Development and Regional Distribution of Methionine Synthetase in Rabbit Brain

The development and regional distribution of methionine synthetase (EC 2.1.1.13) in rabbit brain was determined. In adult rabbits, the specific activity (units per milligram protein) of methionine synthetase in cortex, cerebellum, brain stem, and corpus striatum was comparable to the specific activity in whole brain (0.5 units/mg). In the first few weeks of life, the specific activity of methionine synthetase in whole rabbit brain declined from a value of 1.1 units/mg at 1 day of age to 0.5 units/mg at 6–10 weeks. Two-year-old rabbits had 0.6 units/mg in whole brain. These results show that: (a) methionine synthetase is distributed widely in mammalian brain and (b) methionine synthetase activity in brain declines relatively little with development.     

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.     

Metabolism of Deoxyuridine in Rabbit Brain

Abstract: The metabolism of [³H]deoxyuridine by rabbit brain was investigated in vitro and in vivo . In vitro , brain slices from various regions of brain and from all age groups accumulated [³H]deoxyuridine from artificial CSF. Within the slices, a portion of the accumulated [³H]deoxyuridine was metabolized to [³H]deoxyuridine phosphate, with subsequent conversion to [³H]thymidine phosphate, and ultimately [³H]DNA. The percentage of the [³H]deoxyuridine phosphorylated and subsequently converted into [³H]DNA was highest at birth and declined to adult levels in 3-month-old rabbits. Thymidine, when added to the incubation medium with the [³H]deoxyuridine, was approximately 10 times as potent as unlabeled deoxyuridine in inhibiting the intracellular phosphorylation and conversion of [³H]deoxyuridine to [³H]thymidine phosphate in brain slices. In vivo , 2.5 h after intraventricular injection of [³H]deoxyuridine, over 90% of the [³H]deoxyuridine was cleared from the central nervous system at all ages. However, in both newborn and 3-month-old rabbits, approximately 40 and 12%, respectively, of the ³H remaining in brain was phosphorylated and converted to [³H]thymidine phosphates; and 11 and 4%, respectively, of the ³H remaining in brain was converted to [³H]DNA. These results show that both immature and mature rabbit brain is able to incorporate deoxyuridine into DNA. Thus, all the enzymes involved in this conversion, including thymidylate synthetase (EC 2.1.1.45), are present and active in brain throughout life.     

Thymidylate Synthetase as a Chemotherapeutic Target in the Treatment of Avian Coccidiosis*

SYNOPSIS. Thymidylate synthetase (E.C.2.1.1.45) has been demonstrated in unsporulated oocysts of Eimeria tenella. The properties of this enzyme have also been investigated in Tetrahymena pyriformis, as a protozoan model, and 7-day-old chick embryo, as a host model. The enzymes from E. tenella and chick embryo were inhibited by all concentrations of MnCl₂ and MgCl₂ tested. Tetrahymena pyriformis thymidylate synthetase was stimulated by low concentrations of both these cations but was inhibited by high concentrations. Subsequent data refer to chick embryo, E. tenella and T. pyriformis respectively: the apparent K_m was 5.89 μM, 5.94 μM, and 0.53 M for the substrate dUMP: and 5.13 μM, 1.10 μM and 4.65 μM, respectively for the cofactor N⁵N¹⁰-methylenetetrahydrofolate. The pH optimum for the enzyme from both chick embryo and T. pyriformis was 8.0, with Tris-HCl buffer; activity of E. tenella thymidylate synthetase was still increasing at pH 8.2. The E. tenella enzyme was found to have a molecular weight of 4.6–4.9 × 10⁵ daltons. The effects of nucleotides, inhibitors, and the omission of assay components on each enzyme are presented. Thymidylate synthetase from E. tenella is not greatly different from that of chick embryo, but does not resemble the enzyme from T. pyriformis. A case for using thymidylate synthetase as a chemotherapeutic target in the treatment of Eimeria infections remains. Indeed Eimeria may be considered as a model for infections caused by other protozoan parasites, such as Toxoplasma and Plasmodium, provided that suitable inhibitors can be found that are not toxic to the host.     

Development and Regional Distribution of Deoxyuridine 5''-Triphosphatase in Rabbit Brain

Abstract: The development and regional distribution of deoxyuridine 5'-triphosphatase (dUTPase) in rabbit brain was studied. After partial purification of the dUTPase activity, newborn brain and liver activities fell from V_max values of 1.0 and 6.9 nmol/mg protein/min, respectively, to adult levels of 0.1 (brain) and <0.01 (liver). The dUTPase activity was evenly distributed throughout the entire adult rabbit brain, and there was no difference in the K_m of dUTPase in newborn and adult brain (0.1 μ M ). The low level of dUTPase in adult brain and the lack of activity in adult liver were not due to inhibitors or phenylmethylsulfonyl fluoride-sensitive proteases. dUTPase in brain, as in other tissues, probably inhibits the misincorporation of uracil into DNA.     

Subcellular Distribution of Prolyl Endopeptidase and Cation-Sensitive Neutral Endopeptidase in Rabbit Brain

Abstract: The subcellular distribution of prolyl endopeptidase, and of cationsensitive neutral endopeptidase, two enzymes actively metabolizing many neuropeptides, was determined in homogenates of rabbit brain. The subcellular distribution of both enzymes was more similar to lactate dehydrogenase, a cytoplasmic enzyme marker, than to choline acetyltransferase, a synaptosomal marker. Only 35% of the activity of these two neutral endopeptidases was found in the crude mitochondrial fraction (P₂), the bulk of the remaining activity being associated with the high-speed supernatant. Prolyl endopeptidase and cation-sensitive neutral endopeptidase thus can be regarded as mainly cytoplasmic enzymes in the rabbit brain.     

Succinate Dehydrogenase, Monoamine Oxidase and Glutamine Synthetase in Developing Human Foetal Brain Regions

In human foetal brain ontogeny the cerebral activity of succinate oxidoreductase (EC 1.3.99.1), i.e. succinate dehydrogenase (SDH), is higher than the cerebellar activity. With rise in foetal body weight the activity in all the brain regions gradually declines. SDH in all the brain regions shows two high-activity periods, one at 20-35 g and another at 110-220 g body weight. The enzyme exhibits a craniocaudal pattern of development. At all times of gestation, L-glutamate:ammonia ligase (EC 6.3.1.2), i.e. glutamine synthetase, activity in the spinal cord and medulla is higher than in the other three regions. At 190 g body weight glutamine synthetase shows an activity peak in all brain regions. Monoamine:oxygen oxidoreductase (EC 1.4.3.4). i.e. monoamine oxidase (MAO), is present much before the onset of electrical activity. It develops caudocranially and exhibits a biphasic pattern of development in all the regions. It increases considerably in the medulla and the spinal core towards late gestational periods.     

Glutamine Synthetase of the Human Brain: Purification and Characterization

Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver.     

Ornithine Decarboxylase in Human Brain: Influence of Aging, Regional Distribution, and Alzheimer's Disease

Abstract: Although experimental animal data have implicated ornithine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in brain development and function, little information is available on this enzyme in normal or abnormal human brain. We examined the influence, in autopsied human brain, of postnatal development and aging, regional distribution, and Alzheimer's disease on the activity of ornithine decarboxylase. Consistent with animal data, human brain ornithine decarboxylase activity was highest in the perinatal period, declining sharply (by ∼60%) during the first year of life to values that remained generally unchanged up to senescence. In adult brain, a moderately heterogeneous regional distribution of enzyme activity was observed, with high levels in the thalamus and occipital cortex and low levels in cerebellar cortex and putamen. In the Alzheimer's disease group, mean ornithine decarboxylase activity was significantly increased in the temporal cortex (+76%), reduced in occipital cortex (−70%), and unchanged in hippocampus and putamen. In contrast, brain enzyme activity was normal in patients with the neurodegenerative disorder spinocerebellar ataxia type I. Our demonstration of ornithine decarboxylase activity in neonatal and adult human brain suggests roles for ornithine decarboxylase in both developing and mature brain function, and we provide further evidence for the involvement of abnormal polyamine system activity in Alzheimer's disease.     

Transglutaminase Activity in Rat Brain: Characterization, Distribution, and Changes with Age

The activity of transglutaminase was characterized in the rat brain. In adults, comparable levels of transglutaminase activity are present in all brain regions examined. The activity is present in all subcellular fractions, as studied by differential centrifugation, but the soluble fraction contains the highest specific activity. The endogenous activity (enzyme activity assayed in the absence of the exogenous substrate casein) is very low in all subcellular fractions, except in the synaptosomal fraction where its highest levels are about 40-60% of the activity assayed in the presence of casein. Furthermore, enzyme activity is present on the external surface of synaptosomes. In the soluble fraction, maximal activity can be detected between pH values of 9 and 10 when assayed in the presence of 5 mM CaCl2 (with half-maximal activity requiring 0.75 mM CaCl2) and 0.4 mM putrescine (with an apparent Km for putrescine of 0.1 mM). The activity can be partially inhibited by ZnCl2 (with an IC50 of 4.5 mM) and by AlCl3 (with an IC50 of 5.1 mM). In the cerebellum, where the full span of neuronal development can be studied after birth, the highest specific activity is observed just after birth, thereafter the activity starts to decline and by 14 days, after a reduction of about 65%, it reaches levels observed throughout life.     

Regional Distribution of Glutathione Peroxidase in the Adult Rat Brain

Glutathione peroxidase activity was measured in 10 areas of perfused adult rat brain with the use of a fluorometric assay coupled to NADPH oxidation. The caudate-putamen and the substantia nigra had the highest activities. Cortical areas and several nuclear areas had somewhat lower activity. Activity was lowest in a white matter structure (corpus callosum). High activity of glutathione peroxidase may be related to the need to reduce hydrogen peroxide arising in the course of monoamine metabolism.     

Changes in Rabbit Brain Cytosolic and Membrane-Bound Gangliosides During Prenatal Life

The present study deals with the developmental profile of cytosolic and membrane-bound gangliosides in rabbit whole brain from the 21st day of pregnancy, the time at which brain could be macroscopically recognized and handled, till birth. In this period of prenatal life the content of membrane-bound gangliosides showed a 2.5-fold increase, referred to fresh and dry brain weight and to membrane-bound protein; the content of cytosolic gangliosides reached a maximum at 21-22 days of pregnancy, and then underwent to birth a threefold diminution. The qualitative pattern of membrane-bound gangliosides, in the same period of life, was characterized by an increase of G_D1a and G_M1 (more marked for G_D1a), a decrease of G_T1a, G_T1b and G_Q1b and a constant level of G_D3 and G_D1b. At 21 days of pregnancy the most abundant gangliosides were G_T1b, and G_Q1b, followed by G_D1a and G_D1b; at birth it was G_D1a followed by G_T1b G_D1b, and G_M1 The qualitative pattern of cytosolic gangliosides closely resembled, during the entire period of prenatal life examined, that of membrane-bound gangliosides.     

The Regional Concentrations of S-Adenosyl-l-Methionine, S-Adenosyl-l-Homocysteine, and Adenosine in Rat Brain

Abstract: The concentrations of S -adenosyl- l -methionine (SAM), S -adenosyl- l -homocysteine (SAH), and adenosine (Ado) were determined in whole brain and rat brain regions by HPLC. The whole brain contains, respectively, 22 nmol, 1 nmol, and 64 nmol of SAM, SAH, and Ado per g of wet tissue. Their distribution indicated that SAM and SAH levels are highest in brainstem, whereas the Ado level is highest in cortex. With aging the SAM concentrations decrease in whole brain, brainstem, and hypothalamus (–25%) and SAH levels increase by 90% in striatum and by 160% in cerebellum, while Ado levels are increased in all regions by 100–180%.     

Inhibition of Rabbit Brain Prolyl Endopeptidase by N-Benzyloxycarbonyl-Prolyl-Prolinal, a Transition State Aldehyde Inhibitor

Prolyl endopeptidase cleaves peptide bonds on the carboxyl side of proline residues within a peptide chain. The enzyme readily degrades a number of neuropeptides including substance P, neurotensin, thyrotropin-releasing hormone, and luteinizing hormone-releasing hormone. The finding that the enzyme is inhibited by benzyloxycarbonyl-prolyl-proline, with a Ki of 50 microM, prompted the synthesis of benzyloxycarbonyl-prolyl-prolinal as a potential transition state analog inhibitor. Rabbit brain prolyl endopeptidase was purified to homogeneity for these studies. The aldehyde was found to be a remarkably potent inhibitor of prolyl endopeptidase with a Ki of 14 nM. This Ki is more than 3000 times lower than that of the corresponding acid or alcohol. By analogy with other transition state inhibitors, it can be assumed that binding of the prolinal residue to the S1 subsite and the formation of a hemiacetal with the active serine of the enzyme greatly contribute to the potency of inhibition. The specificity of the inhibitor is indicated by the finding that a variety of proteases were not affected at concentrations 150 times greater than the Ki for prolyl endopeptidase. The data indicate that benzyloxycarbonyl-prolyl-prolinal is a specific and potent inhibitor of prolyl endopeptidase and that consequently it should be of value in in vivo studies on the physiological role of the enzyme.     

Regional Distribution of Homocysteine in the Mammalian Brain

The regional distribution of L-homocysteine (Hcy) was determined in brains from mouse, rat, guinea pig, and rabbit, using a sensitive radioenzymatic assay. Large interspecies variations in the Hcy content in various parts of the brain were observed, but cerebellum contained the highest amount in all species investigated. In the rat the amount of Hcy in cerebellum (6.4 nmol/g) was about sixfold higher than in most other parts of the brain, whereas in the mouse and guinea pig the amount in cerebellum (about 1 nmol/g) was only twofold higher than in the other brain regions. There was a remarkably high level of Hcy in all regions of the rabbit brain (4-10 nmol/g); the highest concentration was found in the cerebellar white matter. In this species the amount of Hcy in all brain regions examined exceeded that in the liver.     

Arachidonoyl-Coenzyme A Synthetase and Nonspecific AcylCoenzyme A Synthetase Activities in Purified Rat Brain Microvessels

Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 microM and 5.8 microM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol X min-1 X mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 microM unlabelled arachidonic acid, with a Ki of 7.1 microM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 microM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 microM unlabelled palmitic acid, with a Ki of 5.0 microM and to a much lesser extent by arachidonic acid (Ki, 23 microM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40 degrees C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

兔脑种植VX2肿瘤动物模型的建立

目的建立兔VX2肿瘤脑内种植动物模型,观察其生长特性。方法采用兔脑内VX2肿瘤组织块种植法将VX2肿瘤组织块种植入24只成年New Zealand大白兔右侧大脑皮质内,用B超检测肿瘤的生长情况,在实验兔在肿瘤种植后第13、171、9、21、232、5天取材,进行组织学观察。并观察实验兔在种植VX2肿瘤后的生存期及出现厌食、偏瘫等神经系统体征和死亡的时间。结果VX2肿瘤组织块种植入脑内后荷瘤兔的中位生存期为24.5 d,平均生存期为24.8 d,肿瘤体积随种植后的时间在对数坐标中接近一条直线。VX2肿瘤种植入兔脑17 d后血供较丰富、呈鱼肉状生长,与正常脑组织边缘界限不清楚,第17~19天肿瘤中心出现坏死并出现腹腔内转移。光镜下从VX2肿瘤种植后第17天开始肿瘤细胞向正常脑组织浸润,并形成瘤巢,瘤周脑组织形成水肿带。结论在缺乏兔源性脑肿瘤的情况下,采用兔VX2肿瘤组织块颅内种植能较好模拟颅内肿瘤生长,为对脑肿瘤的某些实验研究提供条件。     

S-腺苷甲硫氨酸合成酶的组成型表达、产物纯化及鉴定

将大肠杆菌(E.coli K12) S 腺苷甲硫氨酸合成酶(SAMS)基因克隆至质粒pBR322中,获得的重组质粒pBR322-SAMS转入大肠杆菌JM109菌株,构建了能高效组成型表达SAMS的重组菌E.coli JM109 (pBR322-SAMS)。将重组大肠杆菌破碎后上清液经20%~40%硫酸铵分级盐析、Phenyl-Sepharose Fast Flow疏水层析和Q Sepharose Fast Flow离子交换层析,即可得到纯度提高5倍,比活为48.7 μ/mg的SAMS,三步纯化的总回收率为62%,纯度达到92%。SAMS表达量为1 176μ/L,占到菌体可溶性总蛋白的20%。重组酶的最适反应pH为8.5,4℃下在pH 7.5的缓冲液中保温10h酶活性几乎不改变。重组酶反应的最适温度为55℃ ,酶活性稳定的温度范围为20~35℃。重组酶的KmL Met为0.22mmol/L,Vmax L-Met为1.07mmol/(L·h),Km ATP为0.52 mmol/L,Vmax ATP为1.05 mmol/( L·h)。