Characterization and Distribution of Ferritin Binding Sites in the Adult Mouse Brain

Abstract : Studies on iron uptake into the brain have traditionally focused on transport by transferrin. However, transferrin receptors are not found in all brain regions and are especially low in white matter tracts where high iron concentrations have been reported. Several lines of research suggest that a receptor for ferritin, the intracellular storage protein for iron, may exist. We present, herein, evidence for ferritin binding sites in the brains of adult mice. Autoradiographic studies using ¹²⁵I-recombinant human ferritin demonstrate that ferritin binding sites in brain are predominantly in white matter. Saturation binding analyses revealed a single class of binding sites with a dissociation constant ( K _D) of 4.65 × 10^-9 M and a binding site density ( B _max) of 17.9 fmol bound/μg of protein. Binding of radiolabeled ferritin can be competitively displaced by an excess of ferritin but not transferrin. Ferritin has previously been shown to affect cellular proliferation, protect cells from oxidative damage, and deliver iron. The significance of a cellular ferritin receptor is that ferritin is capable of delivering 2,000 times more iron per mole of protein than transferrin. The distribution of ferritin binding sites in brain vis-à-vis transferrin receptor distribution suggests distinct methods for iron delivery between gray and whi     

COMPARISON OF THE FATTY ACIDS OF LIPIDS OF SUBCELLULAR BRAIN FRACTIONS

Abstract— Rat brain grey and white matter were fractionated to yield myelin, nerve terminal, synaptic vesicle, nerve terminal 'ghost', and microsomal fractions of white and grey matter. Ester-type glycolipids were found in all fractions except myelin, while cerebrosides occurred in significant concentrations only in myelin and white microsomes. Comparison of the fatty acid profile of the ethanolamine- and serine-containing phospholipids showed marked differences between myelin and the particles from grey matter, while the microsomes of white matter were of intermediate composition. Docosahexaenoic acid, a minor acid in myelin, was a major fatty acid in microsomes of grey and white matter. The fatty acid composition of sphingomyelin was distinctly different in the fractions derived from grey and white matter, clustering about stearate and nervonate in the latter, but only about stearate in the grey. Marked differences in the positional distribution of fatty acids were seen within phosphatidyl choline from myelin and nerve terminals. Ribonucleic acid was found in nerve terminal and synaptic vesicle fractions. The sphingosine found in the ganglioside from microsomes of both grey and white matter was similar with respect to distribution of the C₁₈ and C₂₀ homologues.
The possibility is discussed that microsomes furnish characteristic lipids for the synthesis or renewal of specific membranes, and that these lipids are accumulated somewhat before being released.     

Copper distribution in the normal human brain

Copper concentration was determined in samples from 38 areas of 7 normal human brains. The grey matter contained higher concentrations of copper than the white matter. Identical areas of the grey and white matter of the cerebral cortex showed significant differences between individuals. In the caudate nucleus the highest concentrations of copper were found in the tail followed by the body and the head, respectively. A negative linear regression between age and brain copper levels was demonstrated.     

Lipid Investigation of Central and Peripheral Nervous System in Connatal Pelizaeus-Merzbacher's Disease

Abstract: Brain grey and white matter of a case of Pelizaeus-Merzbacher disease (connatal type Seitelberger) of a 19-month-old boy were analysed with respect to lipids. Cerebrosides and sulfatides were totally absent in the pathological brain. In comparison to control, differences in gangliosides could be detected in grey and white manner. C_18:1, fatty acids were markedly reduced in the main glycerophospholipids of white matter. In sphingomyelin of cortex and white matter 90% of fatty acids were C_18:0; longer chains were absent. In contrast: PNS (nervus fernoralis) lipids contained the main galactolipids. However, these a s well as all other lipid classes showed a 20% reduction compared with values obtained from nervus femoralis of an infant of the same age. The fatty acid patterns of all lipid classes were determined. The only marked deviations from normal were observed in the C₂₄-chains of cere-brosides and sulfatides. The formalin-fixed brain of an older brother (same disease) was analysed only with respect to glycolipids: neither cerebrosides nor sulfatides could be detected.     

Immunofluorescence and Histochemical Methods for Neural M1 Pyruvate Kinase Localization

Abstract: The distribution of pyruvate kinase (ATP pyruvate phosphotransferase, EC 2.7.1.40) in the nervous system has been studied by both immunofluorescence and a histochemical procedure using nitro blue tetrazolium. The localization in various parts of rat central nervous system in situ , cerebellar and cerebral cortex, was compared to that found in vitro in cultures of cerebellum, spinal ganglia, cerebral astrocytes, and skin fibroblasts. (1) Pyruvate kinase was found predominantly in the cytoplasm of neuronal cell bodies. (2) Large neurons were better visualized than small ones. (3) No glial localization was clearly demonstrated in situ , although this does not rule out the presence of some M₁ pyruvate kinase. (4) Regions expected to be rich in nerve terminals, such as the cerebellar glomeruli or the cerebellar molecular layer, showed intense staining even when the cell bodies themselves were negative. This was expected, owing to the previous demonstration of the presence of M₁ pyruvate kinase in nerve ending by subcellular fractionation methods. (5) The localization was similar in situ and in tissue culture, except that nerve processes were better seen in the latter and astrocytes were sometimes stained in vitro. (6) Variation in intensity of staining was observed in similar cell types in the same section or in the same culture. This could represent different metabolic or functional or maturational states.     

Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins

Abstract: Proteins in peripheral nervous system and central nervous system myelin and homogenates of sciatic nerve and brain from young and adult mice and rats were characterized with affinity-purified anti-P₂ and anti-myelin basic protein sera after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Using this method we have identified a component of rodent peripheral nervous system myelin as P₂ protein. Peripheral nervous system myelin also showed the presence of four basic proteins in addition to P₂ protein. These were found to be analogous to the 14, 17, 18.5, and 21.5K species found in the central nervous system myelin. A number of high-molecular-weight proteins were also detected with anti-myelin basic protein serum in peripheral nervous system, as well as central nervous system myelin. In addition, we report the presence of a high-molecular-weight P₂ cross-reactive protein in rodent brain stem homogenates, but not in central nervous system myelin.     

Adult human brain expresses four different molecular forms of neural cell adhesion molecules

Using antibodies to rat neural cell adhesion molecules (NCAM), we analyzed the NCAM of adult human brain. Various regions of the brain were analyzed quantitatively by Western blot. Grey matter showed four bands of NCAM with apparent molecular weights of 180,000, 170,000, 140,000 and 120,000. White matter showed one major band with an apparent M_r of 120,000 and a minor band of 180,000. Cerebellar grey matter contained mainly 170,000, 140,000 and 120,000, white cerebellar white matter had only 180,000 and 120,000 M₁ NCAMS. Spinal cord showed mainly 120,000 M_r NCAM. Deglycosylation using N-glycanase resulted in 170,000, 160,000, 130,000 and 110,000 M_r proteins, suggesting that the four forms of human NCAM are derived from individual polypeptides. The presence of 170,000 M₁ NCAM is unique to human brain.     

IMMUNOCHEMICAL AND IMMUNOFLUORESCENCE STUDIES OF BETA-TRACE PROTEIN IN DIFFERENT SPECIES AND ORGANS, WITH SPECIAL REFERENCE TO THE CENTRAL NERVOUS SYSTEM

—The localization of beta-trace protein in different human organs and in the brains of other mammals has been investigated with immunochemical and immunofluorescence methods. With the double immunodiffusion technique, the immunological identity between beta-trace protein in human CSF, brain, femoral nerve, testis, epididymis, spermatic cord, ovary and fallopian tube and in monkey brain could be demonstrated. By means of single radial immunodiffusion on agar, the highest concentrations of beta-trace protein were found in preparations of water-soluble proteins of brain and genital organs, especially in white CNS matter, testis and epididymis. With the indirect immunofluorescence technique, bright fluorescence was found in CNS, especially in white matter, and in the stroma of epididymis. The content of beta-trace protein in femoral nerve was found to be considerably lower than that in the CNS. The results indicate a connexion between beta-trace protein and the CNS, probably in the myelin or glial cells, and also between beta-trace protein and the genital system, possibly to cells involved in steroid metabolism. A specificity for highly developed mammals, i.e. man and monkey, is proposed for beta-trace protein, as no precipitation could be found when brain extracts from calves, rabbits or mice were incubated with anti-serum against human beta-trace protein.     

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.     

THE REGIONAL DISTRIBUTION OF SIALOGLYCOPROTEINS, GANGLIOSIDES AND SIALIDASE IN BOVINE BRAIN

Abstract— Sialoglycoproteins and gangliosides were characterized in various bovine brain regions by determining the amount of sialic acid. Expressed per g dry weight, the gangliosidic sialic acid ranged from 11·20 to 1·93 μmol and the glycoprotein sialic acid from 8·93 to 1·84 μmol in grey and white matter respectively (values not corrected for incomplete release and breakdown during hydrolysis). Both the sialoglycoproteins and the gangliosides occur in highest concentration in areas predominating in neuronal cell bodies (cerebral grey, cerebellar grey, caudate nucleus). The lowest concentrations are found in those areas, consisting largely of myelinated fibre tracts and glial cells (pons, medulla, corpus callosum, cerebral white). Relative to the gangliosides the sialoglycoproteins are somewhat more concentrated in white matter.
The sialidase activity was investigated with endogenous substrate as well as with additional gangliosides or sialoglycopeptides. In all conditions the activity was much greater in grey matter than in white matter. The regional sialidase distribution more or less parallels the distribution of sialic acid in the various regions. At high substrate level the sialoglycopeptides inhibit the sialidase activity. There are indications that gangliosides are a far better substrate for brain sialidase than glycoproteins or glycopeptides. The possible significance of this phenomenon is discussed.     

beta-Trace protein in human cerebrospinal fluid: a diagnostic marker for N-glycosylation defects in brain.

As carbohydrate-deficient glycoprotein syndromes (CDGS) are multisystemic disorders with impaired central nervous function in nearly all cases, we tested isoforms of beta-trace protein (beta TP), a 'brain-type' glycosylated protein in cerebrospinal fluid (CSF) of nine patients with the characteristic CDGS type I pattern of serum transferrin. Whereas the serum transferrin pattern did not discriminate between the various subtypes of CDGS type I (CDGS type Ia, type Ic, and patients with unknown defect), beta TP isoforms of CDGS type Ia patients differed from that of the other CDGS type I patients. The percentage of abnormal beta TP isoforms correlated with the severity of the neurological symptoms. Furthermore, two patients are described, who illustrate that abnormal protein N-glycosylation can occur restricted to either the 'peripheral' serum or the central nervous system compartment. This is the first report presenting evidence for an N-glycosylation defect restricted to the brain. Testing beta TP isoforms is a useful tool to detect protein N-glycosylation disorders in the central nervous system.     

Differential expression of apolipoprotein d in human astroglial and oligodendroglial cells.

Apolipoprotein D (Apo D) is a secreted lipocalin in the nervous system that may be related to processes of reinnervation and regeneration. Under normal conditions, Apo D is present in the central nervous system in oligodendrocytes, astrocytes, and some scattered neurons. To elucidate the regional and cellular distribution of Apo D in normal human brain, we performed double immunohistochemistry for glial fibrillary acidic protein (GFAP) and Apo D in samples of postmortem human cerebral and cerebellar cortices. Most of the GFAP-positive cells in the gray matter had features of protoplasmic astrocytes and were mainly Apo D-positive. Apo D staining was mostly confined to the cell soma and proximal processes, whereas GFAP extended to a rich and extensive array of processes. The fibrous astrocytes in the white matter were immunoreactive for GFAP but not for Apo D. In the white matter, Apo D was mainly detected in oligodendrocytes and extracellularly in the neuropil. The results of the present study support a specific behavior for each astrocyte type. These findings suggest that Apo D expression may be cell-specific, depending on the particular tissue physiology at the time of examination.     

Identification of Endothelin Receptor Subtype (ETB) in Human Cerebral Cortex Using Subtype-Selective Ligands

Abstract: Specific endothelin (ET) binding sites were characterized in membranes prepared from human cerebral cortices using binding assay and cross-linking analysis. The presence of immunoreactive (IR) ET-1 was studied by radioimmunoassay. Saturation binding experiments revealed that the K _D and B _max for ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes from gray matter were 25 ± 6 pM and 115 ± 15 fmol/mg of protein and 24 ± 5 p M and 108 ± 13 fmol/mg of protein, respectively. Similar results were obtained for white matter. In the presence of 10 n M sarafotoxin-6c, which is selective for ET_B receptors, ¹²⁵I-ET-1 and ¹²⁵l-ET-3 binding was totally abolished. However, in the presence of 1 μ M BQ123, which is selective for ET_Areceptors, both bindings were not affected. These results suggest that the human cerebral cortex contains only ET_Breceptors. Cross-linking of ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes with disuccinimidyl suberate resulted in the labeling of two bands of 48 and 31 kDa. Concentrations of IR-ET-1 in the gray and white matter were 7.0 ± 3.2 and 2.5 ± 1.7 fmol/g wet weight, respectively. The demonstration of high-affinity ET_B receptors and the presence of IRET-1 suggest that the peptide may act as a neurotransmitter or neuromodulator in the human cerebral cortex.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

Benzodiazepine Antagonist Ro 15–1788: Binding Characteristics and Interaction with Drug-Induced Changes in Dopamine Turnover and Cerebellar cGMP Levels

Abstract: The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [³H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [³H]diazepam binding in vitro (IC₅₀= 2.3 ± 0.6 nmol/liter). [³H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation constant ( K _D) of 1.0 ± 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [³H]Ro 15-1788 from its binding site was of the same rank order as found previously in [³H]diazepam binding. Autoradiograms of [³H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [³H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.     

The distribution of molecular species of phosphatidylinositol in ox brain and its subcellular fractions

1. The phosphatidylinositol content of white and grey matter of ox cerebral hemispheres did not differ. The phosphatidylinositol from grey matter was slightly enriched in palmitic acid and arachidonic acid, and that from white matter was enriched in eicosatrienoic (C(20:3)) acid. These regional differences were apparently due to the greater content of myelin in the white matter, since the same tendencies were observed when combined myelinic and non-myelinic subcellular fractions prepared from the cerebral hemispheres were compared. 2. Purified phosphatidylinositol was converted into its triacetylated methylated derivative and resolved to its molecular species by t.l.c. on AgNO(3)-impregnated silica gel. The tetraenoic molecular species was predominant in phosphatidylinositol from ox cerebral hemispheres, and this feature characterized all the phosphatidylinositol samples extracted from its regions or subcellular fractions. The grey matter was more enriched in the tetraenoic species and the white matter in the trienoic species. 3. The molecular-species composition of phosphatidylinositol from the subcellular fractions of ox cerebral hemispheres was studied. The trienoic species constituted nearly one-fifth of the phosphatidylinositol from two myelinic fractions. ;Large myelin' was more enriched in this species than was ;small myelin'. Both fractions also contained greater concentrations of the dienoic species than the non-myelinic subcellular fractions. The latter fractions, one containing nuclei and the other nerve endings plus mitochondria, were enriched in the monoenoic and tetraenoic species of phosphatidylinositol. The post-mitochondrial supernatant exhibited a pattern of distribution of phosphatidylinositol species intermediate between the myelinic and non-myelinic fractions.     

THE DISTRIBUTION OF GLYCINE, GABA, GLUTAMATE AND ASPARTATE IN RABBIT SPINAL CORD, CEREBELLUM AND HIPPOCAMPUS

The distribution of glycine, GABA, glutamate and aspartate was measured among about 60 subdivisions of rabbit spinal cord, and among the discrete layers of cerebellum, hippocampus and area dentata. A more detailed mapping for GABA was made within the tip of the dorsal horn of the spinal cord. Spinal ventral horn and dorsal root ganglion cell bodies were analyzed for the amino acids and for total lipid. The distribution of lipid and lipid-free dry weight per unit volume was also determined in spinal cord. Calculated on the basis of tissue water, glycine in the cord is highest in lateral and ventral white matter immediately adjacent to the ventral grey. The distribution of GABA is almost the inverse of that of glycine with highest level in the tip of dorsal horn. It is most highly concentrated in the central 75% of Rexed layers III and IV. Aspartate in the tip of ventral horn is 4-fold higher than in the tip of the dorsal horn and 3 times the average concentration in brain. Glutamate was much more evenly distributed and is relatively low in concentration with slightly higher levels in dorsal than in ventral grey matter. Large cell bodies in both ventral horn and dorsal root ganglion contained high levels of glycine. As reported by others, GABA was found to be high in cerebellar grey layers, area dentata, and regio inferior of hippocampus. Glycine was moderately high in cerebellar layers but moderate to low in hippocampus and area dentata.     

Grey plumage colouration in the duck is genetically determined by the alleles on two different, interacting loci

Based on the observation of a grey phenotype in the F₁ generation from a cross between two white plumage duck varieties, the white Kaiya and the white Liancheng , we hypothesized a possible interaction between two autosomal loci that determine grey plumage. Using the parental and F₁ individuals, seven testing combinations including five different F₁ intercrosses (F₂) and two different backcrosses (BC₁ and BC₂) were designed to test our hypothesis. It was demonstrated by chi-squared analysis that six test matings produced offspring in the expected ratios between the grey and white, with P- values ranging from 0.50 to 0.99. Another mating, where all white offspring were expected, produced 33 white individuals. These results verified that the interaction between two loci produced the grey phenotype. The C locus, which carries the recessive allele ( c ), was previously thought to be the only gene responsible for white plumage in the duck. This is the first report that an allele ( t ), carried by the white Liancheng at a different autosomal locus, also determines white plumage in ducks. Furthermore, the dominant alleles at both loci can interact with each other to produce the grey phenotype, and a new dark phenotype, observed in some F₂ individuals, can be attributed to the dosage effect of the T allele.     

Activation of G Proteins Bidirectionally Affects Apoptosis of Cultured Cerebellar Granule Neurons

Abstract: Cultured cerebellar granule neurons maintained in depolarizing concentrations of K⁺ (25 m M ) and then switched to physiological concentrations of K⁺ (5 m M ) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apoptosis of cultured rat cerebellar granule neurons. Stimulation of G_s with cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K⁺, whereas stimulation of G_o/G_i with the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K⁺. Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan. By contrast, pertussis toxin, cell-permeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CNS.     

IMMUNOCHEMICAL STUDIES ON THE BRAIN SPECIFIC PROTEIN

Abstract— In the soluble brain proteins of various species-man, ox, cat, rabbit, rat, mouse, hen, snake, frog and fish–there is a protein group which migrates more slowly than Moore's S-100 protein and faster than the albumin fraction on disc electrophoresis. The protein group is absent from any organs other than brain, and has a different pattern and number of fractions in different species. Immunochemically, the protein fraction group of the mammalian brains shows some common and identical distinctive antigenic determinants compared with the brain protein of the other animals-hen, snake, frog and fish. The protein group was designated the 'SPR' proteins, which were separated to 'P_II,', 'P_III', 'P_IV' and 'P_v' fractions. Common antigenic determinants are found in these fractions. The protein group is found in human brain in larger amounts in grey matter than in white matter and in small amounts in the cellular nuclei of human and bovine brain.