New paradigms on the transport functions of maturation-stage ameloblasts.

Fully matured dental enamel is an architecturally and mechanically complex hydroxyapatite-based bioceramic devoid of most of the organic material that was essential in its making. Enamel formation is a staged process principally involving secretory and maturation stages, each associated with major changes in gene expression and cellular function. Cellular activities that define the maturation stage of amelogenesis include ion (e.g., calcium and phosphate) transport and storage, control of intracellular and extracellular pH (e.g., bicarbonate and hydrogen ion movements), and endocytosis. Recent studies on rodent amelogenesis have identified a multitude of gene products that appear to be linked to these cellular activities. This review describes the main cellular activities of these genes during the maturation stage of amelogenesis.

Surface integrity governs the proteome of hypomineralized enamel.

Growing interest in the treatment and prevention of Molar/Incisor Hypomineralization (MIH) warrants investigation into the protein composition of hypomineralized enamel. Hypothesizing abnormality akin to amelogenesis imperfecta, we profiled proteins in hypomineralized enamel from human permanent first molars using a biochemical approach. Hypomineralized enamel was found to have from 3- to 15-fold higher protein content than normal, but a near-normal level of residual amelogenins. This distinguished MIH from hypomaturation defects with high residual amelogenins (amelogenesis imperfecta, fluorosis) and so typified it as a hypocalcification defect. Second, hypomineralized enamel was found to have accumulated various proteins from oral fluid and blood, with differential incorporation depending on integrity of the enamel surface. Pathogenically, these results point to a pre-eruptive disturbance of mineralization involving albumin and, in cases with post-eruptive breakdown, subsequent protein adsorption on the exposed hydroxyapatite matrix. These insights into the pathogenesis and properties of hypomineralized enamel hold significance for prevention and treatment of MIH.

ToothPrint, a proteomic database for dental tissues.

Increasing demand exists to disseminate and integrate proteomic data as proteome analysis assumes a commanding role in the postgenome era. Databases on the World Wide Web are an effective means to share information obtained from two-dimensional gels and allied proteomic approaches. Here we report the establishment of ToothPrint, a proteomic database for dental tissues accessed at http://toothprint.otago.ac.nz. Using developing rat enamel as a prototype, ToothPrint provides a variety of functionally relevant data (ligand binding, subcellular localisation, developmental regulation) in addition to protein identification maps. Features designed to enhance usability of the website and simplify its computing requirements are also outlined. Customized for mineralizing tissues, ToothPrint should prove to be an effective bioinformatic resource for investigations of dental biology.

Endoplasmic reticulum Ca2+-ATPase pump is up-regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA2b.

Dental enamel-forming cells face a major challenge to avoid the cytotoxic effects of excess calcium. We have characterized sarcoplasmic/endoplasmic reticulum calcium-ATPase pumps (SERCA) in rat enamel cells to address the proposal that non-mitochondrial calcium stores play a dominant role in transcellular calcium transport. A single major isoform, SERCA2b, was detected during the protein-secretory and calcium-transport stages of enamel formation using reverse-transcriptase PCR, cDNA cloning, Northern analysis and immunoblotting. Most importantly, SERCA2b exhibited a specific 3-fold up-regulation to high expression levels during calcium transport, as determined by quantitative immunoblotting and ATPase assays. Sensitivity of the calcium-dependent ATPase to thapsigargin and three other SERCA inhibitors was characterized. These findings indicate that enamel cells are well-equipped to sequester calcium in endoplasmic reticulum stores and so protect against calcium toxicity, associate SERCA with transcellular calcium transport for the first time, and establish SERCA2b as a molecular and pharmacological target for future investigations of calcium transcytosis. The observed physiological regulation in enamel cells contradicts the widespread perception that SERCA2b is restricted to general housekeeping duties.

Evaluation and management of the patient co-infected with human immunodeficiency virus and hepatitis C.

The emerging presence of hepatitis C viral (HCV) infection in the United States has been the focus of much attention among health care providers and the general population. Among patients infected with human immunodeficiency virus (HIV), there has been a dramatic increase in hepatitis C disease. During the 1980s and early 1990s, hepatitis C was viewed as a disease for which little could be done, both because of ineffective treatment and the severity and lack of adequate treatments for acquired immune deficiency syndrome (AIDS) itself. Treatment with interferon had poor effect on hepatitis C in the co-infected population, especially for those with advanced immunosuppression. The regimen was difficult to tolerate even with dose reductions. With the advent of highly active antiretroviral therapy (HAART) and effective treatment and prophylaxis for opportunistic infections, a substantial portion of HIV-infected patients are living long enough to have their health compromised by hepatic failure or hepatocellular carcinoma owing to hepatitis C, rather than by AIDS-related illness. New treatments are available for hepatitis C, with preliminary research yielding promising results. The role of these medications in managing HIV/HCV co-infection is currently under study, with implications for many. Health care providers are increasingly faced with the challenges of caring for people infected with the hepatitis C virus, and the growing number of individuals co-infected with hepatitis C and HIV. The purpose of this article is to provide an overview of hepatitis C, especially in the presence of HIV infection, and to detail the recognition and management of the care of this emerging population.

Calbindin28kDa is specifically associated with extranuclear constituents of the dense particulate fraction.

Recent attempts to understand the function of calbindin28kDa, a widely expressed calcium-binding protein, are confounded by uncertainties over its subcellular location. Using immunoblot analysis of rat brain subregions, we found that the proportion of particulate calbindin28kDa (24-43% of total) was independent of expression level and location. The association of calbindin28kDa with particulate structures appeared to be specific, since it persisted when soluble calbindin28kDa was sequestered by antibodies added before tissue disruption. Moreover, when exogenous calbindin28kDa was added during homogenisation of brain from calbindin28kDa-nullmutant mice, only 10% partitioned to the particulate fraction compared with 33% of endogenous calbindin28kDa in wildtype controls. Confocal microscopy showed that calbindin28kDa was predominantly extranuclear in all tissues analysed (i.e. various brain regions, isolated neurons, and dental enamel epithelium). Dual-label microscopy of neural dense particulate fractions confirmed the extranuclear location of calbindin28kDa and also showed that it partly colocalised with synaptosome and microtubule markers. Using sucrose step gradients, calbindin28kDa was separated from nuclei in parallel with synaptosome and endoplasmic reticulum markers. However, no association with the marker proteins (synaptophysin, ERp29, alpha/beta-tubulin) was detected by calbindin28kDa-immunoprecipitation analysis. Together these findings provide the first consistent picture that calbindin28kDa is located predominantly outside of the nucleus, irrespective of tissue type (neuronal vs. non-neuronal) and experimental approach (biochemical vs morphological). The evidence of a substantial, strong and specific association with insoluble cellular structures challenges the widely held view of calbindin28kDa as a mobile calcium buffer, and supports the existence of important alternative roles that involve target proteins.

Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells. Evidence for a unique role in secretory-protein synthesis.

UNLABELLED: Recently we cloned and described ERp29, a novel 29-kDa endoplasmic reticulum (ER) protein that is widely expressed in rat tissues. Here we report our original isolation of ERp29 from dental enamel cells, and the comprehensive sequence analysis that correlated ERp29 with its cognate cDNA, both in enamel cells and liver. Fractionation of enamel cells using a new freeze-thaw procedure showed that ERp29 partitioned with known reticuloplasmins, and not with soluble proteins from mitochondria or cytosol. The absence of ERp29 in secreted enamel matrix indicated that the C-terminal tetrapeptide (KEEL motif) confers effective ER-retention in enamel cells. ERp29 behaved as a single species (approximately 40 kDa) during size-exclusion chromatography of liver reticuloplasm, suggesting that most ERp29 is not stably associated with other proteins. Immunoblot analysis showed that ERp29 was up-regulated during enamel secretion and expressed most highly in secretory tissues, indicative of a role in secretory-protein synthesis. Unlike other reticuloplasmins, ERp29 was down-regulated during enamel mineralization and thereby dissociated from a calcium-handling role. Tissue-specific variations in ERp29 molecular abundance were revealed by quantification of reticuloplasmin mole ratios. IN CONCLUSION: (a) ERp29 is a novel reticuloplasmin of general functional importance; (b) a unique role in protein processing is implicit from the distinctive expression patterns and molecular structure; (c) ERp29 is primarily involved in normal protein secretory events, not the ER stress response; (d) a major role is likely in tissues where ERp29 was equimolar with established molecular chaperones and foldases. This study implicates ERp29 as a new member of the ER protein-processing machinery, and identifies tissues where the physiological role of ERp29 is most likely to be clearly manifested.

Calcium transport across the dental enamel epithelium.

Dental enamel is the most highly calcified tissue in mammals, and its formation is an issue of fundamental biomedical importance. The enamel-forming cells must somehow supply calcium in bulk yet avoid the cytotoxic effects of excess calcium. Disrupted calcium transport could contribute to a variety of developmental defects in enamel, and the underlying cellular machinery is a potential target for drugs to improve enamel quality. The mechanisms used to transport calcium remain unclear despite much progress in our understanding of enamel formation. Here, current knowledge of how enamel cells handle calcium is reviewed in the context of findings from other epithelial calcium-transport systems. In the past, most attention has focused on approaches to boost the poor diffusion of calcium in cytosol. Recent biochemical findings led to an alternative proposal that calcium is routed through high-capacity stores associated with the endoplasmic reticulum. Research areas needing further attention and a working model are also discussed. Calcium-handling mechanisms in enamel cells are more generally relevant to the understanding of epithelial calcium transport, biomineralization, and calcium toxicity avoidance.

Human ERp29: isolation, primary structural characterisation and two-dimensional gel mapping.

Recently we characterised a novel 29 kDa endoplasmic reticulum protein that is widely expressed in rat tissues, and named it ERp29. Several ERp29-like gene products have been reported in human tissues but uncertainty surrounds their relationships with each other and rat ERp29. To clarify these issues, ERp29 was isolated from human liver and characterised by primary structural analysis and two-dimensional gel mapping. Comparisons with rat ERp29 revealed striking homologies both in sequence and physical properties. Characterisation of the isoelectric heterogeneity and anomalous mass on two-dimensional gels enabled two reported homologues (UL35 and ERp31) to be identified as ERp29. Resolution of a sequence discrepancy led to unequivocal correlation of human ERp29 with the cognate cDNA previously named ERp31 and ERp28. Consequent links established to human genome and proteome projects showed that ERp29 is encoded by a gene on chromosome 12 that is expressed universally in human tissues. Together, these findings unified various ERp29 homologues as products of a single gene orthologous to rat ERp29 and established ERp29 as the only known member of a new protein class. Investigations of ERp29 function in human health and disease should benefit from the integrated links between genome, proteome and murine model organisms established here.

Proteomic analysis of enamel cells from developing rat teeth: big returns from a small tissue.

Poor understanding of the molecular links between disturbed calcium regulation in cells and disease remains a problem of considerable biomedical importance. Dental enamel cells might provide useful insights to this problem since they handle calcium in bulk without suffering its cytotoxic effects. Two practical challenges hindered investigation of calcium handling mechanisms in enamel cells--paucity of molecular information and limited availability of sample from developing rat teeth, the experimental system of choice. This paper outlines the microscale proteomic approaches we applied to overcome these difficulties and reviews several major findings that ensued from initial characterization of the enamel cell proteome. Enamel cells are now established as a powerful model for fundamental calcium research and have provided outcomes of broad biological relevance, including the discovery of a new endoplasmic reticulum protein. Future proteomic approaches that might benefit understanding of function are discussed.

Distribution of Borrelia burgdorferi s.l. spirochaete DNA in British ticks (Argasidae and Ixodidae) since the 19th century, assessed by PCR.

The distribution of Borrelia burgdorferi sensu lato, the Lyme borreliosis agent, was surveyed in British ticks in the collection of the Natural History Museum, London. Alcohol-preserved specimens of eight species of ticks known to attack humans were studied: Ixodes ricinus, I. hexagonus, I. uriae, I. trianguliceps, Dermacentor reticulatus, Haemaphysalis punctata, Rhipicephalus sanguineus and Argas vespertilionis. The sample comprised all life stages and originated from a wide range of host species, collection dates (1896-1994) and geographical localities in England, Scotland and Wales. Borrelia burgdorferi s.l. DNA, detected by a polymerase chain reaction that targeted the outer surface protein A gene, was found in all eight species. The overall proportion of PCR-positive specimens ranged from 7.8% for I. hexagonus (mostly from mustelids and hedgehogs) to 98.3% for I. uriae (mostly from seabirds). Borrelia burgdorferi s.l. DNA was found for the first time in the bat parasite A. vespertilionis (85.3%). The spirochaete is newly recorded in British populations of I. trianguliceps (97.4%, mostly from voles, mice and shrews), D. reticulatus (12.5% from dog and man) and R. sanguineus (30% from dogs and human dwellings). Of the four tick species with larvae available for testing, examples of I. ricinus, I. uriae and A. vespertilionis were PCR positive, as were significantly more nymphs than adults of I. ricinus, I. hexagonus and A. vespertilionis. Analyses showed that B. burgdorferi s.l. has been consistently present in British tick populations since at least 1897. Ticks positive for B. burgdorferi s.l. DNA were collected in all months of the year, throughout Britain, and were found on a wide range of mammal and bird species. PCR positivity does not prove vector or reservoir competence, but the use of archived material has demonstrated an extensive range of host-tick relationships involving B. burgdorferi s.l. in Britain for > 100 years.

Enamel cell biology. Towards a comprehensive biochemical understanding.

Enamel cells ultimately determine the properties of dental enamel. Surprisingly little is known about enamel cell functions at the biochemical and molecular levels. Understanding of both normal and abnormal enamel formation should benefit from elucidation of this area. This paper reviews our recent efforts to establish microscale biochemical analyses of rat enamel cells, and the ensuing initial findings about their protein phenotype (i.e., proteome) and calcium-handling mechanisms. A perspective of the current status of enamel cell research, and where it might head, is also given.

Lysozyme and alpha-lactalbumin from the milk of a marsupial, the common brush-tailed possum (Trichosurus vulpecula).

Lysozyme and alpha-lactalbumin have been identified using N-terminal sequence analysis of whey proteins from the common brush-tailed possum, Trichosurus vulpecula after separation by two-dimensional denaturing electrophoresis. Both proteins were purified from pooled possum milk using ion exchange chromatography and gave mass values of 14,896 and 13,985 Da respectively by MALDI-TOF mass spectrometry. Clones containing the full coding sequences of the genes for both proteins were isolated from a possum mammary cDNA library and the DNA sequence of the coding region determined. The inferred protein sequences were used in phylogenetic analysis of both protein classes. These showed that the T. vulpecula alpha-lactalbumin, along with other marsupial alpha-lactalbumins, formed a family distinct from the eutherian alpha-lactalbumins and the alpha-lactalbumin of a monotreme, the platypus, consistent with the separate evolution of the marsupials. By contrast the T. vulpecula lysozyme was shown to be similar to the ruminant stomach lysozymes and primate lysozymes and quite distinct from the Ca2+-binding lysozymes found in the milk of the echidna and horse.

Molecular cloning of ERp29, a novel and widely expressed resident of the endoplasmic reticulum.

We have isolated a full-length cDNA clone for a novel 29 kDa protein that is highly expressed in rat enamel cells. The clone encodes a 259-residue protein, here named ERp29, with structural features (signal peptide and a variant endoplasmic reticulum-retention motif, KEEL) that indicate it is a reticuloplasmin. ERp29 has limited homology with protein disulfide isomerase and its cognates, but lacks their characteristic thioredoxin-like catalytic moiety and calcium-binding motifs. ERp29 mRNA was expressed in all rat tissues tested, and a homologous transcript was detected in other animal livers (primate, ruminant, marsupial). In human hepatoma cells, ERp29 mRNA expression was not increased by stresses (tunicamycin, calcium ionophore) that induced other reticuloplasmins. We conclude that ERp29 is a new, highly conserved member of the reticuloplasmin family which is widely expressed. The apparent lack of both calcium binding properties and stress responsiveness distinguish ERp29 from all major reticuloplasmins characterised to dates.

Mitochondrial ATP synthase F1-beta-subunit is a calcium-binding protein.

Mitochondrial ATP synthase is responsive to changes in cytosolic calcium concentration, but the regulatory mechanisms are unclear. Here we identified a major 52 kDa calcium-binding protein in rat enamel cells as the mitochondrial ATP synthase F1-beta-subunit. The F1-beta-subunit behaved as a low affinity and moderate capacity calcium-binding protein during comparative 45Ca overlay analyses. Equivalent behavior was shown by the F1-beta-subunit from rat liver mitochondria, but not by the homologous F1-alpha-subunit, supporting the specificity of calcium binding. Evidence that the catalytic F1-beta-subunit binds calcium specifically introduces new mechanistic possibilities for regulating ATP synthase, and thereby coordinating ATP production with demand for ATP-fuelled calcium pump activity.

Abundant calcium homeostasis machinery in rat dental enamel cells. Up-regulation of calcium store proteins during enamel mineralization implicates the endoplasmic reticulum in calcium transcytosis.

UNLABELLED: Enamel cells handle large amounts of calcium, particularly during the developmental phase (termed maturation) when dental enamel is hypermineralized. The extent of intracellular calcium burden, and the nature of calcium homeostasis machinery used to accommodate it, are largely unknown. Here, the calcium-binding capacity of enamel cell cytosol was found to increase during development, in parallel with the putative transcellular flux of calcium. At maturation, the abundance of calcium-binding proteins in enamel cells exceeded that in brain and other established calcium-oriented tissues, which implies a large calcium burden. A search for likely cytosolic calcium transporters revealed only one high-affinity calcium-binding protein (12 kDa, distinguished from alpha-parvalbumin) that was up-regulated during maturation, but its low abundance (0.02% of soluble protein) precluded a major calcium transport or cytoprotective role. Two low-affinity calcium-binding proteins up-regulated during maturation (by 1.8-fold and 2.1-fold respectively) were identified as calreticulin and endoplasmin, both residents of the endoplasmic reticulum. Together, calreticulin and endoplasmin constituted an exceptionally high proportion (5%) of soluble protein during maturation, which gives an inferred calcium capacity 67-fold higher than that of the principal cytosolic calcium-binding protein. 28-kDa calbindin. Evidence that endoplasmin expression varied inversely with serum calcium concentration, and that the inositol trisphosphate receptor also was highly expressed during maturation, supported the novel hypothesis that non-mitochondrial calcium stores play a major role in transcellular calcium transport. IN CONCLUSION: (a) enamel cells contain a general high abundance of calcium homeostasis proteins, consistent with a heavy intracellular calcium burden; (b) the expression pattern (phenotype) of calcium-binding proteins varies with enamel cell function; (c) enamel cells appear to contain unusually large non-mitochondrial calcium stores; (d) contrary to the prevailing view that calcium passes mainly through the cytosol of calcium-transporting cells, the findings imply a route through the endoplasmic reticulum. This study gives novel information about how a highly calcium-oriented tissue avoids calcium toxicity, and provides a new focus for investigations into the mechanisms of transcellular calcium transport.

Articular debridement versus washout for degeneration of the medial femoral condyle. A five-year study.

In a prospective randomised trial 76 knees with isolated degenerative changes in the medial femoral condyle of grades 3 or 4 were treated by either arthroscopic debridement (40) or washout (36). All knees were followed up for at least one year and 58 for five years. The mean follow-up time was 4.5 years in the debridement group and 4.3 years in the washout group. At one year 32 of the debridement group and five of the washout group were painfree and at five years 19 of a total of 32 survivors in the debridement group and three of the 26 in the washout group were also free from pain. The mean improvement in a modified Lysholm score was 28 for the debridement group at one year and 21 at five years. In the washout group it was only 5 at one year and 4 at five years. For knees with lesions of the medial femoral condyle of grades 3 or 4, arthroscopic debridement appears to be the treatment of choice with over half the patients free from pain after five years.

Calbindin28kDa and calbindin30kDa (calretinin) are substantially localised in the particulate fraction of rat brain.

Calbindin28kDa is implicated in cytosolic calcium transport and calciprotection functions, principally as a mobile calcium buffer. Using immunoblotting, we have found that 36% of total calbindin28kDa is in the particulate fraction of rat brain. Particulate calbindin28kDa was located both within and outside organelles and required detergent for solubilisation. Equivalent observations were made for calbindin30kDa, 27% of which was insoluble. These findings indicate that a substantial proportion of calbindin does not function as a mobile calcium buffer, and perhaps instead has a calcium signalling role through target ligands in the insoluble cellular fraction.

Calbindin28kDa and calmodulin are hyperabundant in rat dental enamel cells. Identification of the protein phosphatase calcineurin as a principal calmodulin target and of a secretion-related role for calbindin28kDa.

Enamel cells are likely to experience heavy demands for intracellular calcium homeostasis during the secretion and hypermineralization of dental enamel. Here, the two major high-affinity calcium-binding proteins in rat enamel epithelium were identified as calbindin28kDa and calmodulin, using a microscale approach. Both proteins were hyperabundant, totalling up to 2% of the soluble protein and surpassing the amounts in cerebellum, the benchmark tissue. Calbindin28kDa and calmodulin accounted for 26% of the total calcium-binding capacity in enamel cell cytosol, under near physiological conditions. Numerous calmodulin-binding proteins were detected with an overlay assay, indicating that calmodulin has multiple major targets in enamel cells. The calcium/calmodulin-regulated protein phosphatase, calcineurin, was identified as a principal calmodulin target constituting 0.1% of the soluble protein. Calmodulin and calcineurin were expressed constitutively, implying continued heavy usage of calcium/calmodulin-based and phosphorylation-based signalling events throughout enamel cell development. Calbindin28kDa, in contrast, was expressed at fourfold higher levels in secretion-phase cells than during the calcium-intensive hypermineralization phase, unexpectedly pointing to an important role associated with secretion. Supporting this notion, immunoblots revealed that 33% of total (SDS-soluble) calbindin28kDa was in the particulate fraction and predominantly associated with the Triton-insoluble cytoskeleton. Solubilisation of cytoskeletal calbindin28kDa required high concentrations of NaCl or urea, indicating the existence of a high-affinity target ligand. The unusual abundance of calmodulin, calbindin28kDa and calcineurin demonstrated here provides the first molecular evidence that enamal cells possess a strong capability for intracellular calcium homeostasis. Since none of these proteins was up-regulated during enamel hypermineralization, it appears that other calcium-binding proteins are primarily involved in the putative transcellular passage of calcium.