Brain-specific proteins S 100 and reception of neuromediators by membrane preparations from the rat brain

Study on specific binding of ligands of various nervous cell membrane neuromediator receptors revealed that brain-specific proteins S100 modulated in a dose-dependent fashion the level of specific binding. Proteins S100 (but not BSA or blood gamma-globulins) changed the Kd values of the ligand-receptor complexes and the number of binding sites. The effects of proteins S100 were specifically blocked by antibodies to S100. Proteins S100 influenced the functions of acetylcholine, serotonin, dopamine, noradrenaline and GABA receptors but not of opiate, imipramine, histamine and benzodiazepine receptors.     

Amino acid, peptide, and protein hydroperoxides and their decomposition products modify the activity of the 26S proteasome

Proteins are major biological targets for oxidative damage within cells because of their high abundance and rapid rates of reaction with radicals and singlet oxygen. These reactions generate high yields of hydroperoxides. The turnover of both native and modified/damaged proteins is critical for maintaining cell homeostasis, with this occurring via the proteasomal and endosomal-lysosomal systems; the former is of particular importance for intracellular proteins. In this study we have examined whether oxidation products generated on amino acids, peptides, and proteins modulate 26S proteasome activity. We show that oxidation products, and particularly protein hydroperoxides, are efficient inhibitors of the 26S proteasome tryptic and chymotryptic activities, with this depending, at least in part, on the presence of hydroperoxide groups. Removal of these species by reduction significantly reduces proteasome inhibition. This loss of activity is accompanied by a loss of thiol residues, but an absence of radical formation, consistent with molecular, rather than radical, reactions being responsible for proteasome inhibition. Aldehydes also seem to play a role in the inhibition of chymotryptic activity, with this prevented by treatment with NaBH(4), which reduces these groups. Inhibition occurred at hydroperoxide concentrations of ≥1μM for oxidized amino acids and peptides and ≥10μM for oxidized proteins, compared with ca. 100μM for H(2)O(2), indicating that H(2)O(2) is a much less effective inhibitor. These data indicate that the formation of oxidized proteins within cells may modulate cell function by interfering with the turnover of native proteins and the clearance of modified materials.     

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.     

Possible role of brain-specific proteins S100 in the regulation of phosphorylation-dephosphorylation of endogenous substrates of the nerve tissue

The effects of brain-specific proteins S100 (BSP S100) and their endogenous protein ligands (both cationic and anionic) on the phosphorylation of macromolecular substrates from rat brain and liver were studied. It was shown that BSP S100 significantly affects both the phosphorylation and dephosphorylation of various substrates, depending on BSP S100 endogenous protein ligands.     

Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses.

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.     

NMR investigation of the heme electronic structure in deoxymyoglobin possessing a fluorinated heme

The heme electronic structures of deoxymyoglobins (deoxy-Mbs) reconstituted with 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-(trifluoromethyl)porphyrinatoiron(III) (7-PF), 13,17-bis(2-carboxylatoethyl)-3,7-difluoro-2,8,12,18-tetramethylporphyrinatoiron(III) (3,7-DF), and 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethylporphyrinatoiron(III) (2-MF) have been characterized by ¹H and ¹⁹F NMR. The analysis of heme methyl proton shift patterns of the hemes in their bis-cyano forms demonstrated that, owing to the substitution of a strongly electron-withdrawing perfluoromethyl group, CF₃, to porphyrin, the porphyrin -system of 7-PF is more significantly distorted from four-fold symmetry than those of the ring-fluorinated hemes, 3,7-DF and 2-MF. The presence of the heme orientation disorder resulted in the observation of the two well-resolved ¹⁹F signals in the spectra of deoxy-Mbs possessing 7-PF and 2-MF. The ¹⁹F signals of deoxy-Mb possessing 7-PF exhibited a relatively large difference in paramagnetic shift (~30 ppm), despite their small paramagnetic shifts (~30 ppm), supporting the significant contribution of a spin delocalization mechanism in this Mb due to the d-electron configuration derived from the ⁵E ground state. On the other hand, ¹⁹F signals of deoxy-Mbs with 3,7-DF as well as 2-MF exhibited large paramagnetic shifts (~250 ppm) with a relatively small difference in the paramagnetic shift (~20 ppm), indicating the predominant contribution of spin delocalization, due to a d-electron configuration derived from the ⁵B₂ ground state. These results demonstrate for the first time that the relative contributions of the orbital ground states derived from ⁵E and ⁵B₂ states to the heme electronic structure in deoxy-Mb are affected by the distortion of the porphyrin -system exerted by chemical properties of the heme peripheral side-chains.Abbreviations 3,7-DF 13,17-bis(2-carboxylatoethyl)-3,7-difluoro-2,8,12,18-tetramethylporphyrinatoiron(III) - 2-MF 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethylporphyrinatoiron(III) - 7-PF 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-(trifluoromethyl)porphyrinatoiron(III) - Mb myoglobin - Mb(7-PF) deoxy-Mb reconstituted with 7-PF - Mb(3,7-DF) deoxy-Mb reconstituted with 3,7-DF - Mb(2-MF) deoxy-Mb reconstituted with 2-MF - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect correlated spectroscopy     

Isolation and characterization of human liver mRNA

Poly(A)-RNA isolated from human liver shows a predominant size in the 18 S region. It can be translated into a broad range of proteins up to molecular weights of more than 100 000 with predominant translation products in the 45 000-55 000 region. Full length cDNA has been transcribed and used for a complexity analysis of this mRNA giving a total sequence complexity of about 7 000 different mRNA species.     

Electron microscopy of 26 S complex containing 20 S proteasome.

A high molecular weight protease complex (26 S complex) involved in the intracellular protein degradation of ubiquitinated proteins was purified from rat liver and studied by electron microscopy. The most prevalent molecular species with best preserved symmetrical morphology had two large rectangular terminal structures attached to a thinner central one having four protein layers. We concluded that they were the closest representation of the 26 S complex so far reported. The central structure was identified as 20 S proteasome and the terminal one as recognition units for ubiquitinated proteins.     

A new large form of transcarboxylase with six outer subunits and twelve biotinyl carboxyl carrier subunits.

A new form of transcarboxylase has been isolated which has a molecular weight of 1,200,000, an s20,w of 26 S, and contains 12 biotinyl groups. Transcarboxylase as isolated previously has a molecular weight of 790,000, an s20,w of 18 S, and contains six biotinyl groups. The larger species of enzyme consists of a central hexameric subunit with six dimeric outer subunits attached to it by biotinyl carboxyl carrier proteins, three each at the opposite faces of the central subunits. This larger species is stable at pH 5.5, but dissociates to the 18 S species at pH values near neutrality with loss of a set of three of the outer subunits with two of the biotinyl carboxyl carrier proteins still attached to each of these subunits. The dissociation to the 18 S form occurs by several rapidly reversible steps and under certain conditions of centrifugation multiple peaks are observed as a consequence of the occurrence of different forms of enzyme with variable numbers of the outer subunits attached to the 18 S enzyme. The s20,w value of the so-called 26 S enzyme varies with conditions. Isolated 18 S enzyme has been combined with isolated outer subunits to form active 26 S enzyme. The newly enzyme is a normal form but has not been isolated previously because of its dissociation to the 18 S form at neutral pH. A procedure is described for the isolation of the 26 S form in a highly purified state. The molecular weight of the enzyme has been determined by high speed meniscus depletion. In addition, a procedure is described for dissociation of the 26 S form of the enzyme and isolation of the resulting outer subunits with the biotinyl subunits still attached to it. Evidence is presented that all six outer subunits participate in the enzymatic reaction which includes the demonstration that; (a) all 12 biotins of the 26 S form of the enzyme can be carboxylated with [3-14C]methylmalonyl coenzyme A; (b) there is an increase in enzymatic activity when the outer subunits are combined with the normal 18 S enzyme with formation of the 26 S enzyme; and (c) a 26 S form of the enzyme is active which is prepared by combination of inactive 18 S trypsin-treated transcarboxylase with the outer subunits. The trypsin-treated 18 S enzyme is inactive because trypsin removes the biotin as biotinyl peptides and the 26 S enzyme is active because of the second set of active outer subunits.     

Characterization of genes with a putative key role in the parasitic lifestyle of the nematode Strongyloides ratti

SUMMARY Parasitic nematodes are significant pathogens of humans and other animals. The molecular and genetic basis of animal parasitism is not yet fully understood. Strongyloides spp. are a genus of gastrointestinal nematodes of which species infect approximately 100–200 million people worldwide. S. ratti is a natural parasite of the rat, and a useful and amenable laboratory model. Previous EST and microarray analyses of the S. ratti life cycle have identified genes whose expression was specific, or biased, to the parasitic adult stage, suggesting that they may play a key role in parasitism in this species. Here we have further investigated the expression of these genes (by RT-PCR) throughout the S. ratti life-cycle. We produced recombinant proteins in vitro for a subset of these genes, which were used in Western blot analyses to investigate the distribution of the gene products among different stages of the S. ratti life cycle. We tested the efficacy of these recombinant proteins as anti-S. ratti vaccines. One of the proteins was detected in the excretory/secretory products of the parasitic stages.     

Neuronal and Glial Marker Proteins in the Evaluation of the Protective Action of MK 801

A quantitative dot immunobinding procedure was used to quantify glial [the S-100 protein and the glial fibrillary acidic (GFA) protein] and neuronal (the 68- and 200-kDa neurofilament polypeptides, neuron-specific enolase, and neuronal cell adhesion molecule) markers. A single intraperitoneal administration of 10 mg/kg of MK 801 blocked the increase of glial parameters and the decrease in content of neuronal marker proteins that occurred as the response to an N-methyl-D-aspartate (NMDA) lesion in the rat hippocampus. The degradation products of GFA protein and the 68-kDa neurofilament polypeptide that were induced by the NMDA lesion did not appear after MK 801 treatment. This study shows that brain-specific proteins are a set of precise tools for the evaluation of neuroprotective effects of antagonists to excitatory amino acids.     

A BRAIN-SPECIFIC PROTEIN FROM OCTOPUS VULGARIS, LAM

Abstract— Several major brain-specific proteins have been detected in cephalopods by electrophoretic analysis of the soluble proteins extracted from the optic lobes and other organs of octopus and by 2-dimensional fractionation of the soluble proteins from optic lobes and hepatopancreases of octopus and squid. One of the brain-specific proteins from octopus, identified as 0-1, has been purified by chromatography on DEAE-cellulose, Sephadex G-150, and DEAE-Sephadex. The protein appears to be pure on the basis of several physicochemical criteria. Amino acid analysis indicates a high content of glutamic and aspartic acids or their amides (or both) and the lack of tryptophan. A molecular weight of 17,000 has been calculated from sodium dodecyl sulphate-gel electrophoresis, gel filtration and ultracentrifugation analysis. The preparation of a specific rabbit antiserum against 0-1 has allowed its determination by agar immunodiffusion and complement fixation techniques. With the latter procedure it has been shown that the protein is absent outside the nervous system, is present in a concentration of several mg/g wet weight in octopus brain and is widely distributed within the octopus central and peripheral nervous system and in several molluscan species. It is also present in optic lobes of octopus at early stages of development.     

Characterization of sulfated forms of the pro-opiomelanocortin amino-terminal glycopeptide in rat intermediate lobe cells

Explants of rat neurointermediate lobes were incubated in the presence of radioactive amino acids, sugars or sulfate and the labeled proteins were separated by two-dimensional gel electrophoresis. A double series of acidic peptides (Mr = 16,000-21,500) were identified as variant forms of the amino-terminal glycopeptide of pro-opiomelanocortin (N-POMC). The series of peptides with the higher molecular weights (Mr = 18,000-21,500) contain a tryptic fragment (tentatively identified as the tryptic peptide of the "joining peptide": sequence 77 to 93 of rat POMC) which is absent from the forms of the lower molecular weight series (Mr = 16,000 to 18,000). Pulse-chase studies further showed that the high molecular weight forms of N-POMC could be post-translationally cleaved albeit slowly into the species of Mr = 16,000-18,000 which constitute, at least in part, the final maturation products of the N-terminal region of the precursor molecule. All the variant forms of the N-POMC glycopeptide could be labeled with [35S]sulfate. Our results strongly suggest that most of the sulfate groups are attached to N-linked oligosaccharide side chains of N-POMC. We therefore propose that one of the final maturation products of the N-terminal portion of POMC in rat intermediate lobes is a sulfated glycopeptide (Mr = 16,000-18,000) composed of the 1-74 sequence of rat POMC.     

Limited proteolysis of brain-specific protein S100. Isolation, physico-chemical and immunochemical characteristics of the neuropeptide AT-1-1

Six peptides (presumably products of natural protein S100 catabolism) were isolated from bovine brain extracts by hydrophobic chromatography, affinity chromatography on immobilized antiprotein S100 antibodies, gel filtration and chromatography on TSK HW-40 columns in a methanol: water system. At 10(-12) M, peptide AT-I-I caused a 70% inhibition of the specific binding activity of endogenous benzodiazepine brain receptors. When used at higher concentrations (10(-9)-10(-5) M), AT-I-I inhibited the binding activity of central serotonin, dopamine and m-cholinoreceptors. Immunochemical analysis revealed the presence of identical material in rat brain glial cell nuclei (astrocytes). Using a solid phase immunoenzymatic assay, it was shown that peptide AT-I-I was not identical to any other of the 14 peptides tested (commercial preparations). Data from immunochemical analysis testified to the species non-specificity of AT-I-I. It was concluded that in brain tissue natural proteolysis of proteins S100 leads to the formation of biologically active oligopeptide products that are involved, in particular, in the modulation of the functional activity of central benzodiazepine receptors.     

Purification of Drosophila ribosomal proteins. Isolation of proteins S8, S13, S14, S16, S19, S20/L24, S22/L26, S24, S25/S27, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 7/8, 9, and 11

The proteins of Drosophila melanogaster embryonic ribosomes were separated into seven groups (A80 through G80) by stepwise elution from carboxymethylcellulose with lithium chloride at pH 6.5 by procedures previously described [Chooi, W. Y., Sabatini, L. M., MacKlin, M. D., & Fraser, W. (1980) Biochemistry 19, 1425-1433]. Three relatively acidic proteins, S14, S25/S27, and 7/8, have now been isolated from group A80 by ion-exchange chromatog raphy on carboxymethylcellulose eluted with a linear gradient of lithium chloride at pH 4.2. Fractions containing the relatively basic proteins (groups B80 through G80) were furher combined into a total of 24 "pools". The criterion for combination was the migration patterns in one-dimensional polyacrylamide gels containing sodium dodecyl sulfate (NaDodS04) of every fifth fraction from the carboxymethylcellulose column. Each pool contained between 1 and 12 major proteins. Proteins S8, S13, S16, S19, S20/L24, S22/L26, S24, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 9, and 11 have now been isolated from selected pools by gel filtration through Sephadix G-100. The amount of each protein recovered from a starting amount of 1.8 g of total 80S proteins varied form 0.2 to 10.8 mg. Five proteins had no detectable contamination, and in each of the others the impurities were no greater than 9%. The amino acid composition of the individual purified proteins was determined. The molecular weights of the proteins were estimated by polyacrylamide gel electrophoresis in NaDodSO4.     

S100 proteins in cartilage: Role in arthritis

S100 proteins are low molecular weight calcium binding proteins expressed in vertebrates. The family constitutes 21 known members that are expressed in several tissues and cell types and play a major role in various cellular functions. Uniquely, members of the S100 family have both intracellular and extracellular functions. Several members of the S100 family (S100A1, S100A2, S100A4, S1008, S100A9, S100A11, and S100B) have been identified in human articular cartilage, and their expression is upregulated in diseased tissue. These S100 proteins elicit a catabolic signaling pathway via receptor for advanced glycation end products (RAGE) in cartilage and may promote progression of arthritis. This review summarizes our current understanding of the role of S100 proteins in cartilage biology and in the development of arthritis.     

S100B and S100A6 differentially modulate cell survival by interacting with distinct RAGE (receptor for advanced glycation end products) immunoglobulin domains

S100 proteins are EF-hand calcium-binding proteins with various intracellular functions including cell proliferation, differentiation, migration, and apoptosis. Some S100 proteins are also secreted and exert extracellular paracrine and autocrine functions. Experimental results suggest that the receptor for advanced glycation end products (RAGE) plays important roles in mediating S100 protein-induced cellular signaling. Here we compared the interaction of two S100 proteins, S100B and S100A6, with RAGE by in vitro assay and in culture of human SH-SY5Y neuroblastoma cells. Our in vitro binding data showed that S100B and S100A6, although structurally very similar, interact with different RAGE extracellular domains. Our cell assay data demonstrated that S100B and S100A6 differentially modulate cell survival. At micromolar concentration, S100B increased cellular proliferation, whereas at the same concentration, S100A6 triggered apoptosis. Although both S100 proteins induced the formation of reactive oxygen species, S100B recruited phosphatidylinositol 3-kinase/AKT and NF-kappaB, whereas S100A6 activated JNK. More importantly, we showed that S100B and S100A6 modulate cell survival in a RAGE-dependent manner; S100B specifically interacted with the RAGE V and C(1) domains and S100A6 specifically interacted with the C(1) and C(2) RAGE domains. Altogether these results highlight the complexity of S100/RAGE cellular signaling.     

Replication of Sindbis Virus V. Polyribosomes and mRNA in Infected Cells

Cells infected with wild-type Sindbis virus contain at least two forms of mRNA, 26S and 49S RNA. Sindbis 26S RNA (molecular weight 1.6 x 10(6)) constitutes 90% by weight of the mRNA in infected cells, and is thought to specify the structural proteins of the virus. Sindbis 49S RNA, the viral genome (molecular weight 4.3 x 10(6)), constitutes approximately 10% of the mRNA in infected cells and is thought to supply the remaining viral functions. In cells infected with ts2, a temperature-sensitive mutant of Sindbis virus, the messenger forms also include a third species of RNA with a sedimentation coefficient of 33S and an apparent molecular weight of 2.3 x 10(6). Hybridization-competition experiments showed that 90% of the base sequences in 33S RNA from these cells are also present in 26S RNA. Sindbis 33S RNA was also isolated from cells infected with wild-type virus. After reaction with formaldehyde, this species of 33S RNA appeared to be completely converted to 26S RNA. These results indicate that 33S RNA isolated from cells infected with either wild-type Sindbis or ts2 is not a unique and separate form of Sindbis RNA.