Distribution of the thy-1 antigen in cellular and subcellular fractions of adult mouse brain.

By using a cytotoxicity inhibition assay employing AKR anti-C3H thymocyte antiserum, we have determined the degree of expression of the thy-1 antigen in fractions of adult mouse brain. As expressed as cytotoxicity inhibitory capacity per mg protein with C3H whole brain arbitrarily assigned a value of 1.0, the following values were found: C3H cerebral cortex, 5.8; C3H cerebral cortex synaptosomes, 2.5: C3H whole brain myelin, 0.65; C3H cerebral cortex neurons, 0.16; and C3H cerebral cortex mitochondria 0.10. Neither C1300 neuroblastoma cells nor any AKR neural fraction had detectable levels of thy-1. The findings indicate that the thy-1 antigen is found mainly in mouse cerebral cortex and in synaptosomal fractions, whereas myelin fractions contain lower but perhaps significant amounts of thy-1. Cerebral cortex neurons, isolated by a method requiring a 90-min mild trypsinization at 37 degrees C, did not display significant amounts of the thy-1 antigen. These results lend themselves to further study in the area of differentiation and development of central nervous system components and in the area of central nervous system immunopathology.     

Activity of glutathione reductase and glutathione-S-transferase in rabbit brain tissue in the long-term post-traumatic period

The glutathione-reductase and glutathione-S-transferase activities in the cortex and brain stem tissues of rabbits with post-traumatic epileptic reality (1 year after the light brain injury) was defined. An increase of glutathione-reductase activity in the cortex microsomal and stem mitochondrial fractions, and increase of glutathione-S-transferase activity in cortex and stem mitochondrial fractions was obtained. The conclusion is made that the activation of the anti-oxidant glutathione fermentative system is a long-term metabolic CNS adaptation in the case while mitochondrial oxidation and oxidative phosphorilation are disturbed.     

Increased Cyclic AMP-Dependent Protein Kinase Activity in Postmortem Brain from Patients with Bipolar Affective Disorder

Previous observations of reduced [3H]cyclic AMP binding in postmortem brain regions from bipolar affective disorder subjects imply cyclic AMP-dependent protein kinase function may be altered in this illness. To test this hypothesis, basal and stimulated cyclic AMP-dependent protein kinase activity was determined in cytosolic and particulate fractions of postmortem brain from bipolar disorder patients and matched controls. Maximal enzyme activity was significantly higher (104%) in temporal cortex cytosolic fractions from bipolar disorder brain compared with matched controls. In temporal cortex particulate fractions and in the cytosolic and particulate fractions of other brain regions, smaller but statistically nonsignificant increments in maximal enzyme activity were detected. Basal cyclic AMP-dependent protein kinase activity was also significantly higher (40%) in temporal cortex cytosolic fractions of bipolar disorder brain compared with controls. Estimated EC50 values for cyclic AMP activation of this kinase were significantly lower (70 and 58%, respectively) in both cytosolic and particulate fractions of temporal cortex from bipolar disorder subjects compared with controls. These findings suggest that higher cyclic AMP-dependent protein kinase activity in bipolar disorder brain may be associated with a reduction of regulatory subunits of this enzyme, reflecting a possible adaptive response of this transducing enzyme to increased cyclic AMP signaling in this disorder.     

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.     

Sex hormone receptors in the hypothalamus and their role in the sexual differentiation of the brain of male rats

The content of receptors to estradiol and testosterone was determined in cytoplasmic and nuclear fractions of hypothalamus and brain cortex of male rats in the early postnatal period. Receptors to both estradiol and testosterone were revealed in cytosol and nuclear fractions, with the decrease in their concentration observed from days 1 to 5. The data obtained demonstrate that receptors to sexual hormones take part in the brain differentiation and regulation of hypophysis gonadotropic function by male or female type.     

Epidermal Growth Factor in Synaptosomal Fractions of Mouse Cerebral Cortex

Using a specific and sensitive epidermal growth factor radioimmunoassay (EGF-RIA) we measured EGF concentrations in whole brain, cerebral cortex, and cerebral cortical synaptosomal (pinched-off presynaptic nerve terminals) fractions of 26-day-old mouse brain. The relative EGF concentration in synaptosomal fractions was significantly greater than the growth factor concentrations in whole brain or cerebral cortex. Intracerebral injection, in an amount of EGF, several-fold greater than whole brain EGF content, did not appreciably increase synaptosomal EGF concentration, suggesting that no artifact was involved. The high synaptosomal EGF content suggests a neurotransmitter or a neuromodulator role for EGF in the CNS.     

Experimental alcoholism in rats. Effect of acute ethanol intoxication on the in vitro incorporation of ( 3 H)leucine into neuronal and glial proteins

Abstract— Ethanol administered in vivo or in vitro during incubation of brain slices was studied with respect to its effect on brain protein synthesis. In the in vivo series the rats were given a single intraperitoneal injection of ethanol 3 h before death. Slices of cerebral cortex and liver were incubated in isotonic saline media containing [³H]leucine. Amounts of free and protein-bound radioactivity were determined. Subcellular fractions and fractions enriched in neuronal perikarya and in glial cells were prepared from cortical slices subsequent to incubation, and the specific radioactivity determined for each cell type. The incorporation of [³H]leucine into brain proteins was inhibited while incorporation into liver proteins was stimulated in ethanol-treated rats. The levels of TCA-soluble radio-activity, however, did not differ between the ethanol group and the controls. In the fractionated material from cerebral cortex, the specific radioactivity in the neuronal fraction was unaffected by ethanol, while the radioactivity in the glial fraction was significantly depressed. In vitro administration of ethanol induced a non-linear response in both brain and liver, with depression of leucine incorporation into proteins of cerebral cortex at all concentrations used. When brain slices were exposed to ethanol in vitro, in concentrations corresponding to the in vivo experiments, a similar reduction of the leucine incorporation into the glial fraction was obtained. Incorporation of leucine into subcellular fractions from whole brain cortex was also investigated. The specific sensitivity of the glial fraction to ethanol is discussed in relation to the involvement of the different cell types with transport processes in the brain.     

Regional and subcellular distribution of choline acetyltransferase in the brain of rats

—Homogenates of corpus striatum, cerebral cortex and hypothalamus excised from rat brain were fractionated on discontinuous Ficoll and sucrose density gradients, and the distribution of choline acetyltransferase (ChAc) in the mitochondrial and synaptosomal fractions was determined. In the hypothalamic and cortical regions the fractions enriched in synaptosomes showed much higher activity of ChAc than those containing mainly mitochondria. On the other hand, the corpus striatum showed an equal distribution of ChAc activity in those two fractions. The localization of ChAc was also studied in the postnuclear supernatants obtained from three brain regions, using continuous sucrose density gradients. The distribution of ChAc was compared to that of monoamine oxidase (MAO), potassium and protein. When the pellets obtained from the fractions collected from the gradient were suspended in sucrose, the peak of ChAc activity was close to that of MAO in all three brain regions. When 0.1 mm EDTA +1% butanol was used in order to liberate the occluded form of ChAc, the maximum liberation occurred in lighter fractions, resulting in a shift of the activity peak toward the top of the gradient. This was found with fractions from hypothalamic and cortical regions. In the striatum, the liberated ChAc remained in the same fractions as the occluded enzyme. The results indicate that ChAc is liberated only in those fractions where it is present in synaptosomes. In agreement with the results on the discontinuous gradients this occurs in particles of lower density than mitochondria in cortex and hypo-thalamus, but in particles of similar density to mitochondria in the corpus striatum, indicating regional differences in the distribution of ChAc in the brain. K+ containing particles centrifuged in less dense fractions than those containing ChAc, indicating that synaptosomes are heterogeneous with respect to these two marker substances.     

Binding of Paroxetine to the Serotonin Transporter in Membranes from Different Cells,Subcellular Fractions and Species

The binding of [³H]-paroxetine to membrane serotonin transporter (SERT) has been studied in membranes from different sources and subcellular fractions. From rat were membranes from venous blood platelets, brain total cortex, brain microsomes, brain crude and purified synaptosomes. Membranes were obtained from venous blood platelets from human volunteers and from brain cortex tissue from neurosurgery (cerebral lobectomies following craniocerebral injuries). The main finding was that the K _D of paroxetine binding to the SERT was the same for platelet and nerve ending (synaptosomal) membranes. That parameter was significantly lower in membranes from brain microsomes and cortex total tissue. No species related difference was found, where comparison was possible, between human and rat tissue. The equality of K _D of paroxetine binding to blood platelet membranes and to membranes from nerve endings appears to encourage the use of such membranes as a model for brain SERT. Binding at two different temperatures for several of the fractions suggests that paroxetine–SERT interaction is entropy-driven.     

Subcellular distribution of carbonic anhydrase and Na+,K+-ATPase in the brain of the hyt/hyt hypothyroid mice

Activities of carbonic anhydrase and Na⁺,K⁺-ATPase in tissue homogenates and in subcellular fractions from different brain regions were studied in inherited primary hypothyroid (hyt/hyt) mice. The body weight, the weight of different brain regions, and the plasma thyroxine and triiodothyronine levels of hyt/hyt mice were significantly lower than those of the age-matched hyt/+ controls. In tissue homogenates of cerebral cortex, brain stem and cerebellum of hypothyroid mice, the activity of carbonic anhydrase (units/mg protein) was 59.2, 57.6, and 43.2%, and the activity of Na⁺,K⁺-ATPase (nmol Pi/mg protein/min) was 73.7, 74.4 and 68.7%, respectively, of that in corresponding regions of euthyroid littermates. The decrease in enzyme activity in tissue homogenates was also reflected in different subcellular fractions. In cerebral cortex and brain stem, carbonic anhydrase activity in cytosol, myelin and mitochondrial fractions of hypothyroid mice was about 45–50% of that in euthyroid mice, while in cerebellum the carbonic anhydrase activity in these subcellular fractions of hyt/hyt mice was only 33–38% of that in hyt/+ controls. Na⁺,K⁺-ATPase activity in myelin fraction of different brain regions of hyt/hyt mice was about 34–42% of that in hyt/+ mice, while in mitochondria, synaptosome and microsome fractions were about 44–52, 46–53, and 66–68%, respectively of controls. These data indicate that the activity of both carbonic anhydrase and Na⁺,K⁺-ATPase was affected more in the myelin than other subcellular fractions and more in the cerebellum than cerebral cortex and brain stem by deficiency of thyroid hormones. A reduction in the activity of transport enzymes in brain tissues as a result of thyroid hormone deficiency during the critical period of development may underlie permanent nervous disorders in primary hypothyroidism.     

Localization and causes of lipid peroxidation disorders in the rat cerebral cortex in the early stages of hereditary retinal degeneration]

It is established that previously observed increased rate of the induced lipid peroxidation in brain tissue of rats with hereditary retinal degeneration as compared with normal rats is due to the change of the rate of this process in the microsome cortex brain fraction and was not observed in the mitochondrial-synaptosomal and nuclear fractions. The content of nonheme iron ions in microsome cortex brain fraction of the Campbell rats is decreased by 35% and of the Fe ion was in the reduced form as compared with the Wistar rats. The ratio of Fe2+/Fe3+ in this fraction of the Campbell rats will be 5.21; Wistar rats--0.51. The increase of the reduced form of the Fe ion may be a result of the increased rate of the glucose-6-phosphate dehydrogenase activity in cortex brain tissue of the Campbell rats. We accept change of the content and the forms of the Fe ions in the microsome cortex brain fraction as a cause of the increased rate of induced lipid peroxidation in brain of the Campbell rats. All the observed phenomena are manifested at the early stage of life and indicated that different metabolic disorders can be observed in the Campbell rats not only in the retina and eye pigment epithelium but also in the brain tissue.     

Gangliosides in the human brain development and aging.

In this study, brain gangliosides in prenatal and postnatal human life were analyzed. Immunohistochemically, the presence of "c"-pathway of gangliosides (GQ1c) in embryonic brain was only recorded at 5 weeks of gestation. Biochemical results indicated a twofold increase in human cortex ganglioside concentration between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except cerebellar cortex, which was characterized by increasing GT1b. In this developmental period, GD3 was found to be localized in the ventricular zone of the cortical wall. After birth, GD1b ganglioside in neuropil of granular cell layer corresponding to growing mossy fibers was expressed in cerebellar cortex. Between birth and 20/30 years of age, a cerebral neocortical difference of ganglioside composition was observed, characterized by lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In cerebellar cortex, GD1b and GT1b fractions decreased with aging.     

Na+,K+-ATPase activity of the subcellular fractions of the rat brain in aging

The Na+, K+-ATPase activity in the homogenate and in subcellular fractions of different parts of the brain of adult and old rats was studied in comparison. The content of cholesterol in the above fractions was also determined. In old age the Na+, K+-ATPase activity in the homogenate and microsomal fraction of the cerebral hemispheres' cortex decreases, while the Mg2+-ATPase activity in the cortex microsomal fraction increases. The age-related Na+, K+- and Mg2+-ATPase activity in the myelin of the stem in the synaptic plasma membranes of hemispheres and the brain stem remains unchanged whereas in the myelin fraction of hemispheres it grows. The content of cholesterol in the brain of old rats as compared with adult ones increases in the microsomal fraction and remains unchanged in synaptic membranes. The possible role of age-related modification of lipid component of plasma membranes in the above changes of Na+, K+-ATPase activity is discussed.     

Regional and Subcellular Distribution of Thy-1 in Human Brain Assayed by a Solid-Phase Radioimmunoassay

A solid-phase radioimmunoassay, specific for the monomeric form of human Thy-1, was developed and used for quantitation of the Thy-1 antigen in human brain tissue. Determination of Thy-1 in homogenates of 12 anatomically defined brain regions showed that Thy-1 is present throughout the human brain. However, significant variation was found in the expression of the glycoprotein in different regions. Thy-1 appears to be generally enriched within gray matter: caudate nucleus, cerebral cortex, and putamen were found to contain the highest Thy-1 concentration (approximately 2.5 micrograms Thy-1/mg protein). Interestingly, the cerebellar cortex contained only 25% of the Thy-1 concentration of cerebral gray matter. Cerebral subcortical white matter contained half the amount of Thy-1 compared to cerebral cortex. Determination of Thy-1 in subcellular fractions prepared from human brain biopsy tissue indicated that the highest relative concentration of Thy-1 is associated with synaptosomal membranes and myelin/axonal membrane fractions.     

Aldehyde dehydrogenase activity and level of dopamine in certain sections of the brain of rats preferring and refusing ethanol

Aldehyde dehydrogenase activity (KF 1.2.1.3) of cytosol fractions of brain structures (hypothalamus, midbrain and new cortex) as well as dophamine content in these structures were studied in comparative aspect in rats preferring and rejection ethanol. It has been shown that there were two isoforms of aldehyde dehydrogenases (aldehyde dehydrogenase 1 and aldehyde dehydrogenase 2) in cytosol fractions of all investigated brain structures of animals preferring ethanol while only aldehyde dehydrogenase 2 has been found in the new cotex of rats rejecting ethanol. Thus, aldehyde-dehydrogenase activity is higher in the animals preferring ethanol than in those ones rejecting ethanol. Content of dophamine in the rats preferring ethanol is higher than in those ones rejecting ethanol both in the hypothalamus and new cortex. Differences between the studied groups of animals can underlie the pathologic attraction to alcohol.     

Influence of exogenous gangliosides (GM1, GD1a, GMix) on a Ca-activated Mg-dependent ATPase in cellular and subcellular brain fractions of the djungarian dwarf hamster (Phodopus sungorus)

The influence of 150 nM exogenously-added gangliosides (G_M1, G_D1a, G_Mix) on a Ca²⁺-activated, Mg²⁺-dependent ATPase was investigated in cellular and subcellular fractions (P1-fraction, synaptosomal fraction, synaptic membranes) from whole brain, cortex, cerebellum and brain stem of the djungarian dwarf hamster (Phodopus sungorus). Gangliosides are effective at this concentration in stimulating the enzyme activity in all fractions from whole brain. Inhomogenous results (stimulation, inhibition and no effects), however, were obtained in the different individual brain regions.     

Regional and subcellular distributions of brain neurotensin.

The regional and subcellular distribution of neurotensin were determined using a newly developed radioimmunoassay for this central nervous system tridecapeptide. Neurotensin immunoreactivity in calf brain is high in the hypothalamus and basal ganglia, unevenly distributed through the cerebral cortex, and low in cerebellar cortex and cerebral white matter. Subcellular fractionation of rat hypothalamus reveals a strong association of neurotensin immunoreactivity with synaptosomal and microsomal fractions. These data, taken along with previously described high affinity selective brain membrane receptor binding, are consistent with a neurotransmitter candidate role for neurotensin in the brain.     

NONHISTONE NUCLEAR PROTEINS OF RAT BRAIN

Abstract— The rat brain was dissected into cerebral cortex, cerebellum and the remaining regions. From the nuclei, isolated from these three brain sections, were extracted two fractions of nuclear sap proteins (proteins soluble in 014 M NaCl and proteins soluble in 01 M Tris-HCl buffer pH 7-6) and two fractions of nonhistone chromosomal proteins (one soluble in 0-35 M NaCl and one which is not soluble at this salt concentration). Each of these four fractions of the nonhistone nuclear proteins was further separated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the studied fractions of nuclear proteins are qualitatively identical regardless of the brain section from which the analysed protein fraction was isolated. In addition, there arc no qualitative differences in the electrophoretic patterns of nonhistone chromosomal proteins which are and which are not soluble in 0-35 M NaCl. In contrast to the qualitative similarity of the electrophoretic patterns of proteins from different sections of the brain, the amount of the nonhistone nuclear proteins is characteristic for each studied brain section. The ratio of the total nonhistone nuclear proteins to DNA is highest in the brain cortex and lowest in the cerebellum. The most expressed difference between the nuclei is in the ratio of the nonhistone chromosomal proteins soluble in 0-35 M NaCl to DNA. This ratio is 0-52 in the cortex. 0-38 in the mixture of noncortical and noncerebel-lar regions and only 0-18 in the cerebellum. The amount of the three fractions of nonhistone nuclear proteins in the nuclei of individual brain sections is proportional to the activity of the genome in these nuclei. The only exception are the nonhistone chromosomal proteins which are not soluble in 0-35 M NaCl. These proteins and the histones are present in the same amounts in nuclei isolated from all three studied sections of the brain. The results support a proposal that the nonhistone nuclear proteins are involved in the expression of the genetic activity of the cell, without the majority of the proteins in any of the four fractions being the specific regulatory molecules.     

Cytoplasmic casein kinase 2 and its substrate proteins in the brain in Alzheimer's disease]

The absence of casein kinase 2 on blots of temporal cortex extracts from Alzheimer's disease patients (ADP) was shown using antiserum to casein kinase 2. Casein kinase 2 activity towards endogenous substrates and casein is 2-5 times less in ADP brain in comparison to normal controls. The fractions of heparin-binding proteins, containing protein substrates for phosphorylation, were isolated from temporal cortex of ADP and normal controls. The total amount of heparin-binding proteins from ADP brains is less than from control brains, and the polypeptide composition of these fractions is much more poop.     

Distribution of the Glucose Transporter in the Mammalian Brain

We used [3H]cytochalasin B as a specific ligand to study the glucose transporter of the following tissue preparations: (a) microvessels derived from the cerebral cortex and cerebellum of the rat and pig, (b) particulate fractions of the cerebral cortex and cerebellum of the rat and pig, (c) lateral, third, and fourth ventricular choroid plexus of the pig, and (d) synaptosomes from the pig cerebral cortex. Specific, D-glucose-displaceable binding of [3H]cytochalasin B was present in all the preparations studied. This binding was saturable and displayed the kinetics of a single class of binding sites, similar to the glucose transporter found in other mammalian tissues. The density of the glucose transporter was much higher in cerebral and cerebellar microvessels and choroid plexus than either in crude particulate fractions of the cerebrum and cerebellum or in cerebral synaptosomes. These findings agree with the physiologic function of brain microvessels that transport glucose, not only for their own use, but also for the much greater mass of the entire brain. In the pig, the density of the glucose transporter in cerebral microvessels was significantly higher than in cerebellar microvessels. Irreversible photoaffinity labeling of the glucose transporter of synaptosomal membranes with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis demonstrated a single region of radioactivity that corresponded to a molecular mass of 60,000-64,000 daltons.