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.

Isolation and characterisation of an alternatively-spliced rat amelogenin cDNA: LRAP--a highly conserved, functional alternatively-spliced amelogenin?

A cDNA coding for a 59 amino acid polypeptide containing both the carboxy- and amino-termini, but lacking the central domain, of the rat tooth enamel matrix protein, amelogenin, was cloned and sequenced. The deduced polypeptide sequence indicates that this cDNA was derived from an amelogenin RNA molecule by using an alternative intra-exonic 3' splice acceptor site. This alternatively spliced product is almost identical to products previously identified in both cow and mouse enamel organs: the leucine-rich amelogenin peptide (LRAP). The conservation of this truncated polypeptide across the species suggests that it may have an important role in the formation of tooth enamel.

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.

Expression plasmid vectors containing Escherichia coli tryptophan promoter transcriptional units lacking the attenuator.

Two DNA fragments which contain the Escherichia coli tryptophan promoter operator region but lack the attenuator have been used in the construction of a series of pAT153 based plasmids suitable for the regulated expression of foreign genes in E. coli. The first, a 139-bp HhaI fragment includes 59 bp of the trp leader sequence, ending within the "attenuator peptide" coding sequence, eleven codons from the N-terminus. A fusion-type expression plasmid incorporating this fragment has been constructed. The second, a 99-bp HaeIII-TaqI fragment contains no coding sequence but includes the "attenuator peptide" SD site situated 4 bp upstream of the TaqI site. This fragment has been incorporated in expression vectors which result in the direct expression of cloned gene sequences. To further maximise expression, plasmids with directly repeating trp promoter HaeIII-TaqI units have been constructed.

Sequence analysis of the 4.7-kb BamHI-EcoRI fragment of the equine herpesvirus type-1 short unique region.

To localize gene that may encode immunogens potentially important for recombinant vaccine design, we have analysed a region of the equine herpesvirus type-1 (EHV-1) genome where a glycoprotein-encoding gene had previously been mapped. The 4707-bp BamHI-EcoRI fragment from the short unique region of the EHV-1 genome was sequenced. This sequence contains three entire open reading frames (ORFs), and portions of two more. ORF1 codes for 161 amino acids (aa), and represents the C terminus of a possible membrane-bound protein. ORF2 (424 aa) and ORF3 (550 aa) are potential glycoprotein-encoding genes; the predicted aa sequences contain possible signal sequences, N-linked glycosylation sites and transmembrane domains; they also show homology to the glycoproteins gI and gE of herpes simplex virus type-1 (HSV-1), and the related proteins of pseudorabies virus and varicella-zoster virus. The predicted aa sequence of ORF4 shares no homology with other known herpesvirus proteins, but the nucleotide sequence shows a high level of homology with the corresponding region of the EHV-4 genome. ORF5 may be related to US9 of HSV-1.

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 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 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.

Location of open reading frames coding for equine herpesvirus type-1 glycoproteins with homology to gE and gI of herpes simplex virus.

The DNA fragments representing the entire short unique region and part of the repeat sequences of the equine herpesvirus type-1 genome were cloned into plasmid vectors. The approximate positions of the junctions between the short unique region and the inverted repeats were then located by restriction endonuclease mapping. Two open reading frames coding for potential glycoproteins have been identified within the short unique region, using DNA sequence analysis. The predicted amino acid sequences of these open reading frames had extensive homology to the herpes simplex virus glycoproteins gE and gI and the related glycoproteins of pseudorabies virus and varicella-zoster virus.

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.

Uptake and metabolism of albumin by rodent incisor enamel in vivo and postmortem: implications for control of mineralization by albumin.

The distribution of albumin throughout enamel development in the rat mandibular incisor was investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting employing an anti-rat albumin antibody. Intact albumin was detectable at all stages of enamel development but was most evident during late secretion/transition. Its concentration was subsequently reduced during the maturation stage. Albumin degradation products appeared during the transition/early maturation stage indicating that albumin breakdown preceded its removal. As albumin inhibits apatite crystal growth, its degradation and removal may be a necessary prerequisite for normal enamel crystal growth, perhaps reflecting a general mechanism for removal of residual endogenous matrix or adventitious crystal growth inhibitors. Additional studies revealed that the maturation stage was particularly susceptible to albumin influx postmortem. Albumin could therefore form part of the natural crystal growth control process, which, if not removed, could hamper maturation and lead to white spot hypoplasias.

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.

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.

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.

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.

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.

Molecular cloning and DNA sequence of rat amelogenin and a comparative analysis of mammalian amelogenin protein sequence divergence.

The developing rat incisor is a common model used in the study of enamel development. It has been impossible to study correlation between rat enamel structure and the sequence of the major developing enamel protein in this species as to date a DNA sequence for rat amelogenin has not been reported. This study presents the first cloning of a full-length cDNA copy of rat amelogenin and its deduced primary sequence. Detailed analysis of this sequence provides evidence that the gene has evolved by internal sequence duplication. Comparison of the rat amelogenin primary sequence with those published for other species provides evidence that this protein, while exhibiting extreme levels of sequence conservation, has been subject to significant structural changes that may be related to alterations in enamel structure in different mammalian groups.

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.