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Biomedical subjects

W T Butler

Publications and source records attributed to W T Butler.

At least 37 records · Page 2Linked to original sources
PubMed1997

Improved immunohistochemical staining of osteopontin (OPN) in paraffin-embedded archival bone specimens following antigen retrieval: anti-human OPN antibody recognizes multiple molecular forms.

Studies to assess osteopontin (OPN) localization in adult human bone using immunochemical techniques produce conflicting results due to variations in tissue processing or antibody immunoreactivity. The present study was designed to resolve these discrepancies using well-characterized antibodies and improved antigen detection. An anti-osteopontin (alpha-OPN) antiserum was developed that recognizes various soluble molecular weight forms of human OPN, including monomeric, cleaved, and dimerized products. An affinity column of full length recombinant human OPN (rOPN) coupled to support was used to purify alpha-OPN antibodies. Western analysis showed that the affinity-purified antibodies recognized numerous molecular weight forms of OPN. These antibodies were used to study the distribution of OPN in adult human bone using immunohistochemical techniques combined with an antigen retrieval protocol utilizing a newly developed antigen retrieval solution, Retriev-Alltrade mark (Bronco Technologies Inc, Pasadena, TX). Immunolocalization of OPN in archival bone specimens prior to antigen retrieval produced no demonstrable immunostaining even at high concentrations of alpha-OPN. Use of the antigen retrieval protocol restored OPN immunoreactivity, with strong staining apparent in cement lines, osteoblasts, osteocytes, canaliculi, osteoid, and bone matrix. We conclude that antigen retrieval restores immunochemical recognition of OPN in archival specimens containing bone without increasing nonspecific binding.

Adult↗
PubMed1997

Dentin sialoprotein (DSP) transcripts: developmentally-sustained expression in odontoblasts and transient expression in pre-ameloblasts.

Dentin sialoprotein (DSP), a 53 kDa glycoprotein, is believed to be present exclusively in dentin. Using rat and mouse digoxigenin labeled (DIG)-DSP and 35S-DSP riboprobes, and in situ hybridization techniques, we have studied the presence of DSP mRNA at specific developmental stages of dentinogenesis. In mouse and rat molars and incisors, DSP transcripts were localized in young odontoblasts associated with early stages of predentin formation, as well as in mature odontoblasts, cells with cytoplasmic extensions embedded in the forming dentin. No DSP transcripts were detected in dental pulp, enamel organ, ameloblasts, epithelial root sheath, Meckel's cartilage, alveolar bone or tibia. Furthermore, no DSP mRNA was observed in other soft tissues including heart, lung, kidney, intestine, eye, and muscle. In addition to the intense and prolonged expression by odontoblasts, DSP mRNA was transiently expressed by pre-ameloblasts in both developing molars and incisors. These observations are consistent with the results of previous immunohistochemical studies (1). The transient expression of DSP in pre-ameloblasts across from young odontoblasts suggests an involvement of DSP in epithelial-mesenchymal interactions that are crucial to later stages of tooth development.

Alveolar Process↗
PubMed1996

Partial cDNA sequencing of mouse dentine sialoprotein and detection of its specific expression by odontoblasts.

Dentine sialoprotein (DSP), a 53-kDa acidic glycoprotein, is expressed by odontoblasts and secreted into the dentine extracellular matrix. Although little is known about its biological function, it might play a part in dentinogenesis. Because DSP has only been shown to occur in rat dentine, it is important to demonstrate its existence in another species. Here, the presence of DSP gene in the mouse genome, and the cloning of a mouse DSP cDNA coding for about one-fifth of the molecule with a nucleotide sequence similar to that for rat cDNA, are reported. Using in-situ hybridization, DSP mRNA was uniquely detected in mouse odontoblasts.

Animals↗
PubMed1996

Posttranslational modification in rat bone osteopontin.

Osteopontin is a multiply-phosphorylated glycoprotein which contains an RGD cell adhesion sequence and regions containing a high level of aspartic acid. It is abundant in bone, milk, and urine and is also found in malignant tissues. Peptides generated from rat bone osteopontin by digestion with endoprotease Lys-C were analyzed for post-translational modification by a combination of Edman degradation and matrix-assisted laser desorption mass spectrometry. In the peptides analyzed in this way (accounting for approximately half the protein), eleven sites were found that were variably phosphorylated. Three modified lysines were also detected by virtue of their inability to be cleaved by endoprotease Lys-C (Lys19 and Lys286), by observation of an anomalous pth-amino acid during Edman degradation (Lys19 Lys29 and Lys286) and by a mass difference of 115 +/- 5. Bone osteopontin is extremely heterogeneous, as none of these modifications were found in 100% of peptides examined. In this respect, bone osteopontin differs from bovine milk osteopontin in which 28 residues are completely phosphorylated (Sørensen, et al., (1995) Protein Science 4:2040-2049). While the sequences of bovine and rat osteopontin differ significantly, many conserved serines in the N- and C-terminal regions that are phosphorylated in bovine milk osteopontin are at least partially phosphorylated in the rat bone protein. However, there are also conserved residues in both bovine and rat proteins which are phosphorylated in one species but not the other.

Amino Acid Sequence↗
PubMed1995

Bone matrix proteins in osteogenesis and remodelling in the neonatal rat mandible as studied by immunolocalization of osteopontin, bone sialoprotein, alpha 2HS-glycoprotein and alkaline phosphatase.

The neonatal rat mandible was used as a model to study bone formation, mineralization, quiescence, and resorption, using immunolocalization and a variety of tissue-processing techniques. Monospecific antibodies for osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (AP) and alpha 2HS-glycoprotein (alpha 2HS-GP) were used on fixed paraffin-embedded tissue, fixed frozen tissue and unfixed frozen tissue. Immunostaining was correlated with mineral content by two procedures, the von Kossa and the morin techniques. Morin fluorescence was used with secondary immunostaining to provide a way of closely correlating bone matrix proteins and matrix mineralization. Co-immunolocalization procedures were used to compare the sites of bone proteins in the matrix. AP was found earliest during osteogenic cell differentiation, appearing in the preosteoblasts, followed by OPN and BSP, which first appeared in osteoblasts. alpha 2HS-GP expression was not observed in cells. The results provide clear evidence for the presence of OPN in osteoid, while BSP and alpha 2HS-GP were confined to the mineralized matrix. Immunostaining of bone proteins is highly technique-dependent: immunolocalization investigations required several methods of approach to ensure adequate demonstration of these proteins in cells and matrix. The results support the contention that osteopontin is multifunctional in bone metabolism, and that alpha 2HS-GP, though produced in the liver, is abundant in bone matrix and may also have a function in bone metabolism.

Alkaline Phosphatase↗
PubMed1995

Characterization of cellular responses involved in reparative dentinogenesis in rat molars.

During primary dentin formation, differentiating primary odontoblasts secrete an organic matrix, consisting principally of type I collagen and non-collagenous proteins, that is capable of mineralizing at its distal front. In contrast to ameloblasts that form enamel and undergo programmed cell death, primary odontoblasts remain metabolically active in a functional tooth. When dentin is exposed to caries or by operative procedures, and when exposed dentinal tubules are treated with therapeutic dental materials, the original population of odontoblasts is often injured and destroyed. The characteristics of the replacement pool of cells that form reparative dentin and the biologic mechanisms that modulate the formation of this matrix are poorly understood. Based on the hypothesis that events governing primary dentinogenesis are reiterated during dentin repair, the present study was designed to test whether cells that form reparative dentin are odontoblast-like. Cervical cavities were prepared in rat first molars to generate reparative dentin, and animals were killed at various time intervals. In situ hybridization with gene-specific riboprobes for collagen types I and III was used to study de novo synthesis by cells at the injured dentin-pulp interface. Polyclonal antibodies raised against dentin sialoprotein (DSP), a dentin-specific protein that marks the odontoblast phenotype, were used in immunohistochemical experiments. Data from our temporal and spatial analyses indicated that cells forming reparative dentin synthesize type I but not type III collagen and are immunopositive for DSP. Our results suggest that cells that form reparative dentin are odontoblast-like.

Animals↗
PubMed1995

Dentin matrix proteins and dentinogenesis.

The precise mechanisms involved in dentinogenesis are not understood; however, the information to date suggests that a number of highly controlled extracellular events are involved. Mature odontoblasts secrete collagen at the cell border into predentin. They synthesize and secrete other non-collagenous proteins (NCPs) at the mineralization front, possibly through odontoblastic processes. A collagen-NCP complex is formed at the predentin-dentin border and apatite crystal initiation and growth takes place. One of the research needs is to uncover the nature of this dentin collagen-NCP complex and to understand how it controls mineralization. At least three dentin specific NCPs are known: phosphophoryn(s), dentin sialoprotein (DSP) and AG1 (Dmp1). Other macromolecules are commonly made by osteoblasts and odontoblasts and participate in bone and dentin formation. Some progress in understanding dentin mineralization has been gained by focusing upon the role of phosphophoryns. These highly phosphorylated proteins are secreted at the mineralization front, where a small portion binds in the gap region of type I collagen fibrils. This portion of phosphoproteins probably initiates formation of plate-like apatite crystals. Additional phosphoryns in higher concentrations bind to the growing apatite crystals and slow their growth, possibly influencing their size and shape. Other areas which need careful investigations are those involving the mechanisms involved in odontoblast differentiation, how the synthesis of the dentin specific NCPs is controlled and the precise roles of these macromolecules in dentinogenesis. Future experimentation will focus on the gene structures for these NCPs and the mechanisms of tissue specific gene regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Apatites↗
PubMed1995

Molecular analysis of rat dentin sialoprotein.

Dentin sialoprotein (DSP) is a noncollagenous protein originally isolated from rat dentin. Because it is made by odontoblasts that are actively synthesizing dentin. DSP may play an important role in dentinogenesis. We have isolated a full length DSP cDNA from a rat odontoblast/dental pulp cDNA library (Ritchie et al. [1994] J. Biol. Chem. 269:3698-3702) which codes for a 17 residue signal peptide and a 366 residue, 53 kDa mature protein. In situ hybridization revealed DSP mRNA expression by odontoblasts, but no other cells, in jaws from newborn rat. Northern analysis of various rat tissues demonstrated the presence of DSP transcripts in newborn tooth germs and 21 day old rat incisors. Moreover, multiple transcripts of 4.6 kb and 1.5 kb were found in these two tissues. To better understand the origin of these DSP mRNA multiple transcripts, we have isolated two rat genomic clones. Digestion of each clone with EcoRI followed by Southern analysis revealed that DSP cDNA hybridized to a 4 kb fragment in a lambda dash clone and to a 6 kb fragment in a cosmid clone. Since DSP cDNA hybridized to a 6 kb EcoRI fragment and a 4 kb EcoRI fragment obtained from a rat liver genomic cDNA digested with EcoRI, the multiple DSP mRNA transcripts are most likely derived from two related DSP genes which coexist in the rat genome.

Animals↗
PubMed1995

The nature and functional significance of dentin extracellular matrix proteins.

Odontoblasts are responsible for formation of predentin, which is transformed to dentin when apatite crystals are formed and the fibrillar matrix becomes mineralized. Odontoblasts are specialized cells that synthesize and secrete a unique set of non-collagenous proteins (NCPs), as well as the collagenous matrix largely comprised of type I collagen. The NCPs consist of dentin specific and mineralized tissue specific proteins, as well as other proteins that are found in a variety of tissues. Three dentin specific proteins have been recognized to date: dentin phosphoprotein (DPP), also called phosphophoryn, AG1 (dentin matrix protein 1, Dmp1) and dentin sialoprotein (DSP). DPP appears to be made by odontoblasts and appears at the mineralization front within a short time. It may be secreted via odontoblastic processes. DPP binds to collagen and potentially initiates formation of apatite crystals. A second DPP function appears to be to bind to the 100 face of growing apatite crystals and to inhibit or slow their growth; thus, DPP may play a dual role by initiating mineralization and then affecting the crystal growth and perhaps the habit of the crystals. Although no function has been ascribed to AG1 or DSP, they should prove to be important markers for the odontoblast phenotype. A recent unique finding is that two separate genes appear to code for more than one DSP mRNA; other transcripts may result from differential splicing. Examples of mineralized tissue specific proteins expressed by osteoblasts as well as odontoblasts are bone sialoprotein (BSP) and osteocalcin. Some NCPs expressed by osteoblasts, odontoblasts and several other tissues include osteopontin (OPN) and the chondroitin sulfate containing proteoglycans, decorin and biglycan. We propose that characterization of odontoblasts in tissues and cultures should rely upon utilization of sets of markers for the above NCPs and their mRNAs. Similar approaches are commonly used in investigations on osteoblasts. Finally, dentin (like bone) contains other molecules such as growth factors, and serum derived proteins, found within the matrix; no functional significance has yet been placed upon this finding. Future experiments should focus upon the elucidation of the three dimensional structures of the collagenous fibrillar network and of the NCPs to determine the relationships to mineralization. The role played by odontoblasts in controlling extracellular events, such as by selective secretory routes, will require careful exploration.

Amino Acid Sequence↗
PubMed1994

Cloning and sequence determination of rat dentin sialoprotein, a novel dentin protein.

Dentin sialoprotein (DSP) is a 53-kDa protein isolated from rat dentin. It contains 29.6% carbohydrate (including 9% sialic acid) and has an overall composition similar to that of the bone sialoproteins osteopontin and bone sialoprotein (i.e. rich in Asp, Ser, Glu, and Gly). Using a monospecific anti-DSP polyclonal antibody to screen a rat incisor odontoblast cDNA library, a cDNA clone was isolated and sequenced. This approximately 750-base pair clone contained a DNA sequence corresponding to the NH2-terminal 9 amino acids of DSP. A second cDNA clone was isolated by using the first cDNA as a probe to rescreen the library. This second clone had the full-length DSP coding region. From the sequence, we deduced that the DSP cDNA coded for 366 amino acids, predominantly Asp, Ser, Glu, and Gly. The amino acid composition calculated for this sequence was very similar to that for purified DSP reported earlier; likewise the deduced molecular weight (53,045) was essentially identical to that determined by sedimentation equilibrium. Six potential N-linked glycosylation sites were present in the predicted DSP sequence. No Arg-Gly-Asp sequence was found, and the sequence for DSP was dissimilar to those of osteopontin and bone sialoprotein. Multiple transcripts near 4.6 and 1.5 kilobases were detected by Northern blot analysis in the incisor of 21-day-old rat and the tooth germ of the newborn rat. Consistent with previous immunohistochemical findings, no transcripts were detected in brain, salivary gland, heart, muscle, spleen, kidney, intestine, lung, liver, pancreas, tibia, calvaria, or osteoblast-like osteosarcoma (ROS 17/2.8) cells, indicating that DSP is specifically expressed by odontoblasts and related cells.

Amino Acid Sequence↗
PubMed1994

Immunolocalization of osteopontin, osteocalcin, and dentin sialoprotein during dental root formation and early cementogenesis in the rat.

Using immunohistochemical methods we studied the tissue localization of the extracellular matrix proteins osteopontin (OPN), osteocalcin (OC), and dentin sialoprotein (DSP) during the formation of acellular and cellular cementum in newly born rats. In the layer of acellular cementum of developing incisor and molar teeth we found a very strong staining for OPN but not for DSP or OC. Many cells immediately adjacent to acellular cementum and PDL cells were also positive for OPN but not for DSP or for OC. In contrast, cellular cementum in molar teeth stained strongly for OPN and OC but not for DSP. Consistent with these observations, the cells engaged in the formation of cellular cementum (cementoblasts and cementocytes) reacted strongly for OPN and OC but not for DSP. In advanced stages of dentinogenesis, both crown and root odontoblasts and dentin stained for OPN, OC, and DSP. Cells and matrices of surrounding alveolar bone stained for OPN and OC but not for DSP. We conclude that cementoblasts and cementocytes of cellular cementum produce OPN and OC but not DSP and thus express an osteoblast-like, not an odontoblast-like, phenotype. The cells responsible for the production of acellular cementum are likely cells of the PDL in close contact with the dental root surface. These fibroblast-like cells express OPN but not OC or DSP and accordingly express only a partial osteoblastic phenotype.

Animals↗
PubMed1994

Osteopontin-stimulated vascular smooth muscle cell migration is mediated by beta 3 integrin.

Osteopontin (OPN), a 41-kDa phosphorylated glycoprotein, has been detected in rat aorta and carotid arteries, and expression of its mRNA in blood vessels is strongly increased in response to vascular injury. To investigate the potential role of OPN in vascular pathophysiology, we studied the effect of rat OPN on aortic smooth muscle cell migration and proliferation in vitro. OPN enhanced the migration of rat smooth muscle cells in a time- and concentration-dependent manner with an EC50 value of 46 +/- 11 nmol/liter (n = 5). The maximal increase in cell migration by OPN was 29-fold over basal levels. OPN-induced smooth muscle cell migration was inhibited in a concentration-dependent manner by the monoclonal antibody F11, which recognizes the rat integrin subunit beta 3. In contrast, polyclonal antiserum recognizing the rat integrin beta 1 subunit did not inhibit smooth muscle cell migration in response to OPN, but did block fibronectin-promoted migration. Moreover, OPN-induced smooth muscle cell migration was dependent on the presence of extracellular divalent cations and was significantly inhibited by anti-OPN antibodies. OPN did not stimulate [3H]thymidine incorporation into cultured smooth muscle cells, indicating that it selectively enhanced migration. In view of the pathological significance of arterial smooth muscle cell migration in the formation of intimal thickening, our results suggest that smooth muscle cell recognition of OPN, probably through the vitronectin receptor, alpha v beta 3, could play a role in the cells' response to vascular injury and especially neointima formation.

Animals↗
PubMed1993

Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin alpha v beta 3 potentiate bone resorption.

We have investigated the mechanism by which osteoclasts adhere to and resorb bone. We show that these cells express beta 1 and beta 3 integrins which are involved in attachment to purified bone matrix proteins. Binding to osteopontin and bone sialoprotein is mediated by alpha v beta 3, while a beta 1 integrin is responsible for attachment to fibronectin. Both the rapid attachment by osteoclasts to intact bone particles and their subsequent resorption are blocked by a monoclonal antibody directed to the alpha v beta 3 complex but not by an antibody against beta 1 integrins. Attachment of osteoclasts to bone is also inhibited with soluble osteopontin, Arg-Gly-Asp-containing peptides derived from both osteopontin and bone sialoprotein, or a monospecific polyclonal antibody against osteopontin. We conclude that both osteoclast adherence to bone and subsequent resorption of its matrix are dependent on interactions between the bone matrix proteins osteopontin and/or bone sialoprotein and the integrin alpha v beta 3. Moreover, collagen, which constitutes 90% of its organic matrix, is minimally involved in binding of chicken osteoclasts to bone.

Amino Acid Sequence↗
PubMed1993

Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and alpha 2HS-glycoprotein) proteins in rat bone.

The high-resolution, postembedding protein A-gold immunocytochemical technique was used to visualize the distribution of two noncollagenous bone proteins, osteopontin (OPN) and osteocalcin (OC), and two plasma proteins, alpha 2HS-glycoprotein (alpha 2HS-GP) and albumin (ALB), in sections of Lowicryl K4M-embedded rat tibial and alveolar bone. In the primary spongiosa of the metaphysis, a seam of organic material (lamina limitans) that labeled intensely with OPN and OC antibodies was observed at the bone/calcified cartilage interface just below the zone of vascular invasion of the growth plate. With deposition of bone matrix proper by osteoblasts in this region and its subsequent mineralization, extensive areas of bone were heavily labeled with anti-OPN, anti-OC, and anti-alpha 2HS-GP antibodies, where the majority of gold particles were associated with amorphous, electron-dense patches of organic material throughout the mineralized bone. In the unmineralized osteoid, substantially less labeling was observed, and where occasional mineralization loci were dispersed throughout the osteoid layer, these sometimes showed a concentration of gold particles. ALB labeling, on the other hand, was moderate and generally diffuse throughout the mineralized bone matrix and the osteoid. In alveolar bone, labeling patterns were generally similar to those found in tibial bone. Particularly striking in alveolar bone, however, was an intense anti-OPN labeling of (1) the lamina limitans at cell-lined bone surfaces, including that surrounding cell processes and osteocytes, (2) cement (reversal, resting) lines, and (3) the perilacumar matrix of some osteocytes. In summary, these data suggest that certain plasma proteins, such as alpha 2HS-GP, interact with bone matrix proteins, such as OPN and OC, at sites of tissue mineralization and that the presence of OPN in mineralized bone and at bone surfaces (lamina limitans) and cement lines has a multifunctional role, including regulation of mineralization and mediation of cell dynamics during endochondral and intramembranous bone modeling and remodeling.

Animals↗
PubMed1993

Evidence that a non-RGD domain in rat osteopontin is involved in cell attachment.

The bone sialoprotein osteopontin (OPN) promotes cell attachment and spreading through its RGD (Arg-Gly-Asp) sequence. To study additional regions of OPN involved in cell attachment, peptides of rat OPN were evaluated for their capacity to mediate cell binding to wells in vitro. Human gingival fibroblasts were incubated on microtiter plates coated with either OPN or OPN peptides. A peptide of M(r) 28 kD, obtained after digestion with endoproteinase Arg-C and isolated by reversed-phase HPLC, enhanced cell attachment to a similar degree as OPN. Sequence analysis showed that the amino terminus of the 28 kD peptide starts at Ser142 and therefore does not contain the RGD cell attachment sequence (residues 128-130). Cell attachment mediated through both OPN and the 28 kD peptide was blocked by the addition of GRGDSPA peptides or LM-609, a monoclonal antibody to the integrin alpha V beta 3, a receptor for vitronectin. A variant peptide, GRG-ESPA, did not alter cell attachment. Based on these observations, we conclude that (1) binding of OPN and the 28 kD peptide to fibroblasts involves binding to alpha V beta 3, (2) a site other than the RGD sequence on OPN is also involved in binding to integrins, and (3) the binding of this second site to alpha V beta 3 is inhibited by RGD-containing peptides.

Amino Acid Sequence↗