Synthesis by a clonal line of rat glial cells of a protein unique to the nervous system

The synthesis of a protein unique to the nervous system, the “S100-protein,” has been studied in a clonal line of rat glial cells. It has been shown that these cells do not begin to accumulate “S100-protein” until the cultures enter a phase of density-dependent inhibition of cell proliferation. Further experiments indicate that the regulation of “S100-protein” accumulation resides at least in part in an interaction involving the cell surface.     

Study of bromthymol blue-prealbumin in the blood serum of rats under hyperbaric oxygenation]

Three-fold increase of BTB-prealbumin (Rm 1.0) in rat serum following fierse convulsions under hyperbaric oxygenation (6 ati, 30-35 min) has been proved by disc electrophoresis. Glial S100-protein and 7-fold increase in the all-organ component of brain BTB-prealbumin were found by immunochemistry to appear in the serum of experimental rats. The consequences of disorders in the blood-brain barrier for non-specific, all-organ proteins and potentialities of protein output from the brain into the blood similarly to neurophysins under hyperbaric oxygenation are discussed.     

Structural and functional insights into RAGE activation by multimeric S100B

Nervous system development and plasticity require regulation of cell proliferation, survival, neurite outgrowth and synapse formation by specific extracellular factors. The EF-hand protein S100B is highly expressed in human brain. In the extracellular space, it promotes neurite extension and neuron survival via the receptor RAGE (receptor for advanced glycation end products). The X-ray structure of human Ca(2+)-loaded S100B was determined at 1.9 A resolution. The structure revealed an octameric architecture of four homodimeric units arranged as two tetramers in a tight array. The presence of multimeric forms in human brain extracts was confirmed by size-exclusion experiments. Recombinant tetrameric, hexameric and octameric S100B were purified from Escherichia coli and characterised. Binding studies show that tetrameric S100B binds RAGE with higher affinity than dimeric S100B. Analytical ultracentrifugation studies imply that S100B tetramer binds two RAGE molecules via the V-domain. In line with these experiments, S100B tetramer caused stronger activation of cell growth than S100B dimer and promoted cell survival. The structural and the binding data suggest that tetrameric S100B triggers RAGE activation by receptor dimerisation.     

S100a0 (alpha alpha) protein in cardiac muscle. Isolation from human cardiac muscle and ultrastructural localization

S100 protein, an acidic and calcium-binding protein, was believed to be localized in the nervous tissue, but recently it has been reported to be mainly present in the cardiac and the skeletal muscles of various mammals in the alpha alpha form (S100a0) at much higher levels than the nervous tissues. We isolated here S100 protein from human cardiac muscles. The isolated cardiac muscle S100 protein showed a single band on electrophoresis at the same position as that of human skeletal muscle S100a0. The amino acid composition of the purified S100 protein was quite similar to that of human skeletal muscle S100a0 or bovine brain S100a0. The immunohistochemical study by use of antibodies monospecific to the alpha subunit of S100 protein (S100-alpha) revealed that S100-alpha was strongly labeled in human myocardial cells, whereas the beta subunit of S100 protein (S100-beta) was not detected in the cells. These results suggest that a predominant form of S100 protein in human myocardial cells is not S100a (alpha beta) or S100b (beta beta), but S100a0 (alpha alpha). In order to determine the ultrastructural localization of S100a0 in mouse cardiac muscle, the direct peroxidase-labeled antibody method was employed. S100a0 was mainly localized in the polysomes in the interfibrillar spaces, the fine filamentous structure of the Z line and fascia adherens of the intercalated disc and in the lumen of junctional sarcoplasmic reticulum.     

Identification of a Chymotrypsin-Like Mast Cell Protease in Rat Brain Capable of Generating the N-Terminus of the Alzheimer Amyloid β-Protein

Abstract: Cleavage after Met⁵⁹⁶ of the β-amyloid precursor protein to generate the N-terminus of β-protein indicates the activity of a protease having chymotrypsin-like specificity. A chymotrypsin-like protease is further implicated in Alzheimer's disease by the increased synthesis of the protease inhibitor α₁-antichymotrypsin in pathologically affected brain regions and by the presence in the amyloid deposits of inactivated forms of α₁-antichymotrypsin (indicating irreversible binding to a target chymotrypsin-like protease). In the present report, we have purified from rat brain a chymotrypsin-like protease that (a) binds with high affinity to human α₁-antichymotrypsin, (b) proteolytically generates a β-protein-containing C-terminal fragment from full-length recombinant human β-amyloid precursor protein, and (c) selectively cleaves methoxysuccinyl-Glu-Val-Lys-Met-p-nitroanilide (a substrate modeling the protease recognition domain for the β-protein N-terminal cleavage site). Amino acid sequences of tryptic fragments of the purified rat brain chymotrypsin-like protease indicate an identity with rat mast cell protease I. Moreover, the ontogeny and compartmentalization of rat brain chymotrypsin-like protease are consistent with those of connective tissue-type mast cells in the meningeal and intracortical perivasculature. Because these areas in human brain form extensive β-amyloid deposits in Alzheimer's disease, Down's syndrome, and hereditary cerebral hemorrhage with amyloidosis of Dutch origin, the present findings suggest that a brain mast cell chymotrypsin-like protease may participate in generating perivascular β-protein, which ultimately aggregates into β-amyloid deposits.     

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.     

Extracranial Sources of S100B Do Not Affect Serum Levels

S100B, established as prevalent protein of the central nervous system, is a peripheral biomarker for blood-brain barrier disruption and often also a marker of brain injury. However, reports of extracranial sources of S100B, especially from adipose tissue, may confound its interpretation in the clinical setting. The objective of this study was to characterize the tissue specificity of S100B and assess how extracranial sources of S100B affect serum levels. The extracranial sources of S100B were determined by analyzing nine different types of human tissues by ELISA and Western blot. In addition, brain and adipose tissue were further analyzed by mass spectrometry. A study of 200 subjects was undertaken to determine the relationship between body mass index (BMI) and S100B serum levels. We also measured the levels of S100B homo- and heterodimers in serum quantitatively after blood-brain barrier disruption. Analysis of human tissues by ELISA and Western blot revealed variable levels of S100B expression. By ELISA, brain tissue expressed the highest S100B levels. Similarly, Western blot measurements revealed that brain tissue expressed high levels of S100B but comparable levels were found in skeletal muscle. Mass spectrometry of brain and adipose tissue confirmed the presence of S100B but also revealed the presence of S100A1. The analysis of 200 subjects revealed no statistically significant relationship between BMI and S100B levels. The main species of S100B released from the brain was the B-B homodimer. Our results show that extracranial sources of S100B do not affect serum levels. Thus, the diagnostic value of S100B and its negative predictive value in neurological diseases in intact subjects (without traumatic brain or bodily injury from accident or surgery) are not compromised in the clinical setting.     

The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding

Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S(A)-protein) of 43 kDa. S(A)-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions (a=118 A; b=53 A, and gamma=102 degrees ) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of S(A)-protein with S-proteins from L. helveticus, Lactobacillus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L. crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-terminal one-third (SAC) of S(A)-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of S(A)-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic S(A)-protein. A calculated S(A)-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei.     

Modification of cell surface antigenicity as a function of culture conditions

Adaptation of monolayer cultures of a clonal line of rat glial cells to suspension culture resulted in the nearly complete loss of certain surface antigens. This change in surface antigenicity was paralleled by the loss of the ability of the cells to accumulate in vitro a protein specific to the nervous system (“S100-protein”). In contrast, when glial cells were co-cultivated in monolayer culture with another cell line apparently lacking these surface antigens, the number of these antigens was markedly increased. The possibility of a causal relationship between the changes in the surface antigenicity and the expression of differentiated function is considered.     

Isolation of a new member of the S100 protein family: amino acid sequence, tissue, and subcellular distribution

A low molecular mass protein which we term S100L was isolated from bovine lung. S100L possesses many of the properties of brain S100 such as self association, Ca++-binding (2 sites per subunit) with moderate affinity, and exposure of a hydrophobic site upon Ca++-saturation. Antibodies to brain S100 proteins, however, do not cross react with S100L. Tryptic peptides derived from S100L were sequenced revealing similarity to other members of the S100 family. Oligonucleotide probes based on these sequences were used to screen a cDNA library derived from a bovine kidney cell line (MDBK). A 562-nucleotide cDNA was sequenced and found to contain the complete coding region of S100L. The predicted amino acid sequence displays striking similarity, yet is clearly distinct from other members of the S100 protein family. Polyclonal and monoclonal antibodies were raised against S100L and used to determine the tissue and subcellular distribution of this molecule. The S100L protein is expressed at high levels in bovine kidney and lung tissue, low levels in brain and intestine, with intermediate levels in muscle. The MDBK cell line was found to contain both S100L and the calpactin light chain, another member of this protein family. S100L was not found associated with a higher molecular mass subunit in MDBK cells while the calpactin light chain was tightly bound to the calpactin heavy chain. Double label immunofluorescence microscopy confirmed the observation that the calpactin light chain and S100L have a different distribution in these cells.     

A new astrocytic cell line which is able to induce a blood-brain barrier property in cultured brain capillary endothelial cells

Astrocytes, a member of the glial cell family in the central nervous system, are assumed to play a crucial role in the formation of the blood-brain barrier (BBB) in vertebrates. It was shown that astrocytes induce BBB-properties in brain capillary endothelial cells (BCEC) in vitro. We now established an astroglial cell line of non-tumoral origin. The cloned cell line (A7) shows a highly increased proliferation rate and expresses the astrocytic marker glial fibrillary acidic protein. Furthermore, the clone A7 expresses S-100-protein and vimentin, which are also expressed by primary cultured astrocytes. This cell line therefore shows general astrocytic features. In addition, we were able to show that A7 cells re-induce the BBB-related marker enzyme alkaline phosphatase in BCEC, when these two cell types are co-cultured. Thus we have a cell line which can be readily cultured in large quantities, shows common astrocyte properties and is able to influence BCEC with respect to a BBB-related feature. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quantitative radioimmunohistochemical method using [125I]-protein A to measure the content of methionine enkephalin in discrete rat brain areas

We report a quantitative radioimmunohistochemical method, using [125I]-protein A in combination with a specific antibody to methionine enkephalin (Met-enk), for determination of the content of this peptide in discrete areas of rat brain. After paraformaldehyde fixation, rat brain sections were incubated with a Met-enk polyclonal antibody, followed by incubation with [125I]-protein A. After autoradiography with 3H-sensitive Ultrofilm, optical densities (OD) were quantified by computerized microdensitometry. The OD obtained were compared to a standard curve, constructed after determination by radioimmunoassay of the Met-enk content in corresponding brain areas from adjacent tissue sections. After comparing 15 different brain areas over a ninetyfold range of concentrations, we found a linear relationship between the content of Met-enk, as determined by radioimmunoassay, and the OD generated by autoradiography. The content of Met-enk in other discrete brain areas can be quantified by interpolation of the OD determined by autoradiography in the standard curve. The method allows, for the first time, precise quantification of peptide concentrations in discrete areas and nuclei from thin sections of rat brain. This technique has a more than 100-fold higher sensitivity than classical radioimmunoassays, with the additional advantage of neuroanatomical localization. It also has the potential for application to the quantification of many other antigens present in brain and other tissues.     

S100a9 Knockdown Decreases the Memory Impairment and the Neuropathology in Tg2576 Mice,AD Animal Model

Inflammation, insoluble protein deposition and neuronal cell loss are important features in the Alzheimer''s disease (AD) brain. To investigate the regulatory genes responsible for the neuropathology in AD, we performed microarray analysis with APP_V717I-CT100 transgenic mice, an animal model of AD, and isolated the S100a9 gene, which encodes an inflammation-associated calcium binding protein. In another AD animal model, Tg2576 mouse brain, and in human AD brain, induction of S100a9 was confirmed. The endogenous expression of S100a9 was induced by treatment with Aβ or CT peptides in a microglia cell line, BV2 cells. In these cells, silencing study of S100a9 showed that the induction of S100a9 increased the intracellular calcium level and up-regulated the inflammatory cytokines (IL-1β and TNFα) and iNOS. S100a9 lentiviral short hairpin RNA (sh-S100a9) was injected into the hippocampus region of the brains of 13-month-old Tg2576 mice. At two months after injection, we found that knockdown of S100a9 expression had improved the cognition decline of Tg2576 mice in the water maze task, and had reduced amyloid plaque burden. These results suggest that S100a9 induced by Aβ or CT contributes to cause inflammation, which then affects the neuropathology including amyloid plaques burden and impairs cognitive function. Thus, the inhibition of S100a9 is a possible target for AD therapy.     

CacyBP/SIP,a calcyclin and Siah-1-interacting protein,binds EF-hand proteins of the S100 family

Recently, a human ortholog of mouse calcyclin (S100A6)-binding protein (CacyBP) called SIP (Siah-1-interacting protein) was shown to be a component of a novel ubiquitinylation pathway regulating beta-catenin degradation (Matsuzawa, S., and Reed, J. C. (2001) Mol. Cell 7, 915-926). In murine brain, CacyBP/SIP is expressed at a high level, but S100A6 is expressed at a very low level. Consequently we carried out experiments to determine if CacyBP/SIP binds to other S100 proteins in this tissue. Using CacyBP/SIP affinity chromatography, we found that S100B from the brain extract binds to CacyBP/SIP in a Ca2+-dependent manner. Using a nitrocellulose overlay assay with 125I-CacyBP/SIP and CacyBP/SIP affinity chromatography, we found that this protein binds purified S100A1, S100A6, S100A12, S100B, and S100P but not S100A4, calbindin D(9k), parvalbumin, and calmodulin. The interaction of S100 proteins with CacyBP/SIP occurs via its C-terminal fragment (residues 155-229). Co-immunoprecipitation of CacyBP/SIP with S100B from brain and with S100A6 from Ehrlich ascites tumor cells suggests that these interactions are physiologically relevant and that the ubiquitinylation complex involving CacyBP/SIP might be regulated by S100 proteins.     

Characterization of Human Brain S100 Protein Fraction: Amino Acid Sequence of S100β

Two major components of human brain S100 fraction were purified by HPLC and an amino acid sequence was elucidated for the S100 beta component. Human S100 proteins showed absorption spectra and amino acid compositions similar to S100 alpha and S100 beta from bovine brain. However, the relative amounts of the human proteins were 4% S100 alpha and 96% S100 beta by weight, while the bovine protein distribution was 47% S100 alpha and 53% S100 beta by weight. An amino acid sequence of human S100 beta was established by analysis of overlapping fragments generated by cyanogen bromide and trypsin cleavage. Three amino acid sequence differences between the human and bovine S100 beta were found at residues 7, 62, and 80. These differences were chemically conservative and compatible with minimum single base changes in the codon structures. These results document that S100 beta is a conserved protein among mammals and provide the necessary foundation for current clinical studies.     

Expression and Possible Role of S4(SSM4) Genes in the Styles of Self-incompatible Pear Cultivar and Its Self-compatible Mutant

Stylar soluble proteins in self-incompatible “Nijisseiki” (S2S4), self-compatible “Osa-Nijisseiki” ( S2SSM4, SM means stylar-part mutant) and its progeny were analyzed by isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE).SSM4-allele associated protein, SSM4-protein, existed in the style of “Osa-Nijisseiki” and its progeny. The SSM4-protein expressed only in the stigma of “Osa-Nijisseiki”, whereas in its original variety “Nijisseiki”, S4-protein expressed in the upper and lower parts of the style as well as in the stigma, and its expression amount decreased from the upper part to the lower part. The protein bands analyzed by IEF-PAGE were subjected to RNase activity staining. The results showed that the S4- and the SSM4-proteins have the similar molecular weights (approximately 30 kD) and RNase activity. The specific-activities measured with yeast RNA were similar, equivalent to approximately 275 U·min-1·mg-1 protein. The S SM4-protein showed almost the same inhibitory effects as the S4-protein on the pollen germination and the pollen tube growth with S4- and SSM4-alleles in vitro . From the above results, the reasons of the self-compatibility of “Osa-Nijisseiki” are considered as (1) low expression of the SSM4-gene and (2) the SSM4-gene expression only in stigma.     

S100B promotes the proliferation, migration and invasion of specific brain metastatic lung adenocarcinoma cell line

Brain metastasis frequently occurs in cancer patients and is associated with a poor prognosis. We previously reported that S100B was highly expressed in PC14/B, a specific brain metastatic lung adenocarcinoma cell line, which suggests that it is associated with brain metastasis of lung cancer. However, the role of S100B in brain metastasis remains to be elucidated. In this study, using PC14/B cell line, we found that siRNA mediated depletion of S100B in PC14/B cells led to notable differences in cell proliferation, apoptosis, cell cycle progression, colony formation ability, cell migratory and invasive activity compared with the mock-transfected cells. Therefore, our data suggest that S100B promotes the brain metastasis of lung adenocarcinoma by promoting cell proliferation, preventing apoptosis and increasing cell migration and invasion.     

S100 alpha, CAPL, and CACY: molecular cloning and expression analysis of three calcium-binding proteins from human heart.

Elevated levels of intracellular calcium are a major cause of myocardial dysfunction. To find possible mediators of the deregulated calcium we searched for EF-hand calcium-binding proteins of the S100 family. By PCR technology we identified three members of the S100 protein family (S100 alpha, CACY, and CAPL) in the human heart. We cloned the corresponding cDNAs and examined their expression levels in various human tissues by Northern blot analysis. All three proteins are expressed at high levels in the human heart. Whereas CACY and CAPL mRNAs are expressed ubiquitously, S100 alpha mRNA is restricted to heart, skeletal muscle, and brain. Interestingly, the expression pattern of S100 alpha, CACY, and CAPL in human tissues differs significantly from that in rodent tissues.     

Structural and functional analysis of the S-layer protein crystallisation domain of Lactobacillus acidophilus ATCC 4356: evidence for protein-protein interaction of two subdomains

The structure of the crystallisation domain, SAN, of the S(A)-protein of Lactobacillus acidophilus ATCC 4356 was analysed by insertion and deletion mutagenesis, and by proteolytic treatment. Mutant S(A)-protein synthesised in Escherichia coli with 7-13 amino acid insertions near the N terminus or within regions of sequence variation in SAN (amino acid position 7, 45, 114, 125, 193), or in the cell wall-binding domain (position 345) could form crystalline sheets, whereas insertions in conserved regions or in regions with predicted secondary structure elements (positions 30, 67, 88 and 156) destroyed this capacity. FACscan analysis of L.acidophilus synthesising three crystallising and one non-crystallising S(A)-protein c-myc (19 amino acid residues) insertion mutant was performed with c-myc antibodies. Fluorescence was most pronounced for insertions at positions 125 and 156, less for position 45 and severely reduced for position 7. By cytometric flow sorting a transformant harbouring the mutant S(A)-protein gene (position 125) was isolated that showed an increased fluorescense signal. Immunofluorescence microscopy suggested that the transformant synthesized mutant S(A)-protein only. PCR analysis of the transformant grown in the absence of selection pressure indicated that the mutant allele was stably integrated in the chromosome. Proteolytic treatment of S(A)-protein indicated that only sites near the middle of SAN are susceptible, although potential cleavage sites are present through the entire molecule. Expression in E.coli of DNA sequences encoding the two halves of SAN yielded peptides that could oligomerize. Our results indicate that SAN consists of a approximately 12kDa N and a approximately 18kDa C-terminal subdomain linked by a surface exposed loop. The capacity of S(A)-protein of L.acidophilus to present epitopes, up to approximately 19 amino acid residues in length, at the bacterial surface in a genetically stable form, makes the system, in principle, suitable for application as an oral delivery vehicle.