ABSTRACT
S100A6 (calcyclin) is a member of the large S100 Ca2+-binding protein family, considered to activate several processes along the calcium signal transduction pathway including the regulation of cell growth, proliferation, secretion, and exocytosis. In the present study, the distribution of S100A6 in the rat nervous system was examined by immunohistochemistry with a goat antiserum against recombinant human S100A6, which recognizes the rat S100A6 homologue. The main S100A6-immunoreactive elements were 1) neuronal somata and dendrites in some specific regions of the limbic system (e.g., the basolateral amygdaloid nucleus, ventral tip of the CA1-subicular border region, entorhinal cortex, and parasubiculum), most of which were identified as a subpopulation of pyramidal cells; 2) olfactory receptor cells and olfactory nerve fibers and terminals in the olfactory bulb; 3) some tracts of the hindbrain and spinal cord (e.g., the spinal trigeminal tract, solitary tract, dorsal root fibers, and the tract of Lissauer) and their terminals (e.g., the principal sensory trigeminal nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, marginal zone, substantia gelatinosa, and proper sensory nucleus of the dorsal horn), as well as some sensory neurons of their origins in the dorsal root and trigeminal ganglia; 4) a subpopulation of astrocytes in the white matter (e.g., the corpus callosum, cingulum, external capsule, internal capsule, and fimbria of the hippocampus) and around the ventricles; 5) some ependymal cells, especially around the central canal; and 6) Schwann cells. These results will improve our understanding of the diverse function of Ca2+-binding proteins in the CNS.
Subject(s)
Cell Cycle Proteins , Nervous System/metabolism , Neuroglia/metabolism , Neurons/metabolism , S100 Proteins/metabolism , Animals , Blotting, Western , Brain/cytology , Brain/metabolism , Cerebral Ventricles/cytology , Cerebral Ventricles/metabolism , Ependyma/cytology , Ependyma/metabolism , Humans , Immunohistochemistry , Limbic System/cytology , Limbic System/metabolism , Male , Nervous System/cytology , Olfactory Bulb/metabolism , Olfactory Mucosa/innervation , Olfactory Mucosa/metabolism , Rats , Rats, Wistar , S100 Calcium Binding Protein A6 , Tissue Distribution/physiologyABSTRACT
Unbalanced translocations generating trisomy of 1q are common in Wilms tumor (WT). We present eight unbalanced 1q translocations from seven tumors and a review of the literature. An unbalanced translocation that results in a der(16)t(1q;16q) chromosome represents more than half of the published +1q generating translocations in WT. This translocation is also common to many other tumor types. Four of the tumors presented here contained this chromosome and,in two cases, it was the primary acquired cytogenetic abnormality within the tumor. The other four translocations involved 9q31, 9q34, 17p1?, and 21p11 as the partner to 1q. The chromosome 17 and 21 translocations occurred within the same tumor as apparently independent events. In contrast with the 16q translocations, these other translocations were secondary cytogenetic events, thereby indicating a role in tumor progression rather than initiation. Probes mapping to 1q12 and 1q21 were employed for fluorescence in situ hybridization and it was demonstrated that different 1q breakpoints are possible. In this series, the majority of breakpoints either mapped to 1q12 or were centromeric to this region.
Subject(s)
Chromosomes, Human, Pair 1 , Translocation, Genetic , Trisomy , Wilms Tumor/genetics , Humans , In Situ Hybridization, Fluorescence , KaryotypingABSTRACT
S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S10100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0-5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands.
Subject(s)
Calcium-Binding Proteins/analysis , Cell Cycle Proteins , S100 Proteins/analysis , Sublingual Gland/chemistry , Submandibular Gland/chemistry , Animals , Animals, Newborn , Female , Immunoenzyme Techniques , Male , Rats , Rats, Sprague-Dawley , S100 Calcium Binding Protein A6 , Sublingual Gland/growth & development , Submandibular Gland/growth & developmentABSTRACT
The Ca(2+)-binding proteins S100A1, S100A2, S100A4, S100A6 and S100B were evaluated immunohistochemically in normal skin and skin appendage tumours. Epidermal basal cells, epithelial cells of sebaceous glands, hair follicle sheet epithelia and eccrine duct reacted strongly with an antiserum against human S100A2 but were nonreactive or weakly reactive to S100A1, S100A4, S100A6 and S100B. Varying types of skin appendage tumours and most peripheral cells in tumour nests of basal cell carcinoma and squamous cell carcinoma showed positive S100A2 immunoreactivity in neoplastic cells corresponding to basal cells but were nonreactive or faintly reactive for other S100 proteins. Langerhan's cells and melanocytes were labelled by S100B. Basophilic cells of calcifying epithelioma were occasionally stained with S100A2 antiserum. Eccrine poroma did not react with any S100 antiserum. Mixed tumours of the skin containing neoplastic myoepithelial cells stained strongly for S100A2 and S100B but only faintly for S100A1, S100A4, S100A6. This is the first report on selective evaluation of different S100 proteins in normal skin. These antibodies are valuable tools for better characterization of skin appendage tumours.
Subject(s)
Calcium-Binding Proteins/metabolism , Carcinoma/metabolism , Hair Follicle/metabolism , Sebaceous Gland Neoplasms/metabolism , Skin Neoplasms/metabolism , Sweat Gland Neoplasms/metabolism , Animals , Basophils/metabolism , Carcinoma/pathology , Hair Follicle/pathology , Humans , Immunoenzyme Techniques , Langerhans Cells/metabolism , Melanocytes/metabolism , Rabbits , S100 Proteins , Sebaceous Gland Neoplasms/pathology , Skin/metabolism , Skin Neoplasms/pathology , Sweat Gland Neoplasms/pathologyABSTRACT
S100 Ca2+-binding proteins became of major interest because of their differential expression in tissues and their association with human diseases. Earlier studies showed that 13 S100 genes are located as a cluster on human chromosome 1q21. Since a number of mouse S 100 genes, such as S100A4 and S100A6, have been localized to a syntenic region on mouse chromosome 3, we investigated if the S100 gene cluster exists in mouse and is structurally conserved during evolution. First we identified the cDNA sequences of mouse S100A1, S100A3 and S100A5. Then we isolated a 490 kb mouse YAC clone which gives a specific signal by FISH most likely on chromosome 3. Hybridization studies with different mouse S100 cDNAs revealed that eight mouse S100 genes are arranged in a clustered organization similar to that in human. The linkage relationships between the genes S100A8-S100A9 and S100A3-S100A4-S100A5-S100A6 were conserved during divergence of human and mouse about 70 million years ago. However, the separation of the mouse S100 genes S100A1 and S100A13 in comparison to the human linkage group suggests rearrangement processes between human and mouse. Our data demonstrate that the S100 gene cluster is structurally conserved during evolution. Further studies on the genomic organization of the S100 genes including various species could generate new insights into gene regulatory processes and phylogenetic relationships.
Subject(s)
Multigene Family , S100 Proteins/genetics , Amino Acid Sequence , Animals , Calcium-Binding Proteins/genetics , Chromosome Mapping , Chromosomes, Artificial, Yeast/genetics , DNA, Complementary/analysis , DNA, Complementary/genetics , Evolution, Molecular , Humans , In Situ Hybridization, Fluorescence , Mice , Molecular Sequence Data , Polymerase Chain Reaction , S100 Proteins/chemistryABSTRACT
The 6-pyruvoyl-tetrahydropterin synthase (PTPS) is the second enzyme in the biosynthetic pathway from GTP to tetrahydrobiopterin (BH4). BH4 is an essential cofactor of NO synthases and aromatic amino acid hydroxylases, the latter being responsible for hepatic phenylalanine degradation and monoamine neurotransmitter biosynthesis. BH4 deficiency due to autosomal recessive mutations in the human gene for PTPS leads to a broad range of phenotypes ranging from mild hyperphenylalaninemia to high phenylalanine levels concomitant with neurotransmitter depletion. An animal model to study PTPS deficiency is thus desired to investigate the molecular basis of the disease and its variability. Here, we report on the isolation and recombinant expression of the mouse PTPS gene, Pts. It is located on chromosome 9C-D and contains six exons with an open reading frame of 144 codons. The derived protein monomer has a molecular mass of 16187 Da and shows 82% and 93% identity to its human and rat counterparts, respectively. The mouse PTPS was expressed in bacterial cells and purified to homogeneity. The kinetic properties of the recombinant protein, apparent Km of approximately 10 microM and k(cat) of 0.27 s(-1), were similar to the native mouse enzyme in liver and brain extracts, and to the corresponding human and rat PTPS.
Subject(s)
Chromosome Mapping , Phosphorus-Oxygen Lyases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , Humans , In Situ Hybridization, Fluorescence , Kinetics , Mice , Molecular Sequence Data , Phosphorus-Oxygen Lyases/metabolism , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
The purpose of this study was to evaluate the expression of the Ca(2+)-binding S100 proteins S100A1, S100A2, S100A3, S100A4, S100A6 and S100B in normal skin. These immunohistochemical staining patterns were compared with those in melanocytic lesions. Paraffin-embedded tissue of normal skin adjacent to 26 naevi, 39 primary cutaneous melanomas and 14 cutaneous melanoma metastases was incubated with polyclonal antibodies against the recombinant human S100 proteins (S100A1, S100A2, S100A3, S100A4, S100A6, S100B) using the alkaline phosphatase anti-alkaline phosphatase method. The S100A2 antibody stained the basal layer of the epidermis and hair follicles of normal skin. Four of 39 primary cutaneous melanomas were positive for S100A2, whereas none of the metastases or naevi showed any immunoreactivity. The S100A3 antibody only stained the inner root sheath cuticle of some hair follicles but no melanocytes or melanocytic lesions. Staining of S100A4 was weak and thus omitted to further analysis. S100A6 faintly labelled keratinocytes. Langerhans' cells, melanocytes and sweat glands. S100A6 immunoreaction was found in two of seven junctional naevi, five of seven compound naevi, and all dermal and blue naevi. There was an intense cytoplasmatic reaction for S100A6 in all primary cutaneous melanomas and in nine of 14 (64%) metastases, S100B was positive in melanocytes and Langerhans' cells, all primaries as well as in the metastases, S100A1 protein was not detected on any of the tissue specimens examined. Whereas S100B and S100A6 antibodies are useful markers for malignant melanoma, expression of S100A4 antibody is too low to be used for immunohistochemical staining. S100A1 and S100A3 antibodies are not expressed in melanocytic lesions and S100A2 is only found in selected tumours. The investigated S100 proteins, including S100B and S100A6, are also expressed in selected elements of normal skin. These findings are important for correct interpretation of staining patterns, when S100 antibodies are used as markers for melanoma or other tumours.
Subject(s)
Melanoma/chemistry , S100 Proteins/analysis , Skin Neoplasms/chemistry , Skin/chemistry , Antibodies, Monoclonal , Hair Follicle/chemistry , Humans , Immunohistochemistry , Neoplasm Proteins/analysis , Nevus/chemistryABSTRACT
PURPOSE: Commercially available polyclonal antibodies against a mixture of bovine brain S100 proteins have become an established marker for immunohistochemical characterization of malignant melanoma. However, the commercially available antibodies used are undefined and to date, 13 different human S100 proteins are known. The purpose of this study was to examine the expression of 4 newly available polyclonal antibodies against the human recombinant Ca(2+)-binding S100 proteins, S100A1, S100A2, S100A4 and S100A6, in cutaneous melanoma and to correlate these findings with the standard S100 staining as well as with the metastatic potential of the primary. METHODS: 39 formalin-fixed paraffin-embedded primary cutaneous melanomas were incubated with polyclonal antibodies against recombinant human S100 proteins using the APAAP method. The extent of staining was qualitatively assessed and a staining index was calculated. Findings were correlated to the metastatic potential and the overall survival in all patients. RESULTS: Staining with antibodies against human S100A6 as well as with antibodies against conventional bovine S100 proteins was positive in all specimens. No correlation was found between the extent of protein expression and patients' outcome for standard S100 staining as well as for S100A6. S100A1, S100A2 and S100A4 staining could not be used for statistical analysis due to their low expression in melanoma. CONCLUSION: Staining was positive using S100A6 antibodies in all specimens, the quality being inferior to the commercially available S100 antibody. Highly specific and well characterized antibodies against the individual S100 proteins are now available for future immunohistochemical characterization studies in melanomas.
Subject(s)
Calcium-Binding Proteins/analysis , Melanoma/immunology , Neoplasm Proteins/analysis , Nerve Growth Factors/analysis , S100 Proteins/analysis , Skin Neoplasms/immunology , Adult , Aged , Aged, 80 and over , Female , Follow-Up Studies , Humans , Immunohistochemistry , Male , Middle Aged , Recombinant Proteins/analysis , S100 Calcium Binding Protein beta SubunitABSTRACT
Immunohistochemical distribution of the Ca2+ binding proteins S100A1, S100A2, S100A4, S100A6, S100B, and bone morphogenetic protein (BMP) in chondro-osseous tumors and lesions, both benign and malignant, was investigated using specific anti S100 protein and BMP antibodies. Chondrogenic tumor cells of chondro-osseous lesions were characterized by the presence of marked staining for S100B and BMP, while they were only faintly reactive for S100A1, S100A2, S100A4 and S100A6. Dense fibrous connective tissue in osseous tumor and ossifying fibroma showed moderate immunoreactivity for S100A1, S100A4 and BMP. Immunoreactivity of S100A2, prominent in epidermal basal cells and associated or homologous cells of epidermal tumors or skin appendages tumors, was not found in cartilage and bone forming cells. Biological roles of S100B in chondroid cells may involve Ca2+-signaling in precalcified tissue, cytoskeletal protein organization and matrix formation since glycosaminoglycan synthesis is mediated by calcium ions. S100B positive cells in chondro-osseous structures also strongly expressed BMP. The present study allowed us to conclude that among the S100 proteins, the S100B in particular and S100A1, S100A4 and S100A6 but not S100A2 may be involved in the process of tumorigenesis of chondro-osseous tumors and BMP may have an important role in the chondroid and osseous differentiation. The detailed biological role of S100 proteins in chondro-osseous tumors is under investigation.
ABSTRACT
In adult rodents, proliferating cells in the subventricular zone of lateral ventricle tangentially migrate into the olfactory bulb, where they become the interneurons. The present immunocytochemical analysis revealed that S100A6 (calcyclin), a specific calcium-binding protein of the S100 family, is restrictedly distributed in some astrocytes in the tangential migration pathway of the rat. These results suggest that a particular type of astrocytes containing S100A6 is associated with the tangential migration pathway.
Subject(s)
Astrocytes/chemistry , Calcium-Binding Proteins/analysis , Cell Cycle Proteins , Cell Movement , Cerebral Ventricles/cytology , Neurons/cytology , S100 Proteins , Animals , Antibodies , Astrocytes/cytology , Calcium-Binding Proteins/immunology , Cell Communication , Epidermal Growth Factor/analysis , Epidermal Growth Factor/immunology , Male , Microscopy, Confocal , Olfactory Bulb/cytology , Rats , Rats, Wistar , S100 Calcium Binding Protein A6ABSTRACT
The Ca(2+)-binding S-100 proteins are involved in the regulation of a number of cellular processes and an altered expression has been reported in several neoplastic tissues. Tissue specimens of normal salivary glands (n = 23), pleomorphic adenomas (n = 60), basal cell adenomas (n = 6), canalicular ademomas (n = 2), myoepitheliomas (n = 2), adenoid cystic carcinomas (n = 26) and adenocarcinomas NOS (n = 11) were evaluated for the expression of S-100A1, S-100A2, A-100A4, S-100A6 and S-100B by using highly specific polyclonal and monoclonal antibodies generated against the recombinant human protein. In normal salivary glands, the ductal cells showed mild to intense immunoreactivity for S-100A1, S-100A2, S-100A4 and S-100A6, while S-100B was observed in nerve fibers in the connective tissue. The normal myoepithelial cells were unreactive. In pleomorphic adenoma, the luminal tumor cells of the duct-like structures showed moderate to intense immunoreactivity for S-100A2, while reactivity for S-100A1, S-100A4 and S-100A6 was relatively weak. The non-luminal cells, also termed neoplastic myoepithelial cells, showed immunoreactivity for S-100B, while tumor cells in the solid, myxoid and chondroid areas were immunoreactive for S-100A1, S-100A4, S-100A6 and S-100B. The non-luminally located tumor cells in basal cell adenomas and canalicular adenomas, and numerous tumor cells in clusters in myoepitheliomas were intensely reactive for S-100A2. In adenoid cystic carcinomas and in adenocarcinomas not otherwise specified, the luminal cells forming the tubular or cribriform structures were markedly positive for S-100A2 and/or S-100A6. Squamous metaplastic cells in salivary tumors showed intense immunoreactivity for S-100A2. The results of the present study suggest that the majority of the tumor cells in salivary neoplasms, despite the most heterogeneous tumor cell differentiation, express S-100 proteins more heterogeneously than the normal glandular ducts. The salivary ducts in normal glands, the luminal tumor cells and squamous metaplastic cells in the neoplastic lesions were intensely immunoreactive for S-100A2 as compared to S-100A1, S-100A4 or S-100A6. In contrast, the non-luminal tumor cells showed a rather heterogeneous expression of the S-100 proteins.
Subject(s)
S100 Proteins/classification , Salivary Gland Neoplasms/pathology , Adenocarcinoma/pathology , Adenoma/pathology , Adenoma, Pleomorphic/pathology , Antibodies, Monoclonal , Antibodies, Neoplasm , Carcinoma, Adenoid Cystic/pathology , Cell Differentiation , Connective Tissue/innervation , Connective Tissue/pathology , Epithelium/pathology , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Metaplasia , Myoepithelioma/pathology , Nerve Fibers/pathology , S100 Proteins/analysis , S100 Proteins/genetics , Salivary Ducts/pathology , Salivary Glands/innervation , Salivary Glands/pathologyABSTRACT
The S100 Ca(2+)-binding proteins recently became of major interest because of their differential expression in neoplastic tissues, their involvement in metastatic processes, and the clustered organization of at least 10 S100 genes on human chromosome 1q21, a region frequently rearranged in several tumors. As a first attempt towards a specific and differentiated immunohistochemical classification of human tumors, we produced, purified and characterized a number of human recombinant S100 proteins and raised specific polyclonal antibodies. Their distinct cellular and intracellular localization was examined by immunohistochemical methods in normal and cancerogenic human tissues and cell lines. S100A1 and S100A2 can be detected in a few normal tissues only, whereas S100A4, S100A6, and S100B are expressed at higher levels in cancer tissues. In the future, these S100 antibodies will potentially be of great value in cancer diagnosis and therapy.
Subject(s)
Calcium-Binding Proteins/analysis , Neoplasms/chemistry , S100 Proteins/analysis , Adenocarcinoma/chemistry , Antibody Specificity , Breast Neoplasms/chemistry , Humans , Immunohistochemistry , Recombinant Proteins/analysis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , S100 Proteins/immunology , S100 Proteins/isolation & purification , Tumor Cells, CulturedABSTRACT
Human recombinant alpha-parvalbumin (PVwt) and nine mutant proteins, containing inactivating substitutions at positions essential for Ca2+ binding in the CD Ca(2+)-binding site (PVE62V, PVD51A, PVD51A,62V), the EF site (PVE101V, PVD90A, PVD90A,E101V) or in both (PVE62V,E101V, PVD51A,D90A, PVD51A,E62V,D90A,E101V), were expressed and purified. Flow dialysis revealed that PVwt binds 2 Ca2+ with equal K'Ca, of 2.3 x 10(7) M-1 and that Mg2+ competes with a K'Mg.compet. of 4.9 x 10(3) M-1. The three mutants with an inactivated CD site bind 1 Ca2+ with K'Ca, of 2.0 to 2.3 x 10(7) M-1 and K'Mg.compet. of 3.4 to 4.6 x 10(3) M-1, i.e. very similar to those of PVwt. The mutants with an inactivated EF site bind 1 Ca2+ with K'Ca values of 7.9 x 10(6), 4.5 x 10(6) and 3.6 x 10(6) M-1 for PVD91A, PVE102V and PVE101V,D91A, respectively. The K'Mg.compet values of these mutants are about 4-times lower than in PVwt. The three mutants with both sites inactivated bind neither Ca2+ nor Mg2+. After excitation at 259 nm, human PV, which contains neither Tyr nor Trp, shows maximal fluorescence emission at 283 nm. Binding of either Ca2+ or Mg2+ to PVwt or to mutants with an inactivated EF site lead to a 1.8-fold decrease in fluorescence intensity, whereas the mutants with an inactivated CD show only a very slight decrease upon binding of Ca2+ or Mg2+. Specific antibodies against human alpha-parvalbumin were raised in rabbits. Their reactivity was tested against the mutant proteins, and their potential value for location and functional studies was investigated.
Subject(s)
Calcium/metabolism , Magnesium/metabolism , Parvalbumins/chemistry , Parvalbumins/metabolism , Animals , Antibody Specificity , Binding Sites , Cell Extracts , Cerebellum , Escherichia coli/genetics , Humans , Immune Sera , Isoelectric Point , Molecular Sequence Data , Molecular Weight , Mutagenesis, Site-Directed , Mutation , Parvalbumins/genetics , Parvalbumins/isolation & purification , Protein Binding , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
S100A12 has been isolated from human neutrophils. The molecular weight and the amino acid sequence of S100A12 was determined by electrospray-mass spectrometry, tandem mass spectrometry and Edman microsequence analysis. Interestingly, a sequence comparison of S100A12 with all known human S100 proteins revealed that S100A12 is the most divergent of the S100 proteins.