Biomedical subjects

L W Fisher

Publications and source records attributed to L W Fisher.

At least 19 recordsLinked to original sources

PubMed1995

The X-chromosomal human biglycan gene BGN is subject to X inactivation but is transcribed like an X-Y homologous gene.

We report the mRNA and protein expression levels of human biglycan (BGN) in patients with different numbers of sex chromosomes. BGN maps to the distal long arm of the X chromosome, band Xq28, near the second pseudoautosomal region. BGN expression levels are reduced in 45,X Turner patients and increased in patients with additional sex chromosomes. This is suggestive of a pseudoautosomal gene or a gene that escapes X inactivation and that has an active Y chromosomal copy. However, we also provide evidence from hybrid cell lines that BGN is subject to X inactivation and that there is no homolog on the Y chromosome. This evidence excludes an escape from X inactivation. Moreover, additional Y chromosomes increase BGN expression levels, despite the absence of a Y chromosomal BGN gene. Therefore, another explanation has to be invoked. The "pseudoautosomal expression" of BGN may be attributed to a gene or genes that escape X inactivation and that regulate the transcriptional activity of BGN. This is the first report concerning an X chromosomal gene that does not show the conventional correlation between gene dosage and expression rate known from other X chromosomal genes.

Adult↗

PubMed1995

The enamelin (tuftelin) gene.

This paper reviews the primary structure, characteristics and possible function of tuftelin/enamelin protein. It describes the distribution of tuftelin in the ameloblast cell and in the extracellular enamel matrix, employing high resolution protein-A gold immunocytochemistry. The chromosomal localization of the human tuftelin gene and its possible involvement in autosomally linked Amelogenesis Imperfecta, the most common hereditary disease of enamel, is also discussed.

Ameloblasts↗

PubMed1992

The role of osteonectin in human tooth development: an immunohistological study.

We investigated immunohistologically 160 teeth and dental germs in various stages of tooth development taken from human individuals (13th week of pregnancy to the 24th year of life) to study the osteonectin expression in dental hard tissue. In the course of dentinogenesis, the predentin, the odontoblasts, and their cell processes show a positive osteonectin staining reaction. During cementogenesis, osteonectin is synthesized by cement-producing fibroblasts, cementoblasts, and cementocytes. The expression of osteonectin during dentinogenesis and cementogenesis is closely related to the development of the respective calcified tissue. All cells of the inner and outer enamel epithelium, the cells of the stratum reticulare and stratum intermedium, the ameloblasts, and the enamel substance are osteonectin negative, just as dentin and cement are. The results of this study indicate one important physiological role of osteonectin as a protein associated with the formation of collagen containing mineralizing tissues like human bone, as well as human dentin and cement.

Adolescent↗

PubMed1992

Evaluation of osteonectin as a diagnostic marker of osteogenic bone tumors.

Osteonectin (ON), a 32,000-kd glycoprotein involved in the early steps of mineralization of skeletal tissue, is a recognized differentiation marker of normal osteogenic cells. The expression of ON was evaluated in vitro and in tissue sections by the polyclonal antibody bON II. In different cell cultures immunocytochemistry and molecular biology displayed a nonspecific reaction for the antibody, which showed itself to be useless for the in vitro identification of cells of the osteoblastic lineage. The diagnostic use of bON II antibody was investigated by immunohistochemistry on a series of osteogenic and nonosteogenic bone tumors. A strongly positive stain of the entire neoplastic component of osteosarcoma and osteoblastoma and a weaker stain of the mononuclear component of giant cell tumor and chondroblastoma were observed. On the other hand, stains for chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant fibrous histiocytoma, and brown tumor from hyperparathyroidism were entirely negative. Our results indicate that ON may be helpful in the histologic diagnosis of bone tumors, particularly in differentiating small cell osteosarcoma from other small round cell tumors.

Biomarkers, Tumor↗

PubMed1991

The cDNA cloning and RNA distribution of bovine osteopontin.

We have isolated and sequenced the bovine cDNA (OPN) counterpart of osteopontin. The cDNA is 1356 nucleotides (nt) in length with an open reading frame of 834 nt, encoding a 278-amino acid (aa) protein. Cell-free transcription and translation of OPN RNA resulted in a major species of approx. 40 kDa in size, in agreement with the predicted size of the deduced aa sequence. Northern analysis of bovine OPN RNA indicated the presence of the message in mineralized, as well as soft tissues. A comparison of the deduced aa sequence among various species indicates both regions of similarity and divergence. One prominent region of dissimilarity in bovine OPN compared to all other species is a 22-aa gap which may represent a loss of a potential Ca(2+)-binding loop. Despite the variability among the species, several regions of conservation are apparent, including a hydrophobic leader sequence, a potential site for Asn-linked glycosylation, a stretch of polyaspartic acid residues, and the cell attachment Arg-Gly-Asp tripeptide. Whether bovine OPN enhances cell attachment is unknown. Furthermore, whether the loss of a potential Ca(2+)-binding loop alters the function of OPN would be interesting to determine.

Amino Acid Sequence↗

PubMed1991

Sequencing of bovine enamelin ("tuftelin") a novel acidic enamel protein.

Enamelins are a major group of 28-70-kDa acidic proteins rich in aspartic acid, glutamic acid, serine, and glycine found in developing and mature extracellular enamel; a unique and highly mineralized ectodermal tissue covering vertebrate teeth. They have been associated with the mineralization and structural organization of this tissue. In an attempt to elucidate the primary structure of enamelin, a 2674-base pair cDNA isolated from a bovine ameloblast-enriched, lambda Zap 2 expression library, was sequenced. The identity and localization of the deduced protein was confirmed by amino acid composition, enzyme-linked immunosorbent assay, Western blotting, indirect immunohistochemistry, and high resolution protein-A gold immunocytochemistry. The immunological techniques were employed using antibodies directed against synthetic peptides corresponding to the protein sequence deduced from the cloned cDNA sequence. The results reveal the deduced protein to be a novel acidic enamel protein. It contains 389 amino acids and has a calculated molecular weight of 43,814. Its amino acid composition is similar to that of "tuft" proteins (enamel matrix protein fragments remaining in the mature tissue). It contains one potential N-glycosylation site and 5 cysteine residues. Southern hybridization of the cloned cDNA with genomic bovine DNA indicated the existence of a single gene with one or more introns.

Amino Acid Sequence↗

PubMed1991

Human biglycan gene. Putative promoter, intron-exon junctions, and chromosomal localization.

Biglycan (PG-I, DS-PG-1, PG-S1) is a small cellular or pericellular matrix proteoglycan that is closely related in structure to two other small proteoglycans, decorin (PG-II, PG-S2, DS-PG2, or PG-40) and fibromodulin. The core protein is made up predominantly of a series of 11 tandem repeats that appear to have been used throughout evolution for protein-protein, protein-cell, or cell-cell interactions. The function of biglycan is unclear at this time, but it has been shown to bind transforming growth factor beta in vitro. We have cloned and partially sequenced the approximately 8-kilobase pair human biglycan gene. The gene consists of eight exons including one in the sequence that encodes the 5'-untranslated region of the mRNA. The first and seventh introns are approximately 1 kilobase pair, while the remainder are shorter. With the exception of the first two introns, all of the introns are spread throughout the hydrophobic repeat domain. The 500-base pair 5' to the start of transcription contains several elements that strongly suggest that it contains a significant amount of the gene promoter. The elements include one AP2 and five SP1 consensus sequences. Like in many other genes, the biglycan gene promoter lacks both a CAAT and TATA box but is rich in GC content. Using 3H-labeled cDNA and in situ hybridization and autoradiography of human chromosomes, the human gene was localized to the end of the long arm of the X chromosome (Xq27-ter). The relationship of biglycan to a number of other proteins containing the leucine-rich repeats is discussed with respect to homologies of cysteine regions immediately adjacent to the repeat sequences.

Amino Acid Sequence↗

PubMed1991

Osteogenesis imperfecta: changes in noncollagenous proteins in bone.

The noncollagenous proteins osteonectin, bone sialoprotein, osteocalcin, the small proteoglycan decorin (PG II), and alpha 2-HS glycoprotein (which is synthesized in the liver but highly concentrated in bone) were measured in extracts of cortical bone from 3 type I, 2 type II, 8 type III and 13 type IV patients with osteogenesis imperfecta (OI) and from 7 control subjects. Osteonectin was found to be reduced in the bone of all OI patients. The bone from severely affected type III OI patients contained the lowest levels of osteonectin. In contrast, bone sialoprotein was found to be elevated in the bones of OI patients. The highest levels were found in individuals classified as type IV patients. Osteocalcin and alpha 2-HS glycoprotein concentrations were increased in all OI patients. Decorin levels were not significantly altered in OI bones compared to controls. These changes in the concentrations of the noncollagenous proteins may contribute to the fragility of the OI bone by interfering with complete mineralization and/or normal tissue architecture.

Adolescent↗

PubMed1991

Expression of bone sialoprotein (BSP) in developing human tissues.

Bone sialoprotein (BSP) and its messenger RNA were localized in developing human skeletal and nonskeletal tissues by means of immunohistochemistry and in situ hybridization. Both protein and mRNA were found in mature, bone-forming cells but not in their immature precursors. In addition, osteoclasts displayed positive immunostaining and high densities of autoradiographic grains by in situ hybridization experiments. BSP was expressed in fetal epiphyseal cartilage cells, particularly in hypertrophic chondrocytes of growth plates. Though neither the protein nor the mRNA were identified in a variety of other connective and nonconnective tissues, an unexpected finding was the expression of BSP in the trophoblast cells of placenta. These findings show that BSP is primarily an osteoblast-derived component of the bone matrix expressed at late stages of differentiation. We have also found that osteoclasts produce BSP, possibly as a mediator of cell attachment to bone.

Animals↗

PubMed1991

Decorin interacts with fibrillar collagen of embryonic and adult human skin.

Biglycan (PG-I, BGN) and decorin (PG-II, DCN) are small proteoglycans that have been isolated in cartilage, skin, and bone. Although the function of biglycan is unknown, there is biochemical evidence that decorin interacts with fibrillar collagens (type I, type II). The purpose of this study was to perform immunofluorescence and immunoelectron microscopy and immunoblotting of human embryonic and adult skin with antibodies directed against biglycan and decorin. These antibodies were developed against synthetic peptides of the core proteins of biglycan (amino acid sequence 11-24) and decorin (amino acid sequence 5-17). Immunofluorescence microscopy showed that decorin stained embryonic and adult collagen fibrils. Biglycan did not stain collagen, but it appeared to stain the pericellular matrix of embryonic mesenchymal cells. Immunoelectron microscopy revealed labeling of all collagen fibrils with decorin antibodies regardless of their diameter, often at 60-nm periodicity. Positive stains suggest that most of the labeling was in the gap of the D-period (d and e bands) and also in one of the steps (c band). Decorin was identified by immunoblotting in fetal and adult skin. Also, significant amounts of core protein was identified lacking the dermatan sulfate chain. This study suggests that the core protein of decorin interacts with collagen fibrils although its specific function remains unknown.

Adult↗

PubMed1990

A rat osteogenic cell line (UMR 106-01) synthesizes a highly sulfated form of bone sialoprotein.

The rat osteosarcoma cell line (UMR 106-01) synthesizes and secretes relatively large amounts of a sulfated glycoprotein into its culture medium (approximately 240 ng/10(6) cells/day). This glycoprotein was purified, and amino-terminal sequence analysis identified it as bone sialoprotein (BSP). [35S]Sulfate, [3H]glucosamine, and [3H]tyrosine were used as metabolic precursors to label the BSP. Sulfate esters were found on N- and O-linked oligosaccharides and on tyrosine residues, with about half of the total tyrosines in the BSP being sulfated. The proportion of 35S activity in tyrosine-O-sulfate (approximately 70%) was greater than that in N-linked (approximately 20%) and O-linked (approximately 10%) oligosaccharides. From the deduced amino acid sequence for rat BSP (Oldberg, A., Franzén, A., and Heinegård, D. (1988) J. Biol. Chem. 263, 19430-19432), the results indicate that on average approximately 12 tyrosine residues, approximately 3 N-linked, and approximately 2 O-linked oligosaccharides are sulfated/molecule. The carboxyl-terminal quarter of the BSP probably contains most, if not all, of the sulfated tyrosine residues because this region of the polypeptide contains the necessary requirements for tyrosine sulfation. Oligosaccharide analyses indicated that for every N-linked oligosaccharide on the BSP, there are also approximately 2 hexa-, approximately 5 tetra-, and approximately 2 trisaccharides O-linked to serine and threonine residues. On average, the BSP synthesized by UMR 106-01 cells would contain a total of approximately 3 N-linked and approximately 25 of the above O-linked oligosaccharides. This large number of oligosaccharides is in agreement with the known carbohydrate content (approximately 50%) of the BSP.

Animals↗

PubMed1990

Human bone sialoprotein. Deduced protein sequence and chromosomal localization.

A cDNA encoding the human bone sialoprotein was isolated from a lambda Zap expression library (made from cultured human bone cell poly(A)+ RNA) using radiolabeled rat bone sialoprotein cDNA (Oldberg, A., and Heinegard, D. (1988) J. Biol. Chem. 263, 19430-19432) as a probe. A 5' 1-kilobase EcoRI fragment of the purified 3-kilobase clone was sequenced and found to contain the entire protein-encoding region. The deduced protein sequence revealed a 317-amino acid protein (34,982 Da) containing a 16-amino acid hydrophobic signal sequence and a 33,352-Da protein destined to undergo extensive post-translational modifications before being secreted from the cell. A comparison of the human and rat protein sequences showed extensive (greater than 70%) amino acid identities including the Arg-Gly-Asp (RGD) tripeptide thought to confer the cell attachment activity observed previously for this protein. Also conserved were three regions rich in acidic amino acids and three regions rich in tyrosine. While all three tyrosine-rich regions appear to be composed of a nominal repeat structure, only the two carboxyl-terminal regions that flank the RGD sequence fit all three of the requirements for extensive tyrosine sulfation. Interestingly, human bone sialoprotein, whose final secreted product is approximately 50% carbohydrate, contains no cystines. Northern analysis showed that while bone cells are the major source of bone sialoprotein message production, other tissues may contain trace amounts of this message. Southern hybridization of DNA from human-rodent somatic cell hybrids that have segregated human chromosomes indicated that the gene is located on human chromosome 4. The human bone sialoprotein gene is a single copy gene unlikely to exceed 11.1 kilobases in length. No restriction fragment length polymorphisms were observed with 12 different restriction enzymes in 10 normal individuals.

Amino Acid Sequence↗

PubMed1990

Changes in osteonectin distribution and levels are associated with mineralization of the chicken tibial growth cartilage.

Osteonectin is a calcium-binding matrix protein thought to play a role in regulating calcium distribution in a variety of biologic processes. To examine its role in endochondral bone formation, we examined the distribution of the protein during mineralization of the chicken tibial growth cartilage, using immunohistochemistry and immunoelectron microscopy. The synthesis of osteonectin was also determined in chondrocyte populations isolated from premineralizing and mineralizing regions of growth cartilage and assayed in short-term culture. The results show that a very low level of osteonectin is detectable in the resting, proliferating, and early hypertrophic zones of growth cartilage; in these zones, osteonectin is largely cell-associated. In contrast, a large amount of osteonectin is present in the mineralizing zone where it is associated with the matrix. Biosynthetic data from short-term culture experiments indicate, however, that osteonectin is synthesized and secreted by chondrocytes from both premineralizing and mineralizing zones. As indicated by immunoprecipitation, Northern hybridization, in vitro translation of hybrid-selected messenger RNA (mRNA), and electrophoretic analysis, osteonectin synthesized by chondrocytes of the premineralizing zones is not obviously different in structure from that synthesized by chondrocytes of the mineralizing zone. We conclude that osteonectin is a product of chondrocytes in each zone of growth cartilage but accumulates only in the mineralizing zone. The high affinity of the protein for calcium could favor its retention in calcifying matrix.

Animals↗

PubMed1990

cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN).

A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells isolated at 6-12 weeks of gestation. Immunohistochemical analysis confirmed that OP expression is high in decidua cells as well as in the endometrial glands of a non-pregnant secretory-phase human uterus. Two variants of the OP message were evident on the basis of DNA sequencing and polymerase chain reaction amplification of bone and decidua cell mRNA. The peptides potentially translated by the variant messages differ by the presence (OP1b) or absence (OP1a) of 14 amino acids at residue 58 of the molecule. The deduced human protein sequence shows a conservation between species in the position of the Arg-Gly-Asp (RGD) cell attachment site. Chromosomal mapping of the osteopontin gene (OPN) using human-rodent cell hybrids demonstrated a location on chromosome 4 in the human genome. In situ hybridization of metaphase chromosomes using radiolabeled OP1a as a probe indicated that the gene is located on a region of 4q that is near the centromere. A high-frequency restriction fragment length polymorphism was evident in the DNA from 29 unrelated individuals using the enzyme BglII. Analysis of total genomic DNA by digestion with several restriction enzymes, Southern blotting, and hybridization with the human osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb.

Amino Acid Sequence↗

PubMed1990

Localization of PGI (biglycan, BGN) and PGII (decorin, DCN, PG-40) genes on human chromosomes Xq13-qter and 12q, respectively.

The genes for PGI (biglycan, BGN) and PGII (decorin, DCN) have been assigned to human chromosomes X and 12 by Southern analysis of a panel of human-rodent somatic cell hybrid DNAs with cDNA probes for these related small proteoglycans. Regional localization of BGN to Xq13-qter and DCN to 12p12.1-qter was also obtained by examining hybrids containing spontaneous breaks or well-characterized translocations involving chromosomes X and 12. Biglycan (BGN) is a single-copy gene about 6 kb in length. Hybridization with subfragment cDNA probes suggests the presence of two copies of the decorin (DCN) gene, or related sequences, at the locus on chromosome 12, although there is no evidence for function of more than one DCN gene. Efforts to detect restriction fragment length polymorphisms with these probes were unsuccessful.

Animals↗

PubMed1990

The impact of osteonectin for differential diagnosis of bone tumors. An immunohistochemical approach.

75 osteosarcoma at various grades of histologic differentiation, including chondroblastic and small cell variants, and 5 fibrosarcomas of bone, 5 Ewing's sarcomas, 5 malignant fibrous histiocytomas of bone, 8 chondrosarcomas, and 2 dedifferentiated chondrosarcomas, were investigated immunohistochemically for evidence of osteonectin. According to the results of our study, osteonectin is present in all osteosarcomas, with special topographic preponderance in the osteoblastic and chondroblastic variants. Evidence of osteonectin was also found in all other bone tumors we had analysed so far. In chondrosarcomas, positive reactions appeared only in the vicinity of trabeculae and in dedifferentiated areas. Thus, osteonectin cannot be regarded as a bone-specific protein. Although a high affinity for the osseous matrix is one of its undoubted features, it is therefore unsuitable for differential diagnostic purposes.

Bone Neoplasms↗

PubMed1990

Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues.

The messenger RNAs and core proteins of the two small chondroitin/dermatan sulfate proteoglycans, biglycan and decorin, were localized in developing human bone and other tissues by both 35S-labeled RNA probes and antibodies directed against synthetic peptides corresponding to nonhomologous regions of the two core proteins. Biglycan and decorin expression and localization were substantially divergent and sometimes mutually exclusive. In developing bones, spatially restricted patterns of gene expression and/or matrix localization of the two proteoglycans were identified in articular regions, epiphyseal cartilage, vascular canals, subperichondral regions, and periosteum, and indicated the association of each molecule with specific developmental events at specific sites. Study of non-skeletal tissues revealed that decorin was associated with all major type I (and type II) collagen-rich connective tissues. Conversely, biglycan was expressed and localized in a range of specialized cell types, including connective tissue (skeletal myofibers, endothelial cells) and epithelial cells (differentiating keratinocytes, renal tubular epithelia). Biglycan core protein was localized at the cell surface of certain cell types (e.g., keratinocytes). Whereas the distribution of decorin was consistent with matrix-centered functions, possibly related to regulation of growth of collagen fibers, the distribution of biglycan pointed to other function(s), perhaps related to cell regulation.

Adrenal Glands↗

PubMed1990

Structure and expression of osteonectin mRNA in human tissue.

A cDNA encoding osteonectin was isolated from a human bone cell cDNA library and used to examine osteonectin protein structure, mRNA structure and expression in human tissue. The deduced protein sequence shows complete identity with a recently isolated placental form and extensive homology to mouse and bovine counterparts. The protein is rich in cysteine residues, which are conserved between species except for cys 194 which is only present in the bovine. In the human, osteonectin mRNA is of two sizes, 2.3 and 3.0 kb, the former being dominant in all tissues studied. Human mRNA was detected in the Ewing sarcoma and in non-bone cell and tissue sources. The potential folded structure of osteonectin mRNA was estimated, based on computer predictions, and indicates the presence of a bulge at the 5' end of the message which includes the start of translation. Southern analysis of human genomic DNA using radiolabeled osteonectin cDNA as probe demonstrates a simple banding pattern confirming earlier studies that the osteonectin gene is present in one copy per haploid human genome.

Amino Acid Sequence↗