Human chromosome structure as revealed by an immunoperoxidase staining procedure

Morphological alterations accompanied by an increase of the glia-specific protein S-100 have been shown to occur in a glial cell line (138 MG) of a human brain tumour if serum is removed from the culture medium. The glial S-100 protein was immunologically indistinguishable from S-100 present in human brain.     

Morphological alterations and increase of S-100 protein in cultured human glioma cells deprived of serum

Morphological alterations accompanied by an increase of the glia-specific protein S-100 have been shown to occur in a glial cell line (138 MG) of a human brain tumour if serum is removed from the culture medium. The glial S-100 protein was immunologically indistinguishable from S-100 present in human brain.     

WATER-SOLUBLE AND PENTANOL-EXTRACTABLE PROTEINS IN HUMAN BRAIN NORMAL TISSUE AND HUMAN BRAIN TUMOURS, WITH SPECIAL REFERENCE TO S-100 PROTEIN

Disc electrophoretic separation of water-soluble and pentanol-extractable protein from normal human brain and human brain tumours (glioblastoma, neurinoma and medulloblastoma) on 10 per cent polyacrylamide gels showed minor differences between tissues. After disc electrophoresis ependymomal tumour cells contained high concentrations of a rapidly migrating anodic protein fraction which was immunologically distinct from S-100 protein. After electrophoresis of normal brain grey matter in a continuous buffer system, a rapidly migrating anodic protein fraction which was immunologically distinct from S-100 protein was found, and this protein fraction had a similar relative mobility to that of ependymomal tumour cells. This protein fraction was present to a low extent in human normal white matter, but absent from neurinoma and glioblastoma. In a continuous buffer system at least two separable protein fractions, immunologically equivalent to S-100 protein, were observed in normal human brain. The more anodic of these two fractions was shown to be present in relatively high amounts in neurinomas, and may be of Schwann cell origin. Additional S-100 protein could be extracted from residual material remaining after removal of water-soluble proteins; 2.8-10 per cent of the water-soluble S-100 in normal material, and 0.1-0.6 per cent of that present in tumour material, was extractable from the water-insoluble residue by pentanol.     

Regional changes in CNS levels of the S-100 and 14-3-2 proteins during development and aging of the mouse

The levels of the S-100 and 14-3-2 proteins were determined in a number of regions of mouse brain at intervals from 1 day to 30 months of age. Both S-100 and 14-3-2 were found in measurable amounts as early as the first day of postnatal age but did not begin to accumulate rapidly in the forebrain, brain stem and cerebellum of the mouse brain until some time between the 7th and 14th days. From days 14 to 28 the levels of S-100 and 14-3-2 in each region continued to increase rapidly with the exception of the forebrain where the rate of accumulation of S-100 appeared to lag considerably behind that in the other regions. The proteins continued to accumulate at a rapid rate until approximately 6 months of age. From 6 to 30 months of age, the levels of 14-3-2 remained relatively stable in cerebellum, hippocampus and hypothalamus and appeared to decrease slightly in striatum and cerebral cortex. In the case of S-100, the level of the protein increased in all regions of brain from 6 to 30 months but the increase was most pronounced in the hippocampus, hypothalamus and striatum. The principal conclusion derived from this study is that the biochemical development and aging of the central nervous system are regionally selective processes.     

PURIFICATION OF SOLUBLE GLYCOPROTEINS FROM HUMAN NERVOUS TISSUE AND LIVER

—The isolation of some water-soluble, 50% methanol-soluble glycoproteins from human normal brain, human ependymoma and human liver is reported. One of them (AM protein) has a similar, or even identical, electrophoretic mobility to brain-specific protein S-100 under certain electrophoretic conditions. Although bands of identical mobility are found in both brain and liver samples, we lack experimental evidence, at the present stage of this work, to draw conclusions on the identity (or relatedness) of such proteins. On the other hand, the brain-soluble glycoproteins appear to be antigenically different from brain protein S-100, from brain α₂-glycoprotein and from GP 350 glycoprotein.     

Modulation of brain protein phosphorylation by the S-100 protein

The effects of the nervous system specific protein, S-100, on protein phosphorylation in rat brain is examined. The S-100 protein inhibits the phosphorylation of several soluble brain proteins in a calcium dependent fashion. The most potent effect exhibited by S-100 was on the phosphorylation of a protein having a molecular weight of 73,000. The data suggest that the calcium binding S-100 protein, for which a function has not yet been assigned, may modulate calcium dependent phosphorylation of selected brain proteins.     

Purification and characterization of adipose tissue S-100b protein

We have purified S-100 protein from bovine brain using Ca2+-dependent affinity chromatography on N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)-Sepharose (Endo, T., Tanaka, T., Isobe, T., Kasai, H., Okuyama, T., and Hidaka, H. (1981) J. Biol. Chem. 256, 12485-12489). By essentially the same procedure, W-7-Sepharose binding protein has been purified to apparent homogeneity from bovine abdominal adipose tissue. Electrophoretically, the purified protein from adipose tissue co-migrated with brain S-100b protein both in the presence and absence of sodium dodecyl sulfate and the protein was indistinguishable from brain S-100b region in terms of amino acid composition, two-dimensional tryptic peptide mapping and reactivity with anti-brain S-100b serum. Immunohistochemical analysis confirmed the existence of S-100b protein in the adipose cell where the protein seems to be located in both the nucleus and cytoplasm. Thus, the results indicate that the adipose cells contain the protein possibly identical with brain S-100b protein. In addition, the contents of S-100b protein in various rat tissues were measured by enzyme immunoassay method using the anti-bovine brain S-100b serum. Significant amounts of S-100b protein were found not only in the adipose tissue but also in the peripheral tissue such as trachea and skin. These observations suggest that S-100b protein should no longer be considered as a protein specific to nervous tissues.     

Regional distribution of S-100a0 (αα), S-100a (αβ) and S-100b (ββ) in the bovine central nervous tissue determined with a sensitive enzyme immunoassay system

A sensitive sandwich-type enzyme immunoassay system for separate measurement of 3 forms of bovine S-100 protein, S-100a₀ (αα), S-100a (αβ) and S-100b (ββ), was developed by the use of purified antibodies to the α or the β subunit of bovine S-100 protein. The assay system consisted of polystyrene balls with immobilized antibody (anti-α for S-100a₀ and S-100a assays, and anti-β for S-100b assay) F(ab′)₂ fragments and antibody (anti-α for S-100a, assay, and anti-β for S-100a and S-100b assays) Fab′ fragments labeled with β-d-galactosidase from Escherichia coli. The minimum measurable sensitivity of each assay was less than 10 pg/assay tube. The assay system for S-100a cross-reacted little with S-100a₀ and S-100b. The assay systems for S-100a₀ and S-100b cross-reacted (10 and 17%, respectively) with S-100a which contains α and β subunits in the molecule. However, levels of S-100a₀, S-100a and S-100b in the soluble extract of bovine brain could be determined by correcting the cross-reacted S-100a to the assays of S-100a₀ and S-100b. Various regions of bovine central nervous tissue were found to contain 0.3–1 μg of S-100a₀, 4–14 μg of S-100a, and 8–30 μg of S-100b per mg soluble protein. The percent concentrations of three forms of S-100 protein in the cerebral cortex were about 3, 38, and 59, for S-100a₀, S-100a, and S-100b, respectively, and those in the cerebellar cortex were 2, 21 and 77, respectively. Purified S-100a and S-100b preparations from human and rat brains were also reactive with the respective assay system for bovine S-100 protein, suggesting that the present assay system is applicable to the assay of three forms of S-100 protein in human and rat tissues.     

In vitro phosphorylation of S-100 protein by brain nuclear protein kinases

A three-fold increased ³²P incorporation was observed when S-100 protein was added to a nuclear protein kinase preparation (NPKP) from brain. The specificity of the reaction was indicated by two observations: an increase in ³²P incorporation was not found either with 14–302 protein or when S-100 was added to liver NPKP. SDS-gel analysis shows prominent incorporation of ³²P by brain NPKP into an endogenous brain protein having a molecular weight near 45000 daltons, and, in the presence of S-100, predominantly into S-100 protein itself. Liver NPKP in the presence of S-100, showed an increased incorporation of ³²P into endogenous proteins without any phosphorylation of S-100.     

Specific Binding Sites for S-100 Protein in Isolated Brain Nuclei

Abstract: Isolated brain nuclei possess binding sites for S-100 protein. The interaction of S-100 with these sites is specific and time-, temperature-, and Ca⁺-dependent. The profile of the ¹²⁵I-labelled S-100 binding inhibition is biphasic, displaying a high-affinity component and a low-affinity component. The S-100 binding to brain nuclei is largely irreversible, probably owing to the formation of a tight complex between the protein and its nuclear binding sites. The S-100 binding to brain nuclei is in most aspects similar to that to synaptosomal membranes. Several lines of evidence indicate, however, that the S-100 binding to nuclei is not due to contamination of these structures with plasma membranes. Isolated liver nuclei do not possess the high-affinity component of S-100 binding.     

Neurospecific protein S-100 in the synaptosomes of rat cerebral cortex]

Neurospecific S-100 protein was revealed by the methods of microelectrophoresis in the 15% polyacrylamide gel with a 0.1% sodium dodecylsulfate and by a highly purified S-100 protein "marker" in the composition of low molecular acidic proteins of the rat brain synaptosomes. The S-100 protein conten constitutes about 15-2o% of the low molecular acidic synaptosomal porteins in the rat brain.     

S-100-Mediated Inhibition of Brain Protein Phosphorylation

The effects of the glial-specific, calcium-binding, S-100 protein on brain membrane and supernatant protein phosphorylation were assessed. S-100 concentrations as low as 5 micrograms/ml caused a marked inhibition of the phosphorylation of a soluble brain protein having a molecular weight of 73,000 daltons (73K). This protein was designated the S-100 protein-modulated phosphoprotein (SMP). Half-maximal inhibition of the phosphorylation of SMP by S-100 was obtained at concentrations of 12 micrograms/ml (0.57 microM). The inhibition of SMP phosphorylation by S-100 was calcium-dependent, with a calculated calcium Ka of 2.0 +/- 0.3 microM. SMP phosphorylation was also inhibited by calmodulin, but only partially and with a much lower potency. The inhibition of SMP phosphorylation by S-100 was not inhibited by fluphenazine, whereas the effect of calmodulin was. SMP was found in many brain areas, with the highest levels seen in the corpus callosum. Various peripheral tissues, such as kidney; liver; and pineal, pituitary, and adrenal glands, did not contain detectable SMP levels. At higher S-100 concentrations, greater than 10 micrograms/ml, the phosphorylation of several other soluble proteins was markedly inhibited. These proteins have molecular weights of 56K, 50K, and 47K. The phosphorylation of these proteins was enhanced by calmodulin. These data suggest that the S-100 protein may function to modulate the phosphorylation of brain proteins in a manner analogous to (although in a reciprocal fashion) that of calmodulin.     

PURIFICATION AND SOME PROPERTIES OF S-100 PROTEIN FRACTIONS FROM SHEEP AND PIG BRAINS

Abstract— The S-100 protein fraction of pig and sheep brain was purified in 40 per cent yield by a modification of the procedure of M oore (1965), which avoided selective loss of S-100 components. The S-100 fraction of both pig and sheep is a mixture of proteins as indicated by acrylamide gel electrophoresis and N -terminal amino acid analysis. Differences in amino acid composition, electrophoretic heterogenity and N -terminal analysis were found.
One fraction (fraction A) was isolated by DEAE-Sephadex chromatography from pig brain S-100 protein fraction. It was considered to be a single protein since it migrated as a single band on acrylamide gel electrophoresis and showed a single symmetrical peak during ultracentrifugal analysis. Only one N -terminal amino acid was detected in fraction A. The amino acid composition of this fraction showed minor but significant differences from that of the complete S-100 protein fraction from pig brain. The S-100 protein fraction of both species, as well as fraction A, had similar s _{20, w} values and similar molecular weights (about 20,000) as indicated by gel filtration. These results, together with the immunological data obtained by other authors, suggest that the proteins of the S-100 fraction are closely related; the heterogeneity of the S-100 protein fraction may be of the same type as the lactate dehydrogenase isoenzymes.     

S-100 Modulates Ca2+-Independent Phosphorylation of an Endogenous Protein (Mr= 19K) in Brain

Abstract: A new brain enzyme (tentatively named protein kinase X), which catalyzes protamine phosphorylation modulated by S-100, was reported recently. An endogenous substrate protein (M_r= 19K) for protein kinase × was isolated from brain by means of S-100-Sepharose 4B affinity chromatography. S-100, but not calmodulin, promoted phosphorylation of the 19K M_r protein in a Ca²⁺-independent manner, and this reaction was inhibited by gossypol. The substrate protein, localized in the particulate fraction, was present at a much higher level in brain from adult than neonatal rats (2-day-old), a developmental change similar to that seen for protein kinase X. It is suggested that a protein phosphorylation system modulated by S-100 exists in brain, and that this process may be involved in regulation of certain neural functions.     

SOLUBLE PROTEINS IN NORMAL and DISEASED HUMAN BRAIN

Abstract– Six brain regions (frontal cortex, parts of the basal ganglia, thalamus and substantia nigra) were examined from over 80 human brains obtained at post-mortem. After elimination of patients with evidence of either 'cerebral hypoxia', lingering modes of death or abnormal brain morphology brain extracts were found to contain a characteristic pattern of 6 major soluble-acidic protein bands (neuronin-type proteins). As judged by studies using cortical biopsy specimens these proteins are relatively unaffected by post-mortem changes. Moreover, in adulthood the pattern is not noticeably age-dependant. Two of the protein bands have been identified as S-100 (neuronin S-1 and 2) while a third (neuronin S-5) is similar in most respects to antigen α (14-3-2). S-100 is increased in brains with evidence of marked gliosis. The other protein bands have not been identified. Two of them (neuronin S-3 and 4) are rarely depleted while the concentration of neuronin S-6 is affected particularly in extracortical regions in controls with either lingering modes of death and/or 'cerebral hypoxia' and in all regions in most patients with Alzheimer's disease, senile dementia and mixed senile and vascular dementia.     

Appearance of a brain-specific antigen (S-100 protein) in the developing rat brain

Abstract— Thelevel of the S-100 protein, a brain-specific antigen, wasdetermined by quantitative complement fixation in the brain stem and cerebrum of the rat during postnatal maturation. The content was minimal at birth in the brain stem and rose to its adult value by day 25. Although S-100 protein could not be detected in the cerebrum of the 2-day-old rat, adult values were also present by the 25th day of age. Neither single dose X-irradiation with 750 rd to the head at 2 days of age or single dose X-irradiation at 11 days of age affected the adult level of S-100 protein in the brain stem or cerebrum. Similarly, hypophysectomy at 20 days of age had no effect on the subsequent levels of S-100 protein.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections.

Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehyde-fixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

S-100 protein in the testis

Summary S-100, a protein originally believed to be unique to the nervous system, has recently been found in extraneural cell types. We report here on the presence of S-100 in the testis, namely in Leydig cells and in lymphatic endothelial cells, using immunohistochemical and immunochemical methods. We show that the protein in the testis is immunologically identical to brain S-100. The S-100-labelled cells in the testis exhibit morphological similarities with other cell types in different tissues known to contain S-100.     

The functional role of neurospecific protein S-100 in memory processes]

Elaboration of alimentary conditioned reflex in rats is accompanied by an increase of the level of protein S-100 in the left and right cerebral hemispheres. Amnestic factor M-cholinolytic atropine disturbs the elaborated habit and simultaneously decreases the quantity of protein S-100 up to the level of unlearned animals. The elaboration of conditioned reflex of passive avoidance does not change the content of protein S-100 in the rats brain. Intracisternal injection of antiserum to protein S-100 has an expressed amnestic action. Intracisternal injection of protein S-100 against the background of amnestic action of cholinolytic does not lead to restoration of memory. The cholinolytic and antiserum to protein S-100 mutually potentiate the amnestic effect.     

Predatory aggressiveness of the rat after intraventricular administration of individual brain-specific proteins of the S-100 group and their peptide fragments

Intraventricular injections to rats of the basic fraction of the brain specific protein S-100 in a concentration of 3 mg/ml, significantly facilitates the formation of their predatory aggression induced by the alimentary deprivation and social isolation, expressed in mice killing. This effect is not produced by fragments of S-100 molecules obtained as a result of treatment of the basic protein fraction by proteolytic enzymes. Administration of the minor S-100 fraction, albumin and rats summate brain proteins did not influence animals predatory aggression.