Detection of the neurospecific protein Sy-1 in the human brain and its regional localization in nerve tissue

Using affinity chromatography, a neurospecific protein was isolated from human and rat brain. Both proteins were shown to possess identical physico-chemical properties. However, antisera raised against these proteins exhibited no cross reactions with the corresponding proteins, thus suggesting their species specificity. Immunochemical analysis revealed that the neurospecific protein from rat brain is identical to protein Sy-1 specific for rat brain. Since the human brain protein is analogous to its rat brain counterpart, it may also be considered as protein Sy-1. The uneven distribution of the protein in various structures of human brain was demonstrated by the immunoenzymatic method. Protein Sy-1 is found at relatively low concentrations in the cortex, caudate nucleus and claustrum (2.5-5 micrograms/mg of water-soluble protein), but is predominant in the brain stem (20 micrograms/mg).     

New protein extraction/solubilization protocol for gel-based proteomics of rat (female) whole brain and brain regions

The rat is an accepted model for studying human psychiatric/neurological disorders. We provide a protocol for total soluble protein extraction using trichloroacetic acid/acetone (TCA/A) from rat (female) whole brain, 10 brain regions and the pituitary gland, and show that two-dimensional gel electrophoresis (2-DGE) using pre-cast immobilized pH (4-7) gradient (IPG) strip gels (13 cm) in the first dimension yields clean silver nitrate stained protein profiles. Though TCA/A precipitation may not be "ideal", the important choice here is the selection of an appropriate lysis buffer (LB) for solubilizing precipitated proteins. Our results reveal enrichment of protein spots by use of individual brain regions rather than whole brain, as well as the presence of differentially expressed spots in their proteomes. Thus individual brain regions provide improved protein coverage and are better suited for differential protein detection. Moreover, using a phosphoprotein-specific dye, in-gel detection of phosphoproteins was demonstrated. Representative high-resolution silver nitrate stained proteome profiles of rat whole brain total soluble protein are presented. Shortcomings apart (failure to separate membrane proteins), gel-based proteomics remains a viable option, and 2-DGE is the method of choice for generating high-resolution proteome maps of rat brain and brain regions.     

Analysis of Messenger RNA Coding for S100 Protein in the Mammalian Brain

S100 protein is a brain-specific protein which is absent at birth and first appears in rabbit brain 2–3 days after birth. To determine how the synthesis of this brain-specific protein is regulated, mRNA was isolated from brain polysomes and assayed for S100 protein mRNA activity by in vitro translation in a heterologous cell-free system and immunoprecipitation of released polypeptides with rabbit anti-S I00 protein antiserum. 5100 protein mRNA was detected primarily in small polysomes containing five to eight ribosomes, and virtually no S 100 protein mRNA was present in polysomes containing more than eight ribosomes. S100 protein mRNA was not detected in brain polysomes at stages prior to the induction of synthesis of S100 protein, i.e., in fetal brain or in 1-day neonates. The amount of S100 protein mRNA in polysomes of the cerebral cortex and cerebellum was measured to see if it correlated with the level of S100 protein in the two regions of adult brain. The cerebellum, which contained three to four times the level of S100 protein in the cerebral cortex, contained four times more S100 protein mRNA.     

A tissue culture system for studies of the regulation of ether-linked and ester-linked aliphatic moieties in glycerolipids

A protein has been detected in chick brain which is immunologically identical to the vitamin D-induced calcium-binding protein found in intestinal tissue. It is present in highest concentration in the cerebellum and at lower levels in the remainder of the brain. Brain calcium-binding protein has the same molecular weight and electrophoretic properties as intestinal calcium-binding protein. Although the synthesis of intestinal calcium-binding protein is totally dependent upon a source of vitamin D, this has not yet been shown for brain calcium-binding protein. The total calcium-binding protein content of the cerebellum of chicks fed a vitamin D-free diet continued to increase during growth from 1 to 5 weeks, and is not responsive to exogenous vitamin D or 1,25-dihydroxyvitamin D.     

Effects of adding dietary lysine to a low gluten diet on the brain protein synthesis rate in aged rats

The purpose of this study was to find whether the addition of dietary lysine affected the rate of brain protein synthesis in aged rats fed on a gluten diet. Experiments were done on two groups of aged rats (30 wk) given the diets containing 5% gluten or 5% gluten + 0.3% lysine for 10 d. The fractional rates of protein synthesis in brain, liver, and kidney increased with an addition of dietary lysine. In brain, liver, and kidney, the RNA activity [g protein synthesized/(g RNA x d)] was significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. The results suggest that the addition of the limiting amino acid for the low quality protein elevates the rate of protein synthesis in the brain of aged rats, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.     

Expression and localization of an agmatinase-like protein in the rat brain

Agmatinase catalyzes the hydrolysis of agmatine into putrescine and urea, and agmatine (decarboxylated l-arginine) plays several roles in mammalian tissues, including neurotransmitter/neuromodulatory actions in the brain. Injection of agmatine in animals produces anticonvulsant, antineurotoxic and antidepressant-like actions. Information regarding the enzymatic aspects of agmatine metabolism in mammals, especially related to its degradation, is relatively scarce. The explanation for this is the lack of enzymatically active preparations of mammalian agmatinase. Recently, we have cloned a protein from a cDNA rat brain library having agmatinase activity although its amino acid sequence greatly differs from all known agmatinases, we called agmatinase-like protein. In this work, we analyzed the expression of this enzyme in the rat brain by means of RT-PCR and immunohistochemical analysis using a polyclonal antibody generated against the recombinant agmatinase-like protein. The agmatinase-like protein was detected in the hypothalamus in glial cells and arcuate nucleus neurons, and in hippocampus astrocytes and neurons, but not in brain cortex. In general, detected localization of agmatinase-like protein coincides with that described for its substrate agmatine and our results help to explain several reported effects of agmatine in the brain. Concretely, a role in the regulation of intracellular concentrations of the neurotransmitter/neuromodulator agmatine is suggested for the brain agmatinase-like protein.     

Glial cell aquaporin-4 overexpression in transgenic mice accelerates cytotoxic brain swelling

Aquaporin-4 (AQP4) is a water transport protein expressed in glial cell plasma membranes, including glial cell foot processes lining the blood-brain barrier. AQP4 deletion in mice reduces cytotoxic brain edema produced by different pathologies. To determine whether AQP4 is rate-limiting for brain water accumulation and whether altered AQP4 expression, as occurs in various pathologies, could have functional importance, we generated mice that overexpressed AQP4 in brain glial cells by a transgenic approach using the glial fibrillary acid protein promoter. Overexpression of AQP4 protein in brain by approximately 2.3-fold did not affect mouse survival, appearance, or behavior, nor did it affect brain anatomy or intracranial pressure (ICP). However, following acute water intoxication produced by intraperitoneal water injection, AQP4-overexpressing mice had an accelerated progression of cytotoxic brain swelling, with ICP elevation of 20 +/- 2 mmHg at 10 min, often producing brain herniation and death. In contrast, ICP elevation was 14 +/- 2 mmHg at 10 min in control mice and 9.8 +/- 2 mmHg in AQP4 knock-out mice. The deduced increase in brain water content correlated linearly with brain AQP4 protein expression. We conclude that AQP4 expression is rate-limiting for brain water accumulation, and thus, that altered AQP4 expression can be functionally significant.     

Alzheimer''s disease amyloidogenic glycoprotein: expression pattern in rat brain suggests a role in cell contact.

The cloned cDNA encoding the rat cognate of the human A4 amyloid precursor protein was isolated from a rat brain library. The predicted primary structure of the 695-amino acid-long protein displays 97% identity to its human homologue shown previously to resemble an integral membrane protein. The protein was detected in rodent brain and muscle by Western blot analysis. Using in situ hybridization and immunocytochemistry on rat brain sections, we discovered that rat amyloidogenic glycoprotein (rAG) and its mRNA are ubiquitously and abundantly expressed in neurons indicating a neuronal original for the amyloid deposits observed in humans with Alzheimer's disease (AD). The protein appears in patches on or near the plasma membranes of neurons suggesting a role for this protein in cell contact. Highest expression was seen in rat brain regions where amyloid is deposited in AD but also in areas which do not contain deposits in AD. Since amyloid deposits are rarely observed in rat brain, we conclude that high expression of AG is not the sole cause of amyloidosis.     

Immunoblot Identification of 13.5 Kilodalton Myelin Basic Protein in Goldfish Brain Myelin

Myelin isolated from goldfish brain shows a multilamellar structure with a major dense line and two intraperiod lines. Sodium dodecyl sulfate gel electrophoresis revealed that the protein profile of goldfish brain myelin is distinctly different from that of rat brain myelin. No protein migrating to the position of proteolipid protein or DM-20 was seen in goldfish myelin. Goldfish acclimated to 5 degrees, 15 degrees, and 30 degrees C showed no qualitative differences in myelin proteins. The 13.5 kD protein in goldfish brain myelin and brain homogenate was intensely immunostained with the antiserum to human basic protein by the immunoblot technique. In contrast, none of the proteins of goldfish myelin were immunostained with antiproteolipid protein serum; however, both proteolipid protein and DM-20 of rat brain myelin were immunostained. The significance of the synthesis of myelin proteins by astrocytes in the goldfish brain is discussed.     

Role of glycosylation in hyperphosphorylation of tau in Alzheimer's disease

In Alzheimer's disease (AD) brain, microtubule-associated protein tau is abnormally modified by hyperphosphorylation and glycosylation, and is aggregated as neurofibrillary tangles of paired helical filaments. To investigate the role of tau glycosylation in neurofibrillary pathology, we isolated various pools of tau protein from AD brain which represent different stages of tau pathology. We found that the non-hyperphosphorylated tau from AD brain but not normal brain tau was glycosylated. Monosaccharide composition analyses and specific lectin blots suggested that the tau in AD brain was glycosylated mainly through N-linkage. In vitro phosphorylation indicated that the glycosylated tau was a better substrate for cAMP-dependent protein kinase than the deglycosylated tau. These results suggest that the glycosylation of tau is an early abnormality that can facilitate the subsequent abnormal hyperphosphorylation of tau in AD brain.     

Specific Lymphocyte Sensitization in Cancer: Is There a Common Antigen in Human Malignant Neoplasia?

From human cancer tissue a basic protein can be extracted by the method which yields encephalitogenic factor when applied to human brain. This tumour basic protein (obtained from several different neoplasms) acts as an antigen in the cytopherometric test for malignant neoplasia and in general gives higher results than does brain basic protein. The reverse is true when degenerative disease of the nervous system is studied. The basic protein extractable from brain and from tumours thus has some degree of specificity probably referable to its amino-acid sequence.     

Effects of quantity and quality of dietary protein on the brain polysome profile in aged rats

The purpose of this study was to determine whether the quantity and quality of dietary protein affected the polysome profile of the brain in aged rats. Two experiments were done on three groups of aged rats (30 wk) given the diets containing 20% casein, 5% casein, or 0% casein (experiment 1), and 20% casein, 20% gluten, or 20% gelatin (experiment 2) for 10 d. The aggregation in brain ribosomes declined with a decrease of quantity and quality of dietary protein except in the hippocampus. The RNA concentration (mg RNA/g protein) did not differ among the three groups varying the dietary protein in any brain regions. The results suggest that the higher quantity and quality of dietary protein improves the polysome profile in the brain of aged rats, and that the polysome profile is at least partly related to the mechanism by which the dietary protein affects brain protein synthesis in aged rats.     

The S100-b Protein: Tyrosine Residues Do Not Exhibit an Abnormal Fluorescence Spectrum

The β subunit of the bovine brain S100-b protein (ββ) lacks tryptophyl residue but contains one tyrosine. Our experiments show that this protein is characterized by a typical tyrosine fluorescence spectrum, with a maximum at 303 nm. Identical fluroescence properties were found for the rat brain S100-b protein. Comparison with the fluorescence spectrum of the bovine brain S100-a’ protein (α'β), which contains a tryptophan residue in the α’ subunit, enables us to demonstrate that the recent report describing an abnormal fluorescence spectrum for the bovine brain S100-b protein may result from a contamination of the S100-b by the S100-α’ protein.     

Brain Slice Protein Degradation and Development

Protein degradation rates were measured in brain slices prepared from rats of various ages. This was done by adding the protein synthesis rate, determined by incorporation of a labeled precursor, and the net protein degradation rate, determined by measuring the changes with time of total free amino acids. These rates are about 30% higher than those previously calculated from data on protein synthesis rates and protein accumulation rates in vico. The protein degradation rates in brain slices diminish with age; i.e., 2-day cerebellum > 2-day cerebral hemisphere > 12-day cerebral hemisphere > young adult cerebral hemisphere. Protein degradation rates in slices from young brain are initially slightly higher than protein synthesis rates, resulting in a small net degradation with time. Unlike slices from adult brain, the protein degradation rates in slices from young brain decline only modestly with time for as much as 100 min of incubation. The characteristics of protein degradation in brain slices from young animals are roughly similar to some of the data calculated for protein degradation in vivi. suggesting that this system may prove useful for studying factors which control or affect brain protein degradation.     

Abnormal dentatorubral-pallidoluysian atrophy (DRPLA) protein complex is pathologically ubiquitinated in DRPLA brains.

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a glutamine repeat in DRPLA protein. DRPLA protein undergoes greater complex formation in DRPLA brain tissue, and expanded glutamine repeat enhances complex formation of DRPLA protein. Immunoblots with and without reduction show that the DRPLA protein complex is ubiquitinated only in DRPLA brain tissue. Moreover, immunoblots of regional DRPLA brain tissues reveal that pathological ubiquitination of DRPLA protein complex is found selectively in affected lesions. Double-labeling immunohistochemical studies with antibodies against DRPLA protein and ubiquitin demonstrate that the DRPLA protein is co-localized with ubiquitin in DRPLA neurons and show characteristic neuronal cytoplasmic inclusions with ubiquitinated DRPLA protein complex in the center. Our findings suggest that DRPLA protein undergoes abnormal complex formation with expanded glutamine repeat, and then the complex is pathologically ubiquitinated in DRPLA brain tissue. Pathological ubiquitination of abnormal DRPLA protein complex plays a role in DRPLA pathology.     

Solubilization and Purification of an α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding Protein from Bovine Brain

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) is a selective ligand for an excitatory amino acid receptor subtype in mammalian brain. We have solubilized an AMPA binding protein from bovine brain membranes with 1% Triton X-100 in 0.5 M phosphate buffer and 20% glycerol at 37 degrees C and purified the stable binding sites using a series of chromatographic steps. Scatchard analysis of the purified preparation showed a curvilinear plot with dissociation constants of 10.6 and 323 nM and Bmax values of 670 and 1,073 pmol/mg of protein for the high- and low-affinity sites, respectively. Inhibition constants for several excitatory amino acid analogues were similar to those obtained for other membrane and solubilized preparations. Gel filtration of the soluble AMPA binding protein showed a single peak of [3H]AMPA binding activity at Mr approximately 500,000. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified AMPA binding protein showed a single major band at Mr = 110,000. Previously, we have shown that a monoclonal antibody (KAR-B1) against a frog brain kainate binding protein selectively recognizes an unknown protein in mammalian brain migrating at Mr approximately 100,000. We now show that this antibody recognizes the major component of the purified AMPA binding protein, supporting a structural similarity between the frog brain kainate binding protein and the mammalian AMPA binding protein.     

Ankyrin and synapsin: spectrin-binding proteins associated with brain membranes

Brain membranes contain an actin-binding protein closely related in structure and function to erythrocyte spectrin. The proteins that attach brain spectrin to membranes are not established, but, by analogy with the erythrocyte membrane, may include ankyrin and protein 4.1. In support of this idea, proteins closely related to ankyrin and 4.1 have been purified from brain and have been demonstrated to associate with brain spectrin. Brain ankyrin binds with high affinity to the spectrin beta subunit at the midregion of spectrin tetramers. Brain ankyrin also has binding sites for the cytoplasmic domain of the erythrocyte anion channel (band 3), as well as for tubulin. Ankyrins from brain and erythrocytes have a similar domain structure with protease-resistant domains of Mr = 72,000 that contain spectrin-binding activity, and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg membrane protein, or about twice the number of copies of spectrum beta chains. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes, and it has the potential to attach microtubules to membranes as well as to interconnect microtubules with spectrin-associated actin filaments. Another spectrin-binding protein has been purified from brain membranes, and this protein cross-reacts with erythrocyte 4.1. Brain 4.1 is identical to the membrane protein synapsin, which is one of the brain's major substrates for cAMP-dependent and Ca/calmodulin-dependent protein kinases with equivalent physical properties, immunological cross-reaction, and peptide maps. Synapsin (4.1) is present at about 60 pmol/mg membrane protein, and thus is a logical candidate to regulate certain protein linkages involving spectrin.     

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.