The collagenous matrix of bovine predentine.

Predentine obtained from bovine teeth by microdissection was solubilized by cyanogen bromide cleavage. The electrophoretic mobility of the resultant peptides was established on polyacrylamide gel and the amino acid composition of several peptides was determined. The data clearly indicated that this collagen is entirely of the Type I genetic species. No differences were detected between the predentine and dentine collagens except that the mature tissue was more highly crosslinked. Nevertheless the amount of stable cross-link formed in the predentine was higher than expected for an immature tissue.     

Non-collagenous ECM proteins in blood vessel morphogenesis and cancer

Background

The extracellular matrix (ECM) is constituted by diverse composite structures, which determine the specific to each organ, histological architecture and provides cells with biological information, mechanical support and a scaffold for adhesion and migration. The pleiotropic effects of the ECM stem from the dynamic changes in its molecular composition and the ability to remodel in order to effectively regulate biological outcomes. Besides collagens, fibronectin and laminin are two major fiber-forming constituents of various ECM structures.

Scope of review

This review will focus on the properties and the biological functions of non-collagenous extracellular matrix especially on laminin and fibronectin that are currently emerging as important regulators of blood vessel formation and function in health and disease.

Major conclusions

The ECM is a fundamental component of the microenvironment of blood vessels, with activities extending beyond providing a vascular scaffold; extremely versatile it directly or indirectly modulates all essential cellular functions crucial for angiogenesis, including cell adhesion, migration, proliferation, differentiation and lumen formation. Specifically, fibronectin and laminins play decisive roles in blood vessel morphogenesis both during embryonic development and in pathological conditions, such as cancer.

General significance

Emerging evidence demonstrates the importance of ECM function during embryonic development, organ formation and tissue homeostasis. A wealth of data also illustrates the crucial role of the ECM in several human pathophysiological processes, including fibrosis, skeletal diseases, vascular pathologies and cancer. Notably, several ECM components have been identified as potential therapeutic targets for various diseases, including cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Non-collagenous proteins of rat dentin. Evidence that phosphoprotein is not covalently bound to collagen

The non-collagenous proteins of rat dentin that remain firmly bound to the matrix after demineralization were studied in order to ascertain if they are covalently linked to insoluble dentin collagen. After solubilization with CNBr or with bacterial collagenase, unusually small amounts of dentin phosphoprotein were detected in the matrix. The phosphoprotein obtained by CNBr digestion of the matrix was separated from collagen peptides using two chromatographic steps. Thus even this small quantity of phosphoprotein found in decalcified rat dentin matrix was not covalently bound to collagen.     

Routes to active proteins from transformed microorganisms.

Over-expression of recombinant proteins in microbial hosts results in the formation of active soluble protein or of insoluble aggregates (inclusion bodies). Efficient in vitro refolding strategies have been developed to reactivate inactive proteins from inclusion bodies. Co-expression of molecular chaperones may provide a tool to promote correct structure formation of recombinant proteins in vivo.     

Electrophoretic analysis of proteins from single bovine muscle fibres.

A number of single fibres were isolated by dissection of four bovine masseter (ma) muscles, three rectus abdominis (ra) muscles and eight sternomandibularis (sm) muscles. By histochemical criteria these muscles contain respectively, solely slow fibres (often called type I), predominantly fast fibres (type II), and a mixture of fast and slow. The fibres were analysed by conventional sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the gels stained with Coomassie Blue. Irrespective of the muscle, every fibre could be classed into one of two broad groups based on the mobility of proteins in the range 135000-170000 daltons. When zones containing myosin heavy chain were cut from the single-fibre gel tracks and 'mapped' [Cleveland, Fischer, Kirschner & Laemmli (1977) J. Biol. Chem. 252, 1102-1106] with Staphylococcus proteinase, it was found that one group always contained fast myosin heavy chain, whereas the second group always contained the slow form. Moreover, a relatively fast-migrating alpha-tropomyosin was associated with the fast myosin group and a slow-migrating form with the slow myosin group. All fibres also contained beta-tropomyosin; the coexistence of alpha- and beta-tropomyosin is at variance with evidence that alpha-tropomyosin is restricted to fast fibres [Dhoot & Perry (1979) Nature (London) 278, 714-718]. Fast fibres containing the expected fast light chains and troponins I and C fast were identified in the three ra muscles, but in only four sm muscles. In three other sm muscles, all the fast fibres contained two troponins I and an additional myosin light chain that was more typical of myosin light chain 1 slow. The remaining sm muscle contained a fast fibre type that was similar to the first type, except that its myosin light chain 1 was more typical of the slow polymorph. Troponin T was bimorphic in all fast fibres from a ra muscles and in at least some fast fibres from one sm muscle. Peptide 'mapping' revealed two forms of fast myosin heavy chain distributed among fast fibres. Each form was associated with certain other proteins. Slow myosin heavy chain was unvarying in three slow fibre types identified. Troponin I polymorphs were the principal indicator of slow fibre types. The myofibrillar polymorphs identified presumably contribute to contraction properties, but beyond cud chewing involving ma muscle, nothing is known of the conditions that gave rise to the variable fibre composites in sm and ra muscles.     

Odontoclastic resorption of the superficial nonmineralized layer of predentine in the shedding of human deciduous teeth

Resorption by odontoclasts of a superficial nonmineralized layer of predentine that occurs in prior to the shedding of human deciduous teeth was studied by light and electron microscopy. As resorption of the tooth roots neared completion, multinucleate cells appeared on the predentine surface of the coronal dentine between the degenerated odontoblasts, excavated characteristic resorption lacunae in the nonmineralized predentine. These multinucleate cells had the same ultrastructural characteristics as odontoclasts and histochemical demonstration of tartrate-resistant acid phosphatase activity in the multinucleate cells revealed intense staining in numerous small granules identified as lysosomes. Occasionally, the multinucleate cells simultaneously resorbed both nonmineralized and calcospherite-mineralized matrix in the predentine. The study demonstrates that multinucleate odontoclasts can resorb nonmineralized predentine matrix in vivo, probably in the same way as they resorb demineralized organic matrix in the resorption zone underlying their ruffled border.     

Immunofluorescent localization of amelogenins in developing bovine teeth.

Antiserum was prepared to the proteins (amelogenins) isolated from fetal bovine enamel matrix. This antiserum was used to localize the amelogenins in the developing bovine molar by immunofluorescent microscopy. Amelogenins could be identified in the preameloblasts before enamel matrix deposition had begun as well as in the secretory ameloblasts. The closely adherent layer of stratum intermedium cells also contained some immunoreactive material, suggesting that they may contribute protein to the enamel matrix. The newly deposited enamel matrix consisted of brightly fluorescent particles. Mature enamel matrix did not contain the immunoreactive protein except in a thin layer along the dentino-enamel junction and adjacent to the ameloblasts. No other portion of the tooth bud or other tissues reacted with the specific antiserum.     

The active form of cytochrome P-450 from bovine adrenocortical mitochondria.

Cytochrome P-450 from bovine adrenocortical mitochondria exists in three forms of molecular weight: 850,000 (protein 16), of one-half (protein 8), and of one-quarter of this value (protein 4). The forms of the enzyme are named according to the number of subunits and all appear to be active in converting cholesterol to 3beta-hydroxy-5-pregnen-20-one (side chain cleavage) (Shikita, M., and Hall, P.F. (1973) J. Biol. Chem. 248, 5606). To determine whether all three forms are active at their characteristic molecular weights, the three cytochromes were each layered onto separate sucrose density gradients and centrifuged at 49,000 rpm for 60 min; the gradients contained all the factors necessary for side chain cleavage including one of the following substrates: cholesterol, 20S-hydroxycholesterol, and 20S,22R-dihydroxycholesterol. Regardless of the form of P-450 layered onto the gradient and regardless of the substrate, enzyme activity (side chain cleavage) was observed only in fractions corresponding to a sedimentation coefficient of 20 to 22 S which is that for protein 16. No activity was observed at S values corresponding to either protein 8 or protein 4. These findings indicate that the active form of cytochrome P-450 from adrenocortical mitochondria is that containing 16 subunits, i.e. the form in which the cytochrome is normally isolated from adrenal mitochondria. Forms consisting of eight and four subunits which can be prepared from protein 16 become active only by forming protein 16, at least in an aqueous medium in vitro.     

Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage.

Proteoglycans were extracted from bovine tracheal cartilage by high-speed homogenization, the use of dissociative solvents being avoided. The homogenate was fractionated by gel chromatography, sucrose-density-gradient centrifugation and ion-exchange chromatography. A previously unrecognized protein, cartilage matrix protein, was identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It cofractionated with the proteoglycans in all systems, indicating an interaction. The cartilage matrix protein-proteoglycan complex was dissociated by treatment with 4M-guanidinium chloride. The complex again formed when the guanidine was removed. The cartilage matrix protein has a mol.wt. of more than 200000. On reduction it yields subunits with a mol.wt. of approx. 60000.     

Purification and characterization of two phospholipid exchange proteins from bovine heart.

Two phospholipid exchange proteins from bovine heart have been purified approximately 2000-fold and judged greater than 90% pure. The proteins are similar in molecular weight (both 33,400 by polyacrylamide gel electrophoresis and 23,500 by gel filtration), in amino acid composition, and in specificity, although they differ in isoelectric points, 5.3 and 5.6. The transfer of phospholipids between artificial membranes is catalyzed by these proteins at the following relative rates: 100 for phosphatidylinositol, 35 for phosphatidylcholine, 5 for sphingomyelin, and 0.1 for phosphatidylethanolamine. The use of these exchange proteins in the study of mixed phospholipid vesicle structure is demonstrated. The purified proteins catalyze the substitution of one membrane phospholipid species for another at a rate comparable to true exchange. The phospholipid exchange activity is inhibited by the presence of sphingomyelin, and also by reagents which react with sulfhydryl groups. Evidence is presented for two sites of N-ethylmaleimide binding on these exchange proteins. Reaction with one site has little effect on activity and occurs in the absence of membranes. Reaction with the second site occurs in the presence of phospholipid vesicles and leads to complete, irreversible inhibition of exchange activity.     

Fibrinogen-like proteins and fibrinolytic proteins in a saline eluate from sucrose washed bovine erythrocytes.

Plasminogen and a spectrum of fibrinogen-like proteins are found in a saline eluate of sucrose washed bovine erythrocytes. It seems probable that these proteins are membrane bound in vivo, and, as such, their physiological significance is discussed.     

Purification of an active opioid-binding protein from bovine striatum

We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta-naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat-sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors.