Patchiness of ion-exchanged mica revealed by DNA binding dynamics at short length scales.

The binding of double-stranded (ds) DNA to mica can be controlled through ion-exchanging the mica with divalent cations. Measurements of the end-to-end distance of linear DNA molecules discriminate whether the binding mechanism occurs through 2D surface equilibration or kinetic trapping. A range of linear dsDNA fragments have been used to investigate length dependences of binding. Mica, ion-exchanged with Ni(II) usually gives rise to kinetically trapped DNA molecules, however, short linear fragments (<800 bp) are seen to deviate from the expected behaviour. This indicates that ion-exchanged mica is heterogeneous, and contains patches or domains, separating different ionic species. These results correlate with imaging of dsDNA under aqueous buffer on Ni(II)-mica and indicate that binding domains are of the order of 100 nm in diameter. Shorter DNA fragments behave intermediate to the two extreme cases of 2D equilibration and kinetic trapping. Increasing the incubation time of Ni(II) on mica, from minutes to hours, brings the conformations of the shorter DNA fragments closer to the theoretical value for kinetic trapping, indicating that long timescale kinetics play a role in ion-exchange. X-ray photoelectron spectroscopy (XPS) was used to confirm that the relative abundance of Ni(II) ions on the mica surface increases with time. These findings can be used to enhance spatial control of binding of DNA to inorganic surfaces with a view to patterning high densities arrays.

Identification of ragAB as a temperature-regulated operon of Porphyromonas gingivalis W50 using differential display of randomly primed RNA.

Porphyromonas gingivalis is a gram-negative, black-pigmented anaerobe that has been associated with advanced periodontal disease. The genome of P. gingivalis has the potential to produce a number of virulence determinants including proteases, hemagglutinins, hemolysin, invasion-associated proteins, and products of the pathogenicity island ragAB; however, little is known about how their expression is controlled. Periodontal pockets experience a higher temperature during inflammation, and this elevated temperature may influence the pathogenicity of P. gingivalis by changing its patterns of gene expression. In this study, RNA has been isolated from cells of P. gingivalis grown to steady state at temperatures of 37, 39, and 41 degrees C under hemin excess conditions (pH 7.0) in a chemostat. The RNA was subjected to PCR amplification following reverse transcription, using various combinations of randomly selected oligonucleotide primers. Reproducible RNA fingerprints have been obtained; however, differences were demonstrated in the RNA profiles of cells grown at the three temperatures, indicating differences in gene expression. Several PCR fragments were isolated that appeared to represent temperature-regulated genes. The nucleotide sequence of one of these has been identified as part of the ragAB locus, which codes for both a 55-kDa immunodominant antigen (RagB) and a homologue of the family of TonB-linked outer membrane receptors (RagA). These data indicate that expression of ragAB may be modulated in response to changes in temperature and that this may suggest a mechanism of evading the host response in the inflamed periodontal pocket.

Trefoil factor expression in normal and diseased human salivary glands.

Trefoil factors are wound-healing peptides important in protection and healing of the human gastrointestinal tract. Their potential for therapy of gastrointestinal ulcers has been established. This study investigated the hypothesis that trefoil factors are also present in human salivary gland. Tissues from surgical biopsy specimens were collected fresh into ice and stored in liquid nitrogen. Breast, stomach, and colon constituted positive controls. Trefoil factor mRNAs were detected by reverse transcription polymerase chain reaction (RT-PCR) or by in situ hybridization (ISH) with formalin-fixed, paraffin-embedded sections. Amplified DNA fragments were ligated into pGEM-T Easy vector and used to transform competent Escherichia coli JM109, allowing sequencing to confirm identity of cloned fragments. Generation of amplifiable cDNA was confirmed using primers specific to the ubiquitously expressed abl gene. By RT-PCR, TFF1 (pS2) mRNA was detected in 14 of 15 glands, TFF3 (hITF) mRNA in 13, and TFF2 (hSP) in only 1 gland. ISH of 15 glands (7 of which had been studied by RT-PCR) showed the same pattern of expression and indicated that TFF1 mRNA was usually expressed at low levels by a few mucous cells, whereas TFF3 was produced abundantly by most mucous cells. There was no difference in patterns of expression comparing parotid, submandibular, and minor mucous glands. Nor was there an obvious relationship between trefoil factor expression and pathology, but those glands not expressing TFF1 or TFF3 had evidence of chronic inflammation or atrophy. Trefoil factors are likely to be important in healing, predisposition to, and therapy of, oral diseases.

The chemical composition of tooth enamel in junctional epidermolysis bullosa.

The junctionalis form of epidermolysis bullosa (EBJ) is associated with a number of clinical problems involving tooth enamel, including increased susceptibility to caries. The aim here was to carry out a chemical characterization of the enamel of teeth from EBJ patients compared with that of unaffected controls. The results showed that while protein concentration, amino acid composition and carbonate content were similar in both groups, EBJ enamel contained a significantly reduced mineral per volume content, resulting in enamel hypoplasia. In addition, Western blotting revealed the presence of serum albumin (a known inhibitor of enamel crystal growth) in EBJ enamel. This was not detected in control enamel or in enamel of teeth from patients with the dystrophic form of the disease. It is concluded that EBJ enamel is developmentally compromised and that the enamel defects are commensurate with the reported genetic lesions.

Influence of sample preparation technique on two-dimensional gel electrophoresis of proteins from Porphyromonas gingivalis.

Sample preparation methods were compared for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of cellular proteins from the proteolytic bacterium Porphyromonas gingivalis. Standard solubilization buffer yielded poorly resolved protein spots, but pre-treatment of cells with trichloroacetic acid or inclusion of the protease inhibitor TLCK during solubilization improved definition and separation. The latter approach allowed reliable detection of a 55 kDa immunodominant surface antigen by Western immunoblotting. Further improvements in resolution occurred when SDS was included in the sample preparation. Thus, controlling proteolysis and optimizing protein solubilization were essential for reproducible separations and maximal protein recovery during 2D-PAGE of P. gingivalis.

Expression of beta-defensin genes by human salivary glands.

This study investigated expression of genes encoding human beta-defensins 1 and 2 by human salivary glands. Tissues from surgical biopsies were collected fresh onto ice and stored in liquid nitrogen. Total RNA was extracted using Trizol reagent and human beta-defensin messenger RNA detected by reverse transcriptase polymerase chain reaction amplification. DNA sequencing of amplified fragments, after ligation into pGEM-T Easy vector and transformation of competent Escherichia coli, confirmed identities of cloned fragments. Human beta-defensin 1 messenger RNA was detected in all 25 samples that generated amplifiable cDNA, as assessed using abl-specific primers. Three of 13 submandibular gland samples (two normal, one chronically inflamed), and 2 of 2 minor salivary gland samples (one normal, one chronically inflamed) expressed human beta-defensin 2 messenger RNA. All six parotid gland samples studied were negative for human beta-defensin 2 messenger RNA. Thus, human beta-defensin 1 gene expression occurred in all human major and minor salivary glands studied, whereas human beta-defensin 2 expression occurred only in a small number of gland samples.

Evidence for regulation of amelogenin gene expression by 1,25-dihydroxyvitamin D(3) in vivo.

The unique hereditary enamel defect clearly related to the disturbance of one enamel matrix protein is X-linked amelogenesis imperfecta (AI), in which several mutations of amelogenin gene have been identified. The clinical phenotype of many of these subjects shows similarities with enamel defects related to rickets. Therefore, we hypothesized that rachitic dental dysplasia is related to disturbances in the amelogenin pathway. In order to test this hypothesis, combined qualitative and quantitative studies in experimental vitamin D-deficient (-D) rat model systems were performed. First, Western blot analysis of microdissected enamel matrix (secretion and maturation stages) showed no clear evidence of dysregulation of amelogenin protein processing in -D rats as compared with the controls. Second, the ultrastructural investigation permitted identification of the internal tissular defect of rachitic enamel, the irregular absence of intraprismatic enamel observed in -D animals, suggesting a possible link between prism morphogenesis and vitamin D. In addition, the steady-state levels of amelogenin mRNAs measured in microdissected dental cells was decreased in -D rats and up-regulated by an unique injection of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). The present study shows evidences that amelogenin expression is regulated by vitamin D. This is the first study of an hormonal regulation of tooth-specific genes.

The teaching of molecular biology to undergraduate dental students within the UK.

This work was undertaken to assess the extent and nature of molecular biology teaching to undergraduate dental students in the UK. We surveyed the lecturers of those schools involved in teaching molecular biology and a questionnaire and covering letter were circulated to all 14 UK Dental Schools.

Identification of human serum albumin in human caries lesions of enamel: the role of putative inhibitors of remineralisation.

Carious attack on enamel is not a unidirectional process but involves both demineralisation and remineralisation. The chemistry of carious attack on enamel has, to a large extent, now been clarified as far as mineral components are concerned but little attention, however, has been paid to the identity of organic material in carious lesions and its possible role in the caries process. The only clear information available is that organic material accumulates with time within enamel lesions. The present study was aimed at identifying a specific protein component known to bind to hydroxyapatite (albumin) in carious lesions with a view to investigating its role in the disease process. The distribution of albumin within both white spot and fissure lesions and adjacent sound enamel of extracted human teeth was investigated using SEM immunohistochemistry on undermineralised sections of human enamel and employing a polyclonal antibody to human serum albumin. The nature of the protein, i.e. whether it was in the form of intact molecules or degraded fragments, was investigated by Western blotting, employing the same antibody. The immunohistochemistry revealed the presence of albumin within both interproximal white spot and fissure lesions with little if any present in sound enamel. The Western blotting indicated that the albumin was in the intact form with no evidence of degradation products. The ability of albumin to bind and to inhibit growth of calcium phosphate crystals raises the question as to the possible role of such a molecule in the development of carious lesions.

Enzyme compartmentalization during biphasic enamel matrix processing.

Processing of enamel matrix proteins is essentially biphasic. Secretory stage metalloprotease activity generates a discrete, presumably functional, spectrum of molecules which may also undergo dephosphorylation. Maturation stage serine proteases almost completely destroy the matrix. The present aim was to examine the tissue compartmentalization of these enzyme activities in relation to their possible function. A sequential extraction using synthetic enamel fluid, phosphate buffer and SDS was used to identify enzymes free in the enamel fluid, crystal bound or aggregated with the bulk matrix respectively. Results indicated that the metallo-proteases and alkaline phosphatase were free in the secretory stage enamel fluid while the serine proteases appeared to be largely bound to the maturation stage crystals. The mobility of the metallo-proteases and alkaline phosphatase would ensure efficient initial processing of secretory matrix, while the largely mineral bound serine proteases would ensure retention of protease activity despite massive destruction and protein removal.

Atomic force microscopy studies of crystal surface topology during enamel development.

During the secretory stage of enamel development, the hydroxyapatite crystals appear as thin ribbons which grow substantially in width and thickness during the later maturation stage. In this study, the atomic force microscope (AFM) was used to investigate developmentally-related changes in deproteinized enamel crystal surface topography in normal animals and in those receiving daily doses of fluoride. The AFM revealed previously undescribed surfaces features, some of which may represent growth sites or different crystalline phases. Secretory stage crystals had greater surface rugosity and were more irregular, with spherical sub-structures of 20-30 nm diameter arranged along the "c"-axis. Maturation stage crystals were smoother and larger but revealed both subnanometer steps and lateral grooves running parallel to the "c"-axis. Crystals from fluorotic tissue showed similar features but were more irregular with a higher degree of surface roughness, suggesting abnormal growth. The AFM may prove an important adjunct in determination of the mechanisms controlling crystal size and morphology in skeletal tissues.

Identification of rat enamel organ RNA transcripts using differential-display.

Enamel formation is a complex process which involves the expression of a number of genes, the most obvious being those related to the mineralized extracellular matrix. In this study the differential-display technique, first described by Liang and Pardee, has been used to identify genes specifically expressed in enamel organ cells. By comparing results obtained from RNA derived from rat enamel organ with RNA derived from other cellular sources, a number of differentially expressed transcripts have been identified. The nucleotide sequences of two of these have been analyzed and shown to have no homology with any previously published sequences. Further analysis will provide information on the type of protein that they may encode, their tissue distribution and their potential role in enamel formation.

Enamel maturation.

Enamel maturation is characterized by massive crystal growth in both width and thickness, resulting in the most highly mineralized of all mammalian skeletal tissues. The control of this process is mediated via a carefully orchestrated series of events that are temporally and spatially regulated, and it requires the co-ordinated degradation and removal of the endogenous enamel matrix. This is affected by both neutral metalloproteases and serine proteases, which are developmentally restricted and may be further modulated by changes in the chemistry of the enamel crystals themselves. Failure of these mechanisms, or the adventitious entry of mineral-binding proteins during the later stages of maturation, may result in the incomplete maturation of the enamel crystals and the eruption of dysplastic tissue.

A method for the quantitative site-specific study of the biochemistry within dental plaque biofilms formed in vivo.

The study of plaque biofilms in the oral cavity is difficult as plaque removal inevitably disrupts biofilm integrity precluding kinetic studies involving the penetration of components and metabolism of substrates in situ. A method is described here in which plaque is formed in vivo under normal (or experimental) conditions using a collection device which can be removed from the mouth after a specified time without physical disturbance to the plaque biofilm, permitting site-specific analysis or exposure of the undisturbed plaque to experimental conditions in vitro. Microbiological analysis revealed plaque flora which was similar to that reported from many natural sources. Analytical data can be related to plaque volume rather than weight. Using this device, plaque fluoride concentrations have been shown to vary with plaque depth and in vitro short-term exposure to radiolabelled components may be carried out, permitting important conclusions to be drawn regarding the site-specific composition and dynamics of dental plaque.

Crystal growth in dental enamel: the role of amelogenins and albumin.

Amelogenin-mineral interactions were investigated using an in vitro binding approach. Rat incisor enamel matrix proteins (mainly amelogenins) were dissolved in synthetic enamel fluid and allowed to equilibrate with deproteinised developing enamel crystals. The results showed that amlogenin proteins of 21, 23, 24, 26 and 27-kDa (corresponding to nascent and partially degraded amelogenins) were associated with the crystals whilst the lower Mr amelogenins (< 21 KDa) remained free in the synthetic enamel fluid. These data suggest the nascent and partially degraded amelogenins may interact with developing enamel crystals and could influence their growth. Albumin-mineral interactions were investigated by extracting developing rat incisor enamel with synthetic enamel fluid. Insoluble material (including the enamel crystals) was then further extracted with 0.1 M phosphate buffer (pH 7.4) to desorb any mineral bound proteins. Western blotting using anti-albumin antibodies showed that almost all of the albumin from the secretory stage enamel and a significant proportion of the albumin present in early transition stage was extractable in the synthetic enamel fluid. However, synthetic enamel fluid did not extract albumin from late transition or maturation stage tissue, which could only be removed following further extraction with phosphate buffer. Albumin degradation was apparent during the transition and maturation stages, where it is degraded and ultimately removed. This binding pattern may be related to amelogenin degradation and removal during the transition stage, permitting albumin access to the previously obscured crystal surfaces. That the secretory stage matrix appears to "protect" secretory stage crystals from albumin may be an important consideration in the aetiology of enamel hypoplasias (i.e. incomplete crystal growth) and when using dissociative extraction procedures for the identification of mineral bound proteins.

The rat amelogenin gene--some aspects of evolution and expression.

This study presents data to support the hypothesis that a major portion of the coding sequence of the amelogenin gene may have arisen by tandem duplication of internal sequences which as a consequence has introduced several additional potential RNA splice acceptor sites into the sequence. This duplication of splice sites has led to an increase in the heterogeneity of amelogenin forms found in developing enamel. By screening a rat enamel organ cDNA library for alternatively spliced products, it appears that as much as 20% of the amelogenin mRNA molecules may be alternatively spliced forms.

The chemical composition of tooth enamel in recessive dystrophic epidermolysis bullosa: significance with respect to dental caries.

Previous reports have linked the prevalence of tooth abnormalities with high caries experience in the different types of epidermolysis bullosa (EB). However, it is not known to what extent the apparent susceptibility to enamel caries is due to disease-related altered enamel chemistry in these cases. The aim of this study was to characterize the enamel of teeth from patients suffering from recessive epidermolysis bullosa dystrophica (rEBD) in terms of its mineral content, carbonate content, protein content, and amino acid composition. The results showed that dental enamel from these patients was essentially normal in terms of its chemistry. It is therefore concluded that the high caries experience in recessive dystrophic epidermolysis bullosa patients is probably related to other factors, such as compromised oral hygiene and prolonged oral clearance due to extensive oral soft tissue damage and a cariogenic diet.

The chemistry of enamel development.

The central problems of enamel biochemistry are the mechanisms concerned with initiation and development of the mineral crystals, together with their architectural arrangement within the tissue. These processes are mediated by the extracellular matrix as well as the composition of the mineral itself. Initial mineral deposition occurs at the dentine surface, nucleated either by dentinal components or early enamel matrix, possibly non-amelogenin molecules. The early crystals are small in size and rich in magnesium and carbonate resulting in relatively poor crystallinity. This is in spite of the fact that fluoride is high at this stage. Crystal development includes a reduction in magnesium, carbonate and fluoride as crystals increase in length following the retreating ameloblasts from the dentine. The matrix acquires increasing concentrations of amelogenin and albumin. Prismatic structure begins to develop together with some growth of crystals in width and thickness. Degradation of amelogenin and non-amelogenin molecules generates a series of specific molecular fragments possibly concerned with modulating crystal growth and morphology and the creation of prismatic and interprismatic structures. Towards the end of secretion, matrix, now almost completely degraded, is replaced by fluid followed by massive crystal growth during maturation. Degradation of albumin also occurs at this stage, probably as a result of comprehensive destruction of molecules which might impair crystal growth. Selective acquisition of magnesium and fluoride at this stage may reflect the hydrated state of the tissue as well as cell changes. Fluid is displaced as crystals grow and the enamel acquires concentrations of mineral characteristic of mature tissue.

The effect of glycosylaminoglycans on the mineralization of sheep periodontal ligament in vitro.

The effect of removal of glycosylaminoglycans on the mineralization of sheep periodontal ligament was determined using enzyme digests followed by incubation in solutions supersaturated with respect to hydroxyapatite at pH 7.4. TEM revealed that control periodontal ligament remained unmineralized. However, tissue from which glycosylaminoglycans had been removed contained plate-like crystals arranged parallel to and within the collagen fibrils. Electron probe and electron diffraction studies suggested that the crystals were apatitic with a similar order of crystallinity to dentine, and a Ca:P ratio of 1.61. In addition, the glycosylaminoglycan content of periodontal ligament, cementum and alveolar bone was compared using cellulose acetate electrophoresis. Periodontal ligament contained predominantly dermatan sulfate while cementum and alveolar bone contained mostly chondroitin sulfate. A role for glycosylaminoglycans in maintaining the unmineralized state of the periodontal ligament is suggested. Control of expression of specific proteoglycan species on a spatially restricted basis is presumably central to this role.