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Biomedical subjects

A Veis

Publications and source records attributed to A Veis.

At least 73 records · Page 4Linked to original sources
PubMed1991

FTIRS in H2O demonstrates that collagen monomers undergo a conformational transition prior to thermal self-assembly in vitro.

The assembly of type I collagen molecules into native fibrils can be accomplished in vitro in solutions at physiological ionic strength and pH by raising the temperature above 30 degrees C. The thermal self-assembly reaction exhibits a distinct lag phase. This lag phase has been proposed to be evidence for a conformational transition in the monomer. Fourier transform infrared spectroscopy (FTIRS) is a very sensitive probe of the H-bonded states within the triple helix. The carbonyl group spectrum (amide I, 1700-1600 cm-1) has been investigated in collagen/H2O solutions at 1 mg/mL under self-assembly conditions from 4 to 34 degrees C and, in the same range, at a higher ionic strength where self-assembly does not occur. The deconvoluted spectra show three very clear bands at approximately 1660, 1644, and 1630 cm-1. These bands vary in both frequency maxima and relative intensity over the temperature range examined. Spectra were also obtained in the amide II and III regions. Spectral changes were evident in the 22-26 degrees C range, under fibril-forming conditions, which lead to the hypothesis that the triple helix of the semiflexible collagen molecule is actually perfected during the lag phase, facilitating nucleation and intermolecular interaction. Further spectral changes after fibrils do form show that the molecules are once again distorted as they are bent to fit within the fibrils.

Animals↗
PubMed1991

A rat incisor dentin matrix protein can induce neonatal rat muscle fibroblasts, in culture, to express phenotypic products of chondroblastic cells.

Demineralized dentin matrix induces the ectopic formation of bone, in vivo, when implanted subcutaneously or in muscle pouches. In these situations the bone induction follows a chondrogenic pathway. As part of the strategy for the assay and isolation of the factors responsible for initiating induction, we have developed a cell culture system in which the addition of soluble factors extracted from the dentin matrix appears to initiate chondrogenesis. Indicators of chondrogenesis, relative to control cultures, were taken as an increase of 35S-sulfate incorporation into proteoglycan (PG), an altered size of the PG, production of type II collagen, and changes in cell morphology and matrix histochemistry. Our studies have taken two directions: the use of the cell culture system under standard conditions to select fractions inducing one or more of the above indica-tors; and, the purification and characterization of the in vitro chondrogenesis inducing factor(s). Here we report the identification of a peptide fraction which acts in culture to satisfy each of the above indicators of chondrogenesis. An EDTA extract of rat incisor dentin was fractionated by CaCl2 precipitation, Sephacryl S-100 chromatography, and reverse phase HPLC. A single peptide fraction from the HPLC, evidenced by the existence of a single spot on 2-D Gel Electrophoresis, was found to be a potent enhancer of 35S-sulfate incorporation during the standard assay, with maximal activity in the 1-10 ng/ml range. Further detailed studies showed that the heightened incorporation occurred without any increase in cell number. The neonatal rat muscle explant fibroblasts exposed to this fraction for 7 days in monolayer culture formed dense cell nodules which stained intensely with Alcian blue relative to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Concentration-dependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth.

The effect of dentin phosphophoryn on hydroxyapatite formation and growth was studied in an in vitro gelatin gel diffusion system. Phosphophoryn, in low concentrations (0.010-1 microgram/ml) promoted de novo hydroxyapatite formation; at a higher concentration (100 micrograms/ml) in the same system, the dentin matrix protein inhibited hydroxyapatite growth. Similar inhibition of hydroxyapatite growth was seen in solution. The intact phosphophoryn was not essential for either inhibition of seeded growth or promotion of mineralization, since the formic acid degraded protein was comparably effective. Transmission electron microscopy of the precipitates formed at 7 days showed no significant differences in crystallite size distribution in the presence and absence of phosphophoryn. However there was a dose-dependent decrease in the number of mineral clusters formed in the presence of increasing amounts of phosphophoryn, suggesting inhibition of secondary nucleation. These data provide support for the postulated 'multifunctional' role of the dentin phosphoprotein in the mineralization process.

Animals↗
PubMed1990

Apical placement of needle tip with an injection-thermoplasticized gutta-percha technique for root canal obturation.

It is recommended that when injection-thermoplasticized gutta-percha is used as the obturation technique, the root canal space should be prepared in such a way that the 23-gauge injection needle tip can be placed at a distance 3 to 5 mm short of the working length. In this study the apical seal and extent of gutta-percha were evaluated when the 23-gauge tip was placed at a distance 4 mm, 6 mm, and 8 mm short of the working length. Ninety-nine human single-rooted mandibular incisors and premolars divided into 3 equal groups were used. Their crowns were separated at the cementoenamel junction and their roots were stored in sodium hypochlorite for 1 week. The roots were then instrumented from size 20 at the working length to size 70:4 mm short of the working length in group A, 6 mm in group B, and 8 mm in group C. Following instrumentation, 3 randomly selected roots of each group were used as control. The remaining 30 roots of each group were covered with aluminium foil and embedded in acrylic blocks. The root canals were then dried, coated with sealer (Roth 811) and obturated by injection of thermoplasticized gutta-percha using the single-phase technique. Vertical condensation followed. The roots were immersed in dye for 3 days and then demineralized, dehydrated and cleared in xylene. Measurements of linear dye penetration for the apical seal and for underextension were done by 2 independent examiners under a stereomicroscope. The results of this study showed no statistically significant difference in leakage between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Coloring Agents↗
PubMed1990

Phosphorylation of extracellular bone and dentine matrix proteins.

Phosphoproteins appear to be involved in several ways in the regulation of the orderly deposition and crystal growth of mineral within the performed collagenous matrix of bone and dentine. The phosphorylation of these proteins is not yet understood. Potential protein kinases were extracted from an osteoblast-like cell line, ROS 17/2.8. The ROS 17/2.8 line was shown to produce a full complement of known kinases. However, neither bone phosphoproteins (BPP) nor dentine phosphophoryn (DPP) could be phosphorylated by the messenger dependent kinases. DPP and dephosphorylated BPP (dBPP) were substrates for a unique messenger independent kinase distinct from casein kinase II, and dDPP was a still better substrate. Thus, BPP and DPP are phosphorylated by a unique kinase or set of kinases which are messenger independent and have very specific substrate sequence requirements.

Bone and Bones↗
PubMed1990

Rat incisor dentine contains a factor which alters the phenotypic expression and stimulates chondrogenesis in fibroblast-like cells in vitro.

A low molecular weight protein fraction isolated under dissociative conditions during the demineralization of rat incisor dentine has the ability to modulate, in culture, the expression of fibroblast-like cells explanted from neonatal rat muscle. The protein fraction enhances the incorporation of 35S-sulphate into a proteoglycan larger in weight than that produced by the uninduced cells; furthermore it induces the production of type II collagen. These changes take place in the absence of cell proliferation as measured by 3H-thymidine incorporation. The altered fibroblast-like cells form nodules and secrete an abundant extracellular matrix which stains for proteoglycan after 7-9 days in culture. These data show that the dentine matrix does contain a factor which can initiate a mitogenesis-independent alteration in the expression of the muscle-explant outgrowth cells. Those changes are consistent with a shift to a chondrogenic mode.

Animals↗
PubMed1989

Nonenzymatic glycation of human blood platelet proteins.

We studied 11 diabetic patients, all of whom had severe atherothrombotic disease, and 11 normal controls. Overall glycation was assessed by the extent of incorporation of [3H]-NaBH4 into fructosyl lysine separated from whole platelet proteins following aminoacid analysis. Fructosyl lysine represented 5.7% +/- 1.0 S.D. of the total radioactivity in the normal whole platelet samples. Increased glycation was observed in platelets from 5 of the 11 diabetics. Platelet glycation did not correlate with glycation of hemoglobin or albumin. The pattern of glycation of various platelet proteins in whole platelets, as determined by the incorporation of [3H]-NaBH4 into electrophoretically separated proteins did not display selectivity, although myosin and glycoproteins IIb and IIIa showed relatively increased levels of [3H]-NaBH4 incorporation. Artificially glycated platelet membranes exhibited glycation mainly in proteins corresponding to the electrophoretic mobility of myosin, glycoproteins IIb and IIIa.

Amino Acids↗
PubMed1989

The coordinate synthesis and cotranslational assembly of type I procollagen.

Nascent polysome-associated type I procollagen pro-alpha-chains isolated from chick embryo tendon fibroblasts were examined for their proteinase resistance. The distribution of chain sizes and their proteinase resistance were also determined following chain elongation in an in vitro readout system in the absence of chain initiation factors. Chains were labeled with [14C]proline in the cells and with [3H]proline in the readout system. Differences in the ratios of 14C to 3H in the double-labeled nascent chains before and after chymotryptic digestion, determined by slicing and counting polyacrylamide gels after electrophoresis, permitted analysis of the relative stabilities of in vivo and in vitro elongated portions of the chains. In confirmation of earlier work, the polysome-bound nascent procollagen contained chymotrypsin, chymotrypsin plus trypsin, and pepsin-resistant alpha-chain size components. The readout system data showed that the full length chains produced in the cell were more resistant to digestion than the fully elongated readout-completed chains. The protease resistance of the chains was taken to indicate the registration of the chains prior to the induction of helix formation during the isolation procedure. These data support the model in which chain selection and folding are facilitated by the organization of the attachment of the ribosomes to the endoplasmic reticulum surface.

2,2'-Dipyridyl↗
PubMed1989

Periodontal tissue reactions following root canal obturation with an injection-thermoplasticized gutta-percha technique.

The thermoplasticized gutta-percha obturation technique is a relatively recent development in endodontics. In this study we evaluated the possible effects of heat transmitted to the periodontal tissues when the Obtura root canal filling device was used in dog teeth. Two 8-month-old beagle dogs were used; 56 root canals of 36 teeth (maxillary incisors and first, second and third premolars of both jaws) were divided into two groups. In Group A 26 root canals were obturated with the lateral condensation technique, while in Group B 30 root canals were obturated using the Obtura device, without sealer. Observation periods were 1, 3, 7, 28 and 56 days. After routine laboratory procedures, hematoxylin-eosin stained sections were examined under a light microscope. In this experimental model the thermoplasticized gutta-percha obturation technique caused a periapical inflammatory reaction at all observation periods. Inflammatory reaction and destruction of collagen fibers were localized in the area around the apical delta, while the alveolar founding bone, the roots of the teeth and the periodontal ligament at the side of the root surfaces remained normal.

Animals↗
PubMed1989

Sealing ability of thermoplasticized gutta-percha in root canal obturation using a sectional vs. a single-phase technique.

Properly prepared straight root canals of freshly extracted maxillary canines and central incisors were used to evaluate the sealing ability of the sectional thermoplasticized gutta-percha obturation technique, with or without sealer and the single-phase thermoplasticized gutta-percha filling technique. The teeth were divided into 3 groups (A, B, C) of 20 teeth and were obturated using the 3 thermoplasticized gutta-percha techniques. Group A: single phase with sealer; group B: sectional technique without sealer; and group C: sectional technique with sealer. The obturated teeth were immersed in India ink for 3 days followed by clearing procedures. The teeth were then examined under a stereo microscope and the linear leakage was recorded. Statistical unpaired Student's t-tests showed significantly less dye penetration in teeth obturated using the sectional technique with sealer (group C) than in teeth obturated with the single-phase technique with sealer (group A) or the sectional technique without sealer (group B).

Gutta-Percha↗
PubMed1989

Isolation of an acidic protein from cholesterol gallstones, which inhibits the precipitation of calcium carbonate in vitro.

In seeking to identify nucleating/antinucleating proteins involved in the pathogenesis of cholesterol gallstones, a major acidic protein was isolated from each of 13 samples of cholesterol gallstones. After the stones were extracted with methyl t-butyl ether to remove cholesterol, and methanol to remove bile salts and other lipids, they were demineralized with EDTA. The extracts were desalted with Sephadex-G25, and the proteins separated by PAGE. A protein was isolated, of molecular weight below 10 kD, which included firmly-bound diazo-positive yellow pigments and contained 24% acidic, but only 7% basic amino acid residues. The presence of N-acetyl glucosamine suggested that this was a glycoprotein. This protein at concentrations as low as 2 micrograms/ml, but neither human serum albumin nor its complex with bilirubin, inhibited calcium carbonate precipitation from a supersaturated solution in vitro. This protein could be precipitated from 0.15 M NaCl solution by the addition of 0.5 M calcium chloride. Considering that cholesterol gallstones contain calcium and pigment at their centers, and that small acidic proteins are important regulators in other biomineralization systems, this protein seems likely to play a role in the pathogenesis of cholesterol gallstones.

Amino Acids↗
PubMed1989

A search for the osteogenic factor in dentin. Rat incisor dentin contains a factor stimulating rat muscle cells in vitro to incorporate sulfate into an altered proteoglycan.

Demineralized dentin matrix has the capacity to induce bone formation via a chondrogenic pathway when implanted into muscle, in a fashion entirely analogous to bone matrix implants. In this work we have attempted to isolate, from rat incisor dentin, the matrix factor responsible for initiating osteogenesis. Rat incisor dentin was demineralized with EDTA plus 4.0 M guanidine. HCl. The proteins in the extracts were collected and, after a CaCl2 precipitation step, fractionated on Sephacryl S-200 in 6.0 M guanidine. HCl. The primary assay for activity was the incorporation of 35S-sulfate into proteoglycan in cultures of the fibroblast-like outgrowth cells from explants of neonatal rat muscle. Two Sephacryl S-200 fractions showed enhanced sulfate incorporating activity, but only one showed enhanced incorporation without a concomitant increase in cell number. In the presence of this fraction, the cell cultures produced a larger amount of a new small proteoglycan, as compared to controls, and a significant amount of a much larger proteoglycan. The active fraction had proteins in the Mr range from 8,000 to 15,000 as the major components. These data suggest that the fraction identified may contain the factors responsible for initiating the osteogenic response to dentin matrix upon its implantation in muscle in vivo.

Animals↗
PubMed1989

Characterization of the molecular weights of bovine molar, rat incisor, and other phosphophoryns.

The phosphophoryns show anomalous behavior in solution, and are easily degraded during extraction. They appear in varied forms in the teeth of different species and differ in the teeth of the same species in a developmental and age dependent fashion. This set of properties has made the characterization of the phosphophoryns by biochemical means a difficult and controversial subject. Bovine molar phosphophoryn, bPP, has been characterized in detail by a variety of physical methods, and then compared with the PP from other species by DEAE-HPLC, gel electrophoresis, and immunological cross reactivity. The possible existence of a proPP biosynthetic precursor has been investigated by rat incisor organ culture and examination of the 32P and 14C labeled products. These studies all show marked differences in the Mr values for the PP from teeth of different species, even when they are antigenically cross-reactive.

Animals↗
PubMed1988

Phosphoproteins from teeth and bone.

The anionic non-collagenous proteins of the extracellular matrices of bone and dentine have been proposed as important participants in the mineralization of these tissues. Phosphorylated protein components have been implicated as mediators of the specific nucleation of the growth of hydroxyapatite crystals on the matrix collagen fibrils. However, the phosphoproteins of bone and dentine are quite different and it is difficult to postulate a common mechanism for the nucleation. If there is a common mechanism some particular domain on each molecule is likely to be involved. Therefore, we have initiated studies of the domain structure of the most highly phosphorylated dentine protein, phosphophoryn. At least three sequence domains with different character have been found, and studies are under way to relate these domains to the antigenic, collagen-binding and calcium ion-binding properties of phosphophoryn. The collagen- and calcium ion-binding regions appear to be localized within the same domains.

Animals↗