed in the activities of the in vitro protein synthesizing systems. Optical K⁺ concentrations for the 0, 2 and 8 week old systems were 30, 50 and 65 mm, respectively. No such age dependence was observed when NH⁺₄ was used as the sole monovalent cation, with all systems exhibiting maximal activity at 50mm-NH⁺₄. The highest in uitro activities were consistently observed (at all three ages) when NNH⁺₄ was employed as the sole monovalent cation. Under optimal conditions, the newborn in vitro protein synthesizing system was observed to be approx 40% as active as either the 2 week or the 8 week systems which were equivalent in activity. The reduced activity of the newborn system appeared to be a function of both the polyribosomal and pH 5 enzyme preparations.     

Rapid estrogen regulation of DHEA metabolism in the male and female songbird brain

In the songbird brain, dehydroepiandrosterone (DHEA) is metabolized to the active and aromatizable androgen androstenedione (AE) by 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD). Thus, brain 3β-HSD plays a key role in regulating the steroidal milieu of the nervous system. Previous studies have shown that stress rapidly regulates brain 3β-HSD activity in a sex-specific manner. To elucidate endocrine regulation of brain 3β-HSD, we asked whether 17β-estradiol (E₂) regulates DHEA metabolism in adult zebra finch ( Taeniopygia guttata ) and whether there are sex-specific effects. Brain tissue was homogenized and centrifuged to obtain supernatant lacking whole cells and cell nuclei. Supernatant was incubated with [³H]DHEA and radioinert E₂ in vitro . Within only 10 min, E₂ significantly reduced 3β-HSD activity in both male and female brain. Interestingly, the rapid effects of E₂ were more pronounced in females than males. These are the first data to show a rapid effect of estrogens on the songbird brain and suggest that rapid estrogen effects differ between male and female brains.     

Glutamate Activates Phospholipase D in Hippocampal Slices of Newborn and Adult Rats

Abstract: Phospholipase D (PLD) is activated by many neuro-transmitters in a novel signal transduction pathway. In the present work, PLD activity was studied comparatively in hippocampal slices of newborn and adult rats. Basal PLD activity in adult rats was almost three times higher than in newborn rats. In newborn rats, L-glutamate and 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD) time- and concentrationdependently enhanced the formation of [³H]phosphatidylpropanol ([³H]PP) and of [³H]phosphatidic acid in the presence of 2% propanol. N -MethylD-aspartate and kainate (both 1 m M ) caused small, but significant increases (∼50%). whereas α-amino-3-hydroxy-5-methylisoxazole-4-propionate (100 μ M ) was ineffective. Maximally effective concentrations of glutamate (1 m M ) and of 1 S ,3 R -ACPD (300 μ M ) increased the PLD activity to almost 300% of basal activity; the EC₅₀ values were 199 and 47 μ M , respectively. Glutamate receptor antagonists, such as DL-2-amino-3-phosphonopropionic acid (AP3). DL-2-aminc-5-phosphonovalenic acid, and kynurenate (all 1 m M ) did not inhibit the glutamate-evoked increase of PP formation. In slices of adult rats, the response to 1 S ,3 R -ACPD was significant, but small, whereas glutamate was effective only in the presence of the glutamate uptake inhibitor L-aspartate-β-hydroxarnate. It is concluded that glutamate activates PLD in rat hippocampus through an AP3-resistant metabotropic receptor. This effect is subject to ontogenetic development, with one important factor being glutamate uptake.     

Metabolism of Prostaglandin D2 by Human Cerebral Cortex into 9α, 11β-Prostaglandin F2 by an Active NADPH-Dependent 11-Ketoreductase

Abstract: In homogenates of rat cerebral neocortex prostaglandin D₂ (PGD₂) was found to be quantitatively the main PG biosynthesized by a cytosolic PGD synthetase from en-dogenously released arachidonic acid. Amounts of 628 ng/g wet weight were found after 30-min incubation periods compared with basal levels of 2.3 ng/g wet weight. In human cerebral cortex, whether obtained at biopsy or postmortem, only small amounts of PGD₂ (4.5–11.7 ng/g wet weight/30 min) were formed. Furthermore, PGD₂, added to homogenates of human biopsy temporal cortex, was converted efficiently into 9α,11β-PGF₂ by a NADPH-dependent 11-ke-toreductase as has been reported in other human tissues (liver and lung). PGF_2α was determined directly as the fl-butylbo-ronate derivative. It became clear that 9α,11β-PGF₂ was formed in considerably greater amounts than PGF_2α and that other metabolites are also formed. These results can account for the low amounts of PGD₂ found in incubations of human brain tissue. The rat brain does not contain 11-ketoreductase activity. The present results indicate that the 9α, 11β-PGF₂ must be considered along with other eicosanoids in pathophysiological situations in brain.     

UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES

Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min⁻¹. mg⁻¹ cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a K_m of 220 μ m and a V _max of 7.9 nmol. min⁻¹. mg⁻¹ cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca²⁺-independent had a K_m for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.     

l-[35S]CYSTINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The uptake of [³⁵S]cystine at 37°C by synaptosomal fractions isolated from adult rat cerebrum can be divided into two components. About 60% of the uptake is due to binding to synaptosomal proteins while the remainder exists as a free amino acid pool. Chemical analysis of this soluble component indicates that considerable reduction of cystine to cysteine occurs with 75% or more of the labeled molecular species being cysteine. The process involved in the uptake into the soluble pool was composed of two saturable systems with apparent K _m values of 0.14 and 1.4 m m . The low K _m system was sodium and oxygen independent but inhibited by dinitrophenol. Dibasic amino acids, lysine, arginine and ornithine, did not inhibit cystine uptake. The characteristics of cystine uptake by synaptosomes from newborn brain are very similar to those of adult brain.     

Screening for Rabbit Brain Neuropeptide-metabolizing Peptidases. Inhibition of Endopeptidase B by Bradykinin Potentiating Peptide 9a (SQ 20881)

Abstract: Neutral thiol-activated peptidases present in the pH 5-soluble fraction of rabbit brain (separated by step-elution chromatography on diethylaminoethyl cellulose) were screened for the hydrolysis of bradykinin, Lysbradykinin, Met-Lys-bradykinin, angiotensin I, angiotensin II, substance P, luteinizing hormone-releasing hormone (LH-RH) and neurotensin by bioassay. The column effluent was monitored for bradykinin inactivation and arylamidase activity and combined in six pools on the basis of bradykinin inactivation. The pools were characterized by determining the peptide fragments and amino acids released from bradykinin with an amino acid analyzer. Pools 1 through 3 contained 80% of the kininase activity and essentially all of the endopeptidase A and B activity, whereas pools 4 through 6 accounted for 98% of the recovered arylamidase activity. Bradykinin, angiotensin I, angiotensin II and substance P were inactivated by all the pools, whereas LH-RH and neurotensin were inactivated by pools 3 and 4 and pools 3, 4 and 5, respectively. These data show that rabbit brain contains peptidases having some selectivity for the inactivation of neuropeptides. Endopeptidase B purified from pool 3 is inhibited by bradykinin-potentiating peptide 9a (BPP_9a' SQ 20881) (Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro), a competitive inhibitor of the hydrolysis of bradykinin ( K _m = 3.5 ± 10⁻⁵ m , K _i = 3 ± 10^−6m) which also completely inhibits the inactivation of LH-RH.     

Evidence for Two Distinct Forms of Fatty Acid Cyclooxygenase in Brain

Abstract: The enzymatic metabolism of [¹⁴C]arachidonic acid (AA) was studied with microsomes prepared from rabbit medulla. Prostaglandin E₂ (PGE₂) levels, measured either by radiochemistry or radioimmunoassay, rose rapidly and abruptly plateaued within 5 min, while prostaglandin F_2a (PGF_2a) levels continued to rise for 30 min. The rapid termination of PGE2 biosynthesis was not the result of limited cofactor, substrate, or product feedback inhibition, nor was it due to PGE₂-9-ketoreductase activity. Inhibition of the PGH₂→ PGE₂ isomerase by arachidonic acid or its metabolites could not explain the abrupt halt in PGE₂ biosynthesis. Proof for two separate cyclooxygenases comes from our observation that a preincubation of the brain microsomes with unlabeled AA eliminated PGE₂ biosynthesis while PGF2o production continued. Further evidence to suggest two cyclooxygenases in brain is derived from the observation that indomethacin inhibited PGE₂ production at concentrations that did not affect PGF_2a biosynthesis. These results suggest that one fatty acid cyclooxygenase is closely associated with PGH₂→ PGE₂ isomerase and readily undergoes autodestruction and the second cyclooxygenase is associated with a PGH₂→ PGF_2a reductase and is somewhat resistant to arachidonate-induced destruction and to nonsteroidal antiinflammatory agents.     

Cerebrovascular Accumulation and Increased Blood-Brain Barrier Permeability to Circulating Alzheimer's Amyloid β Peptide in Aged Squirrel Monkey with Cerebral Amyloid Angiopathy

Abstract: Senescent squirrel monkey is a valuable model to study pathogenesis of cerebrovascular amyloid angiopathy (CAA). Cerebrovascular sequestration and blood-brain barrier (BBB) permeability to ¹²⁵I-amyloid β(1-40) synthetic peptide (sAβ_1-40) were studied in adult versus aged squirrel monkey 1 h after a single intravenous injection. In aged monkey, the half-time of elimination of sAβ_1-40, t ^e_1/2, was prolonged by 0.6 h, the systemic clearance, Cl _SS, was reduced from 1.8 to 1.1 ml/min/kg, and the mean residence time of intact peptide in the circulation was increased by 1 h (45%). In adult monkey, cerebrovascular sequestration of intact sAβ_1-40 was significant, and the BBB permeability was 18.6-fold higher than for inulin. In aged monkey, the sequestration of intact sAβ_1-40 by cortical and leptomeningeal microvessels and the BBB permeability were increased by 5.9, 1.8-, and 2.1-fold, respectively, in the presence of an unchanged barrier to inulin. In brain parenchyma of aged animals, 76.1% of circulating sAβ_1-40 remained intact versus 45.7% in adult. We conclude that multiple age-related systemic effects, i.e., reduced body elimination and systemic clearance of sAβ_1-40, and reduced peripheral metabolism, may act in concert with BBB mechanisms, i.e., increased transendothelial transport and microvascular accumulation of blood-borne sAβ_1-40, and reduced brain metabolism to enhance the development of CAA.