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M F Young

Publications and source records attributed to M F Young.

At least 55 records · Page 3Linked to original sources
PubMed1994

Cloning and sequence analysis of bovine bone sialoprotein cDNA: conservation of acidic domains, tyrosine sulfation consensus repeats, and RGD cell attachment domain.

We isolated and sequenced a cDNA encoding bovine bone sialoprotein (BSP) using a bovine cDNA library made from mRNA isolated from bone-derived cell cultures and ligated to a phage lambda gt11. One of the cDNA clones isolated from this library had a 1800 base pair long insert and was found to contain the entire protein-encoding region. The deduced protein sequence revealed a 310 amino acid protein containing a signal peptide sequence of 16 hydrophobic amino acids. The protein sequence shows remarkable conservation with previously published human and rat sequences (more than 80% similarity for both species). The potential functional domains of BSP, including three acid amino acid-rich sequences, tyrosine sulfation consensus repeats, and the RGD cell binding sequence, are all present in the bovine sequence. Northern analysis of RNA from different bovine tissues indicated the presence of BSP message in bone but not in other nonmineralized tissues, confirming that bone is the major site of BSP message production.

Amino Acid Sequence↗
PubMed1994

A potential role for tetranectin in mineralization during osteogenesis.

Tetranectin is a protein shared by the blood and the extracellular matrix. Tetranectin is composed of four identical, noncovalently bound polypeptides each with a molecular mass of approximately 21 kD. There is some evidence that tetranectin may be involved in fibrinolysis and proteolysis during tissue remodeling, but its precise biological function is not known. Tetranectin is enriched in the cartilage of the shark, but the gene expression pattern in the mammalian skeletal system has not been determined. In the present study we have examined the expression pattern and putative function of tetranectin during osteogenesis. In the newborn mouse, strong tetranectin immunoreactivity was found in the newly formed woven bone around the cartilage anlage in the future bone marrow and along the periosteum forming the cortex. No tetranectin immunoreactivity was found in the proliferating and hypertrophic cartilage or in the surrounding skeletal muscle. Using an in vitro mineralizing system, we examined osteoblastic cells at different times during their growth and differentiation. Tetranectin mRNA appeared in the cultured osteoblastic cells in parallel with mineralization, in a pattern similar to that of bone sialoprotein, which is regarded as one of the late bone differentiation markers. To explore the putative biological role of tetranectin in osteogenesis we established stably transfected cell lines (PC12-tet) overexpressing recombinant tetranectin as evidenced by Northern and Western blot analysis and immunoprecipitation. Both control PC12 cells and PC12-tet cells injected into nude mice produced tumors containing bone material, as evidenced by von Kossa staining for calcium and immunostaining with bone sialoprotein and alkaline phosphatase antiserum. Nude mice tumors established from PC12-tet cells contained approximately fivefold more bone material than those produced by the untransfected PC12 cell line or by the PC12 cells transfected with the expression vector with no insert (Mann Whitney rank sum test, p < 0.01), supporting the notion that tetranectin may play an important direct and/or indirect role during osteogenesis. In conclusion, we have established a potential role for tetranectin as a bone matrix protein expressed in time and space coincident with mineralization in vivo and in vitro.

Animals↗
PubMed1994

Imagery, action, and young children's spatial orientation: it's not being there that counts, it's what one has in mind.

Young children typically fail when asked to judge how objects would look if they moved or changed shape, and this has been taken to mean that they lack the competencies for dynamic imagery. We used a different approach to study young children's imagination and found evidence of much earlier competence. Across 6 experiments, people were asked to imagine familiar surroundings and anticipate their spatial orientation from different observation points there. In the first 2 experiments, children (2 1/2-9-year-olds) and their parents sat at home and were asked to call to mind knowledge of their (child's) classroom relative to the perspective at their (child's) seat at (pre)school. After this, subjects were asked to judge the perspective at the teacher's seat in each of 2 conditions. In the Locomotion Condition they were asked to imagine walking from their seat to the teacher's seat while physically walking a path that resembled the actual one in the remote classroom. In the Imagination-only Condition the instructions were the same but they were not accompanied with physical walking. Children 3 1/2 years of age and older, like the adults, were accurate and rapid in the Locomotion Condition. In the Imagination-only Condition the children almost never judged perspective correctly; the adults responded accurately but slowly. These findings were replicated and extended across 4 additional experiments designed to clarify the operating principles that link perceiving, imagining, and acting.

Adult↗
PubMed1993

Partial characterization of a novel 'GGA' factor which binds to the osteonectin promoter in bovine bone cells.

Osteonectin (On)/SPARC (secreted protein, acidic and rich in cysteine) is a highly conserved extracellular matrix protein found in bone and other tissues throughout vertebrate evolution. In previous studies, approximately 500 bp of DNA 5' to the transcription start point (tsp) and a part of exon 1, including homopurine (Rn)/homopyrimidine (Yn)-rich sequences (the 'GGA box' and its complements), was demonstrated to be important in upregulation of On gene expression in a cell-specific manner. The purpose of this study was to decipher the transcriptional regulation of On through its cis- and trans-acting elements. DNase I footprinting analysis indicated protein binding which may be related to the transcriptional factors, AP2, SP1 and a novel 'GGA' factor which binds to the 3' end of the promoter (-286 to +43 to the tsp). Comparisons of footprinting between nuclear extracts of bone (On-expressing) cells and Madin and Darby bovine kidney (MDBK) (nonexpressing) cells indicate that 'GGA' factor binding to a purine GGGGA/GGA-rich sequence is cell-type specific and therefore may be involved in the cell-specific expression of this gene. From ultraviolet (UV)-crosslinking experiments, this 'GGA' factor was demonstrated to be a single 40-kDa protein.

Animals↗
PubMed1993

Structure and molecular regulation of bone matrix proteins.

The organic matrix of bone contains several protein families, including collagens, proteoglycans, and glycoproteins, all of which may be extensively modified by posttranslational events, such as phosphorylation and sulfation. Many of the glycoproteins contain Arg-Gly-Asp (RGD), the integrin-binding sequence, within their structure, whereas other constituent proteins contain gamma-carboxyglutamic acid. The deposition of bone matrix by cells in the osteoblastic lineage is regulated by extrinsic factors, such as systemic and local growth factors and physical forces, and factors that are intrinsic to the cell, such as position in the cell cycle, maturational stage, and developmental age of the donor. Recent studies of several bone matrix gene promoters have identified cis- and trans-acting elements that are responsible for gene activity, although the precise sequence of regulatory events is not known. Development of in vitro assays, coupled with studies of the appearance of these proteins during development in vivo, provides insight into the functions of these proteins during the various stages of bone metabolism. Potential roles for these proteins include proliferation and maturation of stem cells, formation of matrix scaffolding elaborated by bone-forming cells, modeling, and remodeling. Changes in the functional properties of the extracellular matrix may be involved in a variety of disease processes, including osteoporosis and oral bone loss.

Bone Matrix↗
PubMed1993

Human decorin gene: intron-exon junctions and chromosomal localization.

All of the protein-encoding exons and the 3' flanking region of the human decorin gene have been cloned and partially sequenced. The locations of the intron-exon junctions within the coding portion of the gene were identical to those found for the homologous human gene, biglycan. The sizes of the introns in the decorin gene, however, were substantially larger than those of the same introns of the biglycan gene. Portions of introns 1, 2, and 3 as well as exon 1 were not found during our extensive screening process. The 5' end of intron 2 was found to have an AG-rich region followed immediately by a CT-rich region. Furthermore, the 5' end of intron 3 was very rich in thymidine, whereas the 3' end of intron 7 was rich in adenosine. Several cDNA clones constructed from cultured human bone cell mRNA were found to contain a different sequence at the 5' end compared to that previously published for mRNA from a human embryonic fibroblast cell line. We were also unable to find the alternate 3' flanking region of the previously published cDNA sequence. We have mapped the human decorin gene by in situ methods to chromosome 12q21.3.

Amino Acid Sequence↗
PubMed1993

The human bone sialoprotein gene (IBSP): genomic localization and characterization.

We have isolated and partially sequenced the human bone sialoprotein gene (IBSP). IBSP has been sublocalized by in situ hybridization to chromosome 4q28-q31 and is composed of six small exons (51 to 159 bp) and 1 large exon (approximately 2.6 kb). The intron/exon junctions defined by sequence analysis are of class O, retaining an intact coding triplet. Sequence analysis of the 5' upstream region revealed a TATAA (nucleotides -30 to -25 from the transcriptional start point) and a CCAAT (nucleotides -56 to -52) box, both in the reverse orientation. Intron 1 contains interesting structural elements composed of polypyrimidine repeats followed by a poly(AC)n tract. Both types of structural elements have been detected in promoter regions of other genes and have been implicated in transcriptional regulation. Several differences between the previously published cDNA sequence (L. W. Fisher et al., 1990, J. Biol. Chem. 265, 2347-2351) and our sequence have been identified, most of which are contained within the untranslated exon 1. Three base revisions in the coding region include a G to T (Gly to Val, amino acid 195), T to C (Val to Ala, amino acid 268), and T to A (Glu to Asp, amino acid 270). In conclusion, the genomic organization and potential regulatory elements of human IBSP have been elucidated.

Amino Acid Sequence↗
PubMed1992

Bone matrix mRNA expression in differentiating fetal bovine osteoblasts.

In the accompanying study, we report an in vitro culture system from bovine bone cells that can be applied to investigate bone cell growth and differentiation. In this system, bovine bone cells placed in mineralization medium formed multilayers (days 2-3), began deposition of mineral (days 5-6), and eventually acquired a mineralized matrix sheet (days 14-20) through the stages of mineralizing nodules and trabecular-like structure. In the current study we used this system to investigate the relative expression of bone matrix genes that may play an important role in bone development and metabolism. alpha 1(I)-collagen, alkaline phosphatase, osteonectin, biglycan (PgI), decorin (PgII), osteopontin, and bone sialoprotein mRNA gene expression were measured on days 0, 2, 6, 10, and 20 (date when the cells were placed in mineralization medium as day 0). Total RNA was purified and analyzed by northern blot using radiolabeled cDNA encoding these genes. To comprehend the relationship between gene expression and mineralization, total calcium content in the cultures was also measured. During the culture period we observed several very different gene expression profiles. The expression of both alpha 1(I)-collagen and biglycan increased 3- to 4-fold by day 6 and then returned to basal levels by day 20. The osteonectin gene was highly expressed throughout the culture, with no significant increase in induction found during any time of culture. A significant induction of alkaline phosphatase (13.8-fold) gene expression was observed by day 6. Osteopontin showed a similar profile to that of alkaline phosphatase but had a much greater level of relative expression (26-fold) compared to day 0. Interestingly, downregulation during mineral accumulation seemed a common occurrence among many of the genes measured. In contrast, the bone sialoprotein gene showed a significant and distinct expression pattern, increasing rapidly after the onset of mineralization on day 6 and ultimately reaching 140-fold that of day 0. Decorin (Pg II) showed an increasing pattern, with the final relative level of induction 5-fold on day 20. These data suggest that the development of the mature osteoblastic phenotype, complete with the ability to produce a thick mineralized matrix, requires the differential regulation of a series of genes and their gene products over the culture period.

Alkaline Phosphatase↗
PubMed1992

Renal tubular epithelial cells express osteonectin in vivo and in vitro.

Osteonectin (SPARC, culture shock protein, BM-40) is a widely distributed glycoprotein which binds calcium and several extracellular matrix proteins, including interstitial collagens and thrombospondin, but whose physiologic role remains undefined. In the present studies, we have demonstrated that immunoreactive osteonectin is present in the distal cortical tubule and medullary tubules of murine kidney. We surveyed the renal epithelial cell lines LLC-PK1, MDCK, and OK for the expression of mRNA encoding osteonectin. We found that osteonectin mRNA is expressed by LLC-PK1 and OK cells but not by MDCK cells, as well as by adult kidney from several species. Calcitonin and vasopressin, agents which increase cAMP in these cells, were found to decrease steady-state osteonectin mRNA concentrations. We found that LLC-PK1 cells produced osteonectin protein, that the protein was localized to intracellular granules, and that the protein bound hydroxyapatite in vitro. Pulse-chase analysis revealed that osteonectin was secreted from the cell layer to the medium after a lag time of four to six hours and was secreted preferentially from the basolateral domain of the cell. The preferential secretion of the calcium-binding protein osteonectin from the renal epithelial cell is consistent with several possible functions, including a structural extracellular matrix protein, a participant in transepithelial ion transport, and an inhibitor of extracellular calcification.

Animals↗
PubMed1992

Structure, expression, and regulation of the major noncollagenous matrix proteins of bone.

The noncollagenous proteins (NCPs) that predominate the bone matrix have recently been the focus of intense investigation because of their potential influence on cell attachment, Ca2+ and hydroxyapatite binding, and the mineralization of bone tissue. With the advent of molecular biology, all of the major NCPs of bone have been cloned and their amino acid sequences completely determined. While each of the proteins has distinct structural properties, some proteins appear to be part of gene families. Examples include the small proteoglycans, decorin and biglycan, as well as the gamma carboxyglutamic acid proteins, such as matrix gla protein and osteocalcin (bone gla protein). Some of the NCPs that are clearly not members of any known gene family still share several common characteristics. One such example of this "convergent evolution" is bone sialoprotein and osteopontin. Both are highly posttranslationally modified glycoproteins that share the cell attachment amino acid sequence RGD (arginine-glycine-aspartic acid), which facilitates the attachment of bone cells in vitro, yet they are clearly not related genetically. Using cDNAs and antisera as probes, the precise temporal localization of NCP expression has been determined, and it has been shown that NCPs are produced in skeletal, and in most cases, nonskeletal tissue as well. This observation implies that the functions of the NCPs are not necessarily limited to bone tissue. Many of the promoters for these genes have been isolated and functional domains determined by a combination of chloramphenicol acetyltransferase assay, gel shift, and footprint analyses. The most extensively studied promoter in the NCP category is osteocalcin, whose sensitivity to 1,25-dihydroxycholecalciferol has been delineated in detail. Future studies on the individual and cooperative activities of the NCPs in bone are likely to involve site-directed mutagenesis of cloned DNA and a combination of in vitro and in vivo functional analyses.

Biglycan↗
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↗