Biomedical subjects

M F Young

Publications and source records attributed to M F Young.

At least 73 records · Page 4Linked to original sources

PubMed1991

Expression of the osteonectin gene potentially controlled by multiple cis- and trans-acting factors in cultured bone cells.

The cis-acting regulatory elements of the osteonectin gene have been studied using a chloramphenicol acetyltransferase (CAT) promoter assay in osteonectin-expressing and nonexpressing cultured cells. When various stretches of the promoter were transiently transfected into fetal bovine bone cells, a positive element was detected in the DNA located between bases -504 and 11 (1 being the start of transcription) and a negative element between bases -900 and -504. The positive element of the promoter also conferred preferential expression of the gene, showing more activity in cells with higher levels of osteonectin mRNA expression. A 1.2 kb fragment of intron 1 displayed a negative effect on CAT expression when inserted 5' to the promoter. An additional regulatory element was found in DNA encoding exon 1, which significantly influenced expression of the gene in fetal bovine bone cells. Gel shift analysis using positive genomic elements located 5' to the start of transcription indicated that one of the nuclear proteins that interacts with the osteonectin promoter may be related to the transcription factor AP2.

Animals↗

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

Diverse forms of stress result in changes in cellular levels of osteonectin/SPARC without altering mRNA levels in osteoligament cells.

The osteonectin/SPARC gene has been shown to possess motifs for a heat shock element and metal responsiveness. Also, the expression of the protein has been associated with culture stress in endothelial cells. In the present study, osteoligament (OL) cells derived from the patellar ligament were subjected to diverse forms of stress that included (a) exposure to sodium arsenite, (b) heat shock, (c) cadmium ion, and (d) the amino acid analog, AZC. Osteonectin/SPARC levels in OL cells were determined by Western blot analyses, and immunoprecipitation using antiosteonectin antibodies. Expression of osteonectin/SPARC mRNA was determined by Northern analysis using a 1.5 kb EcoRI restriction fragment of bovine osteonectin cDNA. These studies reveal that osteonectin/SPARC is produced following diverse forms of stress, however, the levels are lower than observed in unchallenged OL cells. In all instances, the mRNA levels were comparable to control cells. These studies indicate that expression of osteonectin/SPARC mRNA is tightly controlled in OL cells and that the protein may be regulated at the level of protein translation.

Animals↗

PubMed1990

Temporal regulation of hyaluronan and proteoglycan metabolism by human bone cells in vitro.

Osteoblasts elaborate a dynamic extracellular matrix that is constructed and mineralized as bone is formed. This matrix is primarily composed of collagen, along with noncollagenous proteins which include glycoproteins and proteoglycans. After various times in culture, human bone cells were labeled with [35S]sulfate, [3H] leucine/proline, or [3H]glucosamine and the metabolism of hyaluronan and four distinct species of proteoglycans (PGs) was assayed in the medium, cell layer, and intracellular pools. These cells produce hyaluronan (Mr approximately 1,400,000; a chondroitin sulfate PG (CSPG), Mr approximately 600,000; a heparan sulfate PG (HSPG), Mr approximately 400,000; and two dermatan sulfate PGs with Mr approximately 270,000 (biglycan, PG I) and Mr approximately 135,000 (decorin, PG II) that distribute between the medium and cell layer. Two days following subculture, 12 h [35S]sulfate steady-state labeling yielded a composition of 24, 27, 31, and 18% for total CSPG, HSPG, biglycan, and decorin, respectively. While HSPG and decorin levels and distribution between medium and cell layer remained relatively constant during steady-state labeling at different times in culture, CSPG and biglycan levels increased dramatically at late stages of growth, and their distribution changed throughout culture. These results were independent of cell density, media depletion, and labeling pool effects. In contrast, hyaluronan synthesis was uncoupled from PG synthesis and apparently density-dependent. Pulse chase labeling at different stages of culture showed that the CSPG and decorin behaved as secretory PGs. Both HSPG and biglycan underwent catabolism, with HSPG possessing a t1/2 of 8 h and biglycan a t1/2 of 4 h. While the rate of HSPG turnover did not appreciably change between early and late culture, that of biglycan decreased. The mRNA for decorin was constant, while that of biglycan changed during culture. These results suggest that each PG possesses a distinct pattern of cellular and temporal distribution that may reflect specific stages in matrix formation and maturation.

Biglycan↗

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

Characteristics of human periodontal ligament cells in vitro.

Periodontal ligament cells may have a role in the regulation of hard and soft periodontal tissues, but their specific function has yet to be determined. To evaluate further their role in periodontal homeostasis, they were examined for osteoblast-like behaviour; in vitro no characteristic osteoblastic responsiveness was found. Periodontal ligament cells gave a PGE2- and isoproterenol-mediated cAMP response, but did not respond in a similar fashion to calcitonin or PTH. When exposed to PGE2, isoproterenol, or 1,25(OH)2 vitamin D3, they did not exhibit an increase in protein production, as measured by [35S]-methionine incorporation. Immunofluorescent localization indicated that periodontal ligament cells produce a bone-associated protein, osteonectin. In addition, mRNA levels for osteonectin and bone proteoglycan I (biglycan) were detected in these cells, in vitro. This information should help to clarify the role such cells play in the regulation of periodontal tissues.

Biglycan↗

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

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↗

PubMed1989

Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species.

The small proteoglycans (PG) of bone consist of two different molecular species: one containing one chondroitin sulfate chain (PG II) and the other, two chains (PG I). These two proteoglycans are found in many connective tissues and have Mr = 45,000 core proteins with clear differences in their NH2-terminal sequences. Using antisera produced against synthetic peptides derived from the human PG I and PG II NH2 termini, we have isolated several cDNA clones from a lambda gt11 expression library made against mRNA isolated from human bone-derived cells. The clones, which reacted with antisera to the PG II peptide, were sequenced and found to be identical with the PG II class of proteoglycan from human fibroblasts known as PG-40 or decorin. The clones reacting to the PG I antisera, however, had a unique sequence. The derived protein sequence of PG I showed sufficient homology with the PG II sequence (55% of the amino acids are identical, with most others involving chemically similar amino acid substitutions) to strongly suggest that the two proteins were the result of a gene duplication. PG II (decorin) contains one attached glycosaminoglycan chain, while PG I probably contains two chains. For this reason, we suggest that PG I be called biglycan. The biglycan protein sequence contains 368 residues (Mr = 42,510 for the complete sequence and Mr = 37,983 for the secreted form) that appears to consist predominantly of a series of 12 tandem repeats of 24 residues. The repeats are recognized by their conserved leucines (and leucine-like amino acids) in positions previously reported for a diverse collection of proteins (none of which is thought to be proteoglycans) including: two morphogenic proteins (toll and chaoptin) in the fruit fly; a yeast adenylate cyclase; and two human proteins, the von Willebrand Factor-binding platelet membrane protein, GPIb, and a rare serum protein, leucine-rich glycoprotein.

Amino Acid Sequence↗

PubMed1989

Osteonectin promoter. DNA sequence analysis and S1 endonuclease site potentially associated with transcriptional control in bone cells.

To understand the basis of osteonectin (SPARC) transcriptional regulation, we have isolated a bovine genomic clone (lambda Og15) encoding exon 1 and 15 kilobase pairs (kb) of flanking DNA. Direct RNA sequencing of the 5' end of the osteonectin message showed it contained a sequence identical to that of a 2.4-kb EcoRI-BamHI fragment located midway in the clone lambda Og15. The results indicate exon 1 is located 10 kb away from exon 2 in the bovine genome. The DNA sequence unit CCTG is repeated five times in exon 1 which is composed exclusively of untranslated sequence. Sequence analysis of the 5'-flanking DNA revealed the presence of many regulatory motifs including a "GC" box with four overlapping SP1 consensus sequences. Immediately downstream from the GC box is a 72-base pair purine-rich stretch composed primarily of direct repeats of the sequence motifs GGGGA and GGA (GAGA box). Digestion of the flanking DNA in vitro with S1 endonuclease showed a site for the enzyme at position -55 which is just 3' to the GAGA box. Chimeric chloramphenicol acetyltransferase constructs were prepared containing the S1-sensitive site and showed substantial transcriptional activity in UMR-106 and fetal and adult human bone cells which are known to be high producers of the protein. The results indicate a potential regulatory activity of the S1 site in osteonectin gene activation.

Animals↗

PubMed1989

Localization of osteonectin expression in human fetal skeletal tissues by in situ hybridization.

The expression of osteonectin gene was studied in developing human fetuses by Northern analysis and in situ hybridization. The highest levels of osteonectin mRNA were detected in RNA extracted from calvarial bones, growth plates, and skin. Low mRNA levels were present in several parenchymal tissues. In situ hybridization of developing long bones revealed three cell types with high osteonectin mRNA levels: osteoblasts, cells of the periosteum, and hypertrophic chondrocytes. Weaker signals were detected in osteocytes, fibroblasts of tendons, ligaments and skin, and in cells of the epidermis. Apart from the hypertrophic chondrocytes, only low osteonectin mRNA levels were seen in cartilage. The localization of osteonectin mRNA in fetal growth plates is consistent with the hypothesis that the protein plays a role in the mineralization of bone and cartilage matrices.

Bone and Bones↗

PubMed1989

Correlation of scrotal circumference and age in American Brahman bulls.

Scrotal circumference (SC) measurements were taken of 921 American Brahman bulls located on two ranches in southcentral Texas. Measurements were taken at various seasons of the year over an 11-yr time span. Body condition scores, as reflected by fat cover, and a subjective estimate of overall nutritional status were 4 or better, based upon a 1 to 9 scoring system, with 5 representing an animal with an average amount of body fat. Associated body weights were not measured and semen evaluations were not performed. Ages were calculated from exact birthdates and rounded to the nearest whole month. Ages from 8 mo to more than 36 mo were reported. SC measurements ranged from 16.0 cm to 42.5 cm. The relationship of SC and age was positively correlated (r=0.74) (P<0.0001) and could be predicted using the equation y=0.525x + 18.702 where y=SC in centimeters and x=age in months. These data provide evidence that the relationship of scrotal circumference and age of American Brahman bulls is similar to that of other cattle breeds.

Journal Article↗

PubMed1989

Thrombospondin is an osteoblast-derived component of mineralized extracellular matrix.

Thrombospondin, the most abundant protein of platelet alpha granules, is a biosynthetic product of a variety of connective tissue cells and a component of many extracellular matrices. In this study, thrombospondin distribution in bone was investigated using a monoclonal antibody specific for the human protein. Thrombospondin was localized in osteoid of undemineralized, frozen sections of fetal subperiosteal bone, and identified as a component of mineralized bone matrix of neonatal and/or young (growing) bone of many animal species by Western blot analysis. Adult human bone cells were demonstrated to contain mRNA for thrombospondin by hybridization of a cDNA thrombospondin probe to a 6.1 kb mRNA. Pulse-chase experiments indicated that the protein was synthesized and the majority was secreted from osteoblastic cells. Treatment of the cells with TGF-beta (0.01-10 ng/ml) slightly decreased total thrombospondin synthesis, but caused an increase in the retention on newly synthesized thrombospondin in the cell layer/matrix fraction. In cell attachment assays, thrombospondin mediated adhesion, but not spreading of adult human bone cells.

Animals↗

PubMed1989

Opposing influences of glucocorticoid and retinoic acid on transcriptional control in preosteoblasts.

UMR 201 is a nontransformed rat clonal cell line derived from neonatal calvaria with phenotypic characteristics of preosteoblasts. Retinoic acid strongly induces expression of alkaline phosphatase and its mRNA in these cells. Dexamethasone substantially reduced the retinoic acid-induced expression of alkaline phosphatase. This apparent interaction between dexamethasone and retinoic acid effects raised the possibility that interactions may extend to other osteoblast-related phenotypic characteristics in UMR 201 cells. Treatment with dexamethasone resulted in a decrease in the expression of mRNA for pro-alpha 1(I) collagen, but upon coincubation with 1 microM retinoic acid for 24 h, the decrease in mRNA for pro-alpha 1(I) collagen was abrogated. Dexamethasone (Dex) treatment caused a dose-dependent increase in osteonectin mRNA, half maximally effective between 1 nM and 10 nM Dex. One micromolar of retinoic acid alone led to a small increase in expression of osteonectin mRNA but prevented any further increase when Dex was added to retinoic acid-treated cells. To study transcriptional control, osteonectin genomic fragments were linked to the bacterial reporter gene, chloramphenicol acetyltransferase, and introduced by transfection into UMR 201 cells. Dexamethasone increased the transcriptional activity of an osteonectin-chloramphenicol acetyltransferase construct; 100 nM Dex resulted in a 3-fold increase over control cells which was attenuated when 1 microM retinoic acid was added to the incubation, while retinoic acid alone resulted in a 2-fold increase in transcriptional activity. Finally, it was noted that coincubation with retinoic acid and Dex stimulated the proliferation of UMR 201 cells when compared with either treatment alone. This study shows the potential importance of hormonal interactions in the expression of osteoblast function.

Alkaline Phosphatase↗

PubMed1989

Synthetic peptide antisera: their production and use in the cloning of matrix proteins.

Small amounts (0.1-1.0 nmole) of purified (greater than 90%) proteins isolated from small quantities of connective tissues can readily be microsequenced and 20 to 40 N-terminal amino acids determined. A synthetic peptide (10 or more amino acids long) can be produced and either injected directly into rabbits for antiserum production (generally peptides greater than 20 amino acids in length) or conjugated to a carrier protein prior to injection. The antisera have proven useful in the isolation of cDNA clones, for both locating clones producing the protein in expression libraries and in the subsequent confirmation of the sequence (preferably using a part of the sequence not directly used in the production of the original antiserum).

Animals↗