Enamel matrix derivative induces matrix synthesis by cultured human periodontal fibroblast cells

Background: Periodontal wound healing and regeneration require that new matrix be synthesized, creating an environment into which cells can migrate. One agent which has been described as promoting periodontal regeneration is an enamel matrix protein derivative (EMD). Since no specific growth factors have been identified in EMD preparations, it is postulated that EMD acts as a matrix enhancement factor. This study was designed to investigate the effect of EMD in vitro on matrix synthesis by cultured periodontal fibroblasts. Methods: The matrix response of the cells was evaluated by determination of the total proteoglycan synthesis, glycosaminoglycan profile, and hyaluronan synthesis by the uptake of radiolabeled precursors. The response of the individual proteoglycans, versican, decorin, and biglycan were examined at the mRNA level by Northern blot analysis. Hyaluronan synthesis was probed by identifying the isotypes of hyaluronan synthase (HAS) expressed in periodontal fibroblasts as HAS-2 and HAS-3 and the effect of EMD on the levels of mRNA for each enzyme was monitored by reverse transcription polymerase chain reaction (RTPCR). Comparisons were made between gingival fibroblast (GF) cells and periodontal ligament (PDLF) cells. Results: EMD was found to significantly affect the synthesis of the mRNAs for the matrix proteoglycans versican, biglycan, and decorin, producing a response similar to, but potentially greater than, mitogenic cytokines. EMD also stimulated hyaluronan synthesis in both GF and PDLF cells. Although mRNA for HAS-2 was elevated in GF after exposure to EMD, the PDLF did not show a similar response. Therefore, the point at which the stimulation of hyaluronan becomes effective may not be at the level of stimulation of the mRNA for hyaluronan synthase, but, rather, at a later point in the pathway of regulation of hyaluronan synthesis. In all cases, GF cells appeared to be more responsive to EMD than PDLF cells in vitro. Conclusions: EMD has the potential to significantly modulate matrix synthesis in a manner consistent with early regenerative events.

HPP1: A transmembrane protein-encoding gene commonly methylated in colorectal polyps and cancers

Adenomas are the precursors of most colorectal cancers. Hyperplastic polyps have been linked to the subset of colorectal cancers showing DNA microsatellite instability, but little is known of their underlying genetic etiology. Using a strategy that isolates differentially methylated sequences from hyperplastic polyps and normal mucosa, we identified a 370-bp sequence containing the 5' untranslated region and the first exon of a gene that we have called HPP1. Rapid amplification of cDNA ends was used to isolate HPP1 from normal mucose. Using reverse transcription-PCR, HPP1 was expressed in 28 of 30 (93%) normal colonic samples but in only seven of 30 (23%) colorectal cancers (P < 0.001). The 5' region of HPP1 included a CpG island containing 49 CpG sites, of which 96% were found to be methylated by bisulfite sequencing of DNA from colonic tumor samples. By COBRA analysis, methylation was detected in six of nine (66%) adenomas, 17 of 27 (63%) hyperplastic polyps, and 46 of 55 (84%) colorectal cancers. There was an inverse relationship between methylation level and mRNA expression in cancers (r = -0.67; P < 0.001), and 5-aza-2-deoxycytidine treatment restored HPP1 expression in two colorectal cancer cell lines. In situ hybridization of HPP1 indicated that expression occurs in epithelial and stromal elements in normal mucosa but is silenced in both cell types in early colonic neoplasia. HPP1 is predicted to encode a transmembrane protein containing follistatin and epidermal growth factor-like domains. Silencing of HPP1 by methylation may increase the probability of neoplastic transformation.

Diffusion modeling of percutaneous absorption kinetics: 2. Finite vehicle volume and solvent deposited solids

The diffusion model for percutaneous absorption is developed for the specific case of delivery to the skin being limited by the application of a finite amount of solute. Two cases are considered; in the first, there is an application of a finite donor (vehicle) volume, and in the second, there are solvent-deposited solids and a thin vehicle with a high partition coefficient. In both cases, the potential effect of an interfacial resistance at the stratum corneum surface is also considered. As in the previous paper, which was concerned with the application of a constant donor concentration, clearance limitations due to the viable eqidermis, the in vitro sampling rate, or perfusion rate in vivo are included. Numerical inversion of the Laplace domain solutions was used for simulations of solute flux and cumulative amount absorbed and to model specific examples of percutaneous absorption of solvent-deposited solids. It was concluded that numerical inversions of the Laplace domain solutions for a diffusion model of the percutaneous absorption, using standard scientific software (such as SCIENTIST, MicroMath Scientific software) on modern personal computers, is a practical alternative to computation of infinite series solutions. Limits of the Laplace domain solutions were used to define the moments of the flux-time profiles for finite donor volumes and the slope of the terminal log flux-time profile. The mean transit time could be related to the diffusion time through stratum corneum, viable epidermal, and donor diffusion layer resistances and clearance from the receptor phase. Approximate expressions for the time to reach maximum flux (peak time) and maximum flux were also derived. The model was then validated using reported amount-time and flux-time profiles for finite doses applied to the skin. It was concluded that for very small donor phase volume or for very large stratum corneum-vehicle partitioning coefficients (e.g., for solvent deposited solids), the flux and amount of solute absorbed are affected by receptor conditions to a lesser extent than is obvious for a constant donor constant donor concentrations. (C) 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:504-520, 2001.

Can increasing the viscosity of formulations be used to reduce the human skin penetration of the sunscreen oxybenzone?

The effect of adding thickening agents on the penetration of a sunscreen benzophenone-3 through epidermal and a high-density polyethylene membrane was studied using both very thick (infinite dose) and thin tin use) applications. Contradictory results were obtained. Thickening agents retard skin penetration, in a manner consistent with a diffusional resistance in the formulation, when applied as an infinite dose. In contrast, when applied as in thin (in use) doses, thickening agents promote penetration, most likely through greater stratum corneum diffusivity arising from an enhanced hydration by the thicker formulations. The two key implications from this work are (i) a recognition of the danger in the potential extrapolation of infinite dosing to in use situations, and (ii) to recognize that thicker formulations may sometimes enhance the penetration of other topical agents when applied in use.

The structures of Langmuir-Blodgett films of fatty acids and their salts

Recent advances in several experimental techniques have enabled detailed structural information to be obtained for floating (Langmuir) monolayers and Langmuir-Blodgett films. These techniques are described briefly and their application to the study of films of fatty acids and their salts is discussed. Floating monolayers on aqueous subphases have been shown to possess a complex polymorphism with phases whose structures may be compared to those of smectic mesophases. However, only those phases that exist at high surface pressures are normally used in Langmuir-Blodgett (LB) deposition. In single LB monolayers of fatty acids and fatty acid salts the acyl chains are in the all-cans conformation with their long axes normal to the substrate. The in-plane molecular packing is hexagonal with long-range bond orientational order and short-range positional order: known as the hexatic-B structure. This structure is found irrespective of the phase of the parent floating monolayer. The structures of multilayer LB films are similar to the structures of their bulk crystals, consisting of stacked bilayer lamellae. Each lamella is formed from two monolayers of fatty acid molecules or ions arranged head to head and held together by hydrogen bonding between pairs of acids or ionic bonding through the divalent cations. With acids the acyl chains are tilted with respect to the substrate normal and have a monoclinic structure, whereas the salts with divalent cations may have the chains normal to the substrate or tilted. The in-plane structures are usually centred rectangular with the chains in the trans conformation and packed in a herringbone pattern, Multilayer films of the acids show only a single-step order-disorder transition at the malting point, This temperature tends to rise as the number of layers increases. Complex changes occur when multilayer films of the salts are heated. Disorder of the chains begins at low temperatures but the arrangement of the head groups does not alter until the melting temperature is reached, Slow heating to a temperature just below the melting temperature gives, with some salts, a radical change in phase. The lamellar structure disappears and a new phase consisting of cylindrical rods lying parallel to the substrate surface and stacked in a hexagonal pattern is formed, In each rod the cations are aligned along the central axis surrounded by the disordered acyl chains. (C) 2001 Elsevier Science B,V. All rights reserved.

Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia

Henneguya lesteri n. sp, (Myxosporea) is described from sand whiting, Sillago analis, from the southern Queensland coast of Australia. H. lesteri displays a preference for the pseudobranchs and is typically positioned along the afferent blood vessels, displacing the adjoining lamellae and disrupting their normal array, The plasmodia appeared as whitish-hyaline, elliptical cysts (mean dimensions 230 x 410 mum) attached to the oral mucosa lining of the hyoid arch on the inner surface of the operculum. Infections of the gills were also found, in which the plasmodia were spherical, averaged 240 x 240 mum in size and were located on the inner hemibranch margin. The parasites lodged in the gill filament crypts and generated a mild hyperplastic response of the branchial epithelium, In histological sections, the plasmodium wall and adjoining ectoplasm appeared as a finely granulated, weakly eosinophilic layer, Ultrastructurally, this section of the host-parasite interface contained an intricate complex of pinocytotic channels. H. lesteri is polysporic, disporoblastic and pansporoblast forming. Sporogenesis is asynchronous, with the earliest developmental stages aligned predominantly along the plasmodium periphery, and maturing sporoblasts and spores toward the center. Ultrastructural details of sporoblast and spore development are in agreement with previously described myxosporeans. The mature spore is drop-shaped, length (mean) 9.1 mum, width 4.7 mum, thickness 2.5 mum, and comprises 2 polar capsules positioned closely together, a binucleated sporoplasm and a caudal process of 12.6 mum. The polar capsules are elongated, 3.2 x 1.6 mum, with 4 turns of the polar filament. Mean length of the everted filament is 23.2 mum, Few studies have analyzed the 18S gene-of marine Myxosporea. In fact, H. lesteri is the first marine species of Henneguya to be characterized at the molecular level: we determined 1966 bp of the small-subunit (18S) rDNA, The results indicated that differences between this and the hitherto studied freshwater Henneguya species are greater than differences among the freshwater Henneguya species.

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts, This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones, This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types, This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.

The oral medicine of tooth wear

This review illustrates, through a series of case histories, how oral medicine insights aid the diagnosis and management of patients with excessive tooth wear. The cases reviewed are drawn from the records of 500 southeast Queensland patients referred to the author over a 12 year period. Patients most at risk of dental erosion have work and sports dehydration, caffeine addiction, gastro-oesophageal reflux, asthma, diabetes mellitus, hypertension or other systemic diseases or syndromes that predispose to xerostomia. Saliva protects the teeth from the extrinsic and intrinsic acids which cause dental erosion. Erosion, exacerbated by attrition and abrasion, is the main cause of tooth wear. These cases illustrate that teeth, oral mucosa, salivary glands, skin and eyes should be examined for evidence of salivary hypofunction and attendant medical conditions. Based on comprehensive oral medicine, dietary analyses and advice, it would seem patients need self-management plans to deal with incipient chronic tooth wear. The alternative is the expensive treatment of pain, occlusal damage and pulp death required to repair the effects of acute severe tooth wear.

Characterization of mouse Frizzled-3 expression in hair follicle development and identification of the human homolog in keratinocytes

Frizzled genes encode a family of Wnt ligand receptors, which have a conserved cysteine-rich Wnt binding domain and include both transmembrane and secreted forms. Work by others has shown that experimental perturbation of Wnt signaling results in aberrant hair formation, hair growth, and hair structure. To date, however, there is no information on the contribution of individual Frizzled proteins to hair development. We now report that Frizzled-3 expression in skin is restricted to the epidermis and to the developing hair follicle. Northern analysis on total mouse skin mRNA revealed a single Frizzled-3 transcript of 3.7 kb. Reverse transcription-polymerase chain reaction and in situ hybridization analysis revealed Frizzled-3 expression in epidermal and hair follicle keratinocytes. Frizzled-3 transcripts are first detected in discrete foci in the developing epidermis of 13 d embryos and later in the hair follicle placodes of 15 d embryos, suggesting a role for this Frizzled isoform in follicle development. In 17 d embryos and id old newborn mice Frizzled-3 expression is limited to suprabasal keratinocytes and is not seen in pelage follicles until 3 d postpartum. In 7 d old neonatal skin, Frizzled-3 is expressed throughout the epidermis and in the outer cell layers of hair follicles. We have also identified the mRNA encoding human Frizzled-3 in epidermal keratinocytes and in the HaCaT keratinocyte cell line. Human Frizzled-3 mRNA encodes a 666 amino acid protein with 97.8% identity to the mouse protein. The human Frizzled-3 gene was mapped using a radiation-hybrid cell line panel to the short arm of chromosome 8 between the markers WI-1172 and WI-8496 near the loci for the Hypotrichosis of Marie Unna and Hairless genes.

EGF sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia

Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and down-regulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreatment with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Sp1 decreased. A causal relationship was established between these observations by demonstrating that up-regulation of Sp1 DNA binding activity by granulocyte/macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells can be explained by induction of ATM protein and kinase activity with time post-irradiation. Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit.

Gill pathology caused by infestations of adult and preadult Dissonus manteri Kabata (Copepoda : Dissonidae) on coral trout, Plectropomus leopardus (Lacepede), (Serranidae)

Adult and preadult Dissonus manteri attached to the gills of Plectropomus leopardus cause significant pathology in the form of large hyperplastic nodules on the afferent (leading), edges of gill filaments. Nodules result from the dual actions of parasite attachment and feeding. The host response is characterized by severe epithelial hyperplasia, supplemented by fibroplasia and inflammation. Parasites attach close to the gill arch near the base of filaments. They have little effect on gill vasculature as the maxillipeds penetrate the filament superficial to the efferent filament artery and do not interfere with the blood vessels of the secondary lamellae. Tissue proliferation is limited to the wide portion of filament 'edge' epithelium in the proximal third and also does not extend to the secondary lamellae. Nodules are most numerous towards the ends of hemibranchs and are generally absent from the central regions. Leading hemibranchs bear significantly more nodules than their trailing counterparts. Of the total number of nodules, 20.5% are located on the pseudobranchs. Distribution patterns are considered to be primarily the result of D. manteri avoiding strong water currents, although this cannot explain the difference between numbers on leading and trailing hemibranchs.

Transcriptional downregulation of ATM by EGF is defective in ataxia-telangiectasia cells expressing mutant protein

There is evidence that ATM plays a wider role in intracellular signalling in addition to DNA damage recognition and cell cycle control, In this report we show that activation of the EGF receptor is defective in ataxia-telangiectasia (A-T) cells and that sustained stimulation of cells with EGF downregulates ATM protein in control cells but not in A-T cells expressing mutant protein, Concomitant with the downregulation of ATM, DNA-binding activity of the transcription factor Spl decreased in controls after EGF treatment but increased from a lower basal level in A-T cells to that in untreated control cells, Mutation in two Spl consensus sequences in the ATM promoter reduced markedly the capacity of the promoter to support luciferase activity in a reporter assay. Overexpression of anti-sense ATM cDNA in control cells decreased the;basal level of Spl, which in turn was increased by subsequent treatment of cells with EGF, similar to that observed in,A-T cells. On the other hand full-length ATM cDNA increased the basal level of Spl binding in A-T cells, and in response to EGF Spl binding decreased, confirming that this is an ATR I-dependent process. Contrary to that observed in control cells there was no radiation-induced change in ATM protein in EGF-treated A-T cells and likewise no alteration in Spl binding activity. The results demonstrate that EGF-induced downregulation of ATM (mutant) protein in A-T cells is defective and this appears to be due to less efficient EGFR activation and abnormal Spl regulation.

In situ zymography: topographical considerations

In situ gelatin zymography is a simple technique providing valuable information about the cellular and tissue localization of gelatinases. Until recently, the use of this technique has been confined to soft, relatively homogeneous tissue. In this report in situ zymography has been utilized to assess the sub-lamellar location of gelatinases in the hard, semi-keratinized epidermal layer and the adjacent soft connective tissue matrix of the dermis of the equine hoof. We show that alterations in the orientation at which the tissue is dipped and withdrawn from the emulsion cause profound alterations in emulsion thickness. Microscopic Variations in the surface topography of frozen tissue sections also influence emulsion thickness making interpretation of the results difficult. Given these results, researchers must be aware of potential variations in zymographic analysis may be influenced by physical tissue parameters in addition to suspected gelatinase activity. (C) 2001 Elsevier Science B.V. All rights reserved.

HIV gp120 receptors on human dendritic cells

Dendritic cells (DCs) are important targets for human immunodeficiency virus (HIV) because of their roles during transmission and also maintenance of immune competence. Furthermore, DCs are a key cell in the development of HIV vaccines. In both these settings the mechanism of binding of the HIV envelope protein gp120 to DCs is of importance. Recently a single C-type lectin receptor (CLR), DC-SIGN, has been reported to be the predominant receptor on monocyte-derived DCs (MD-DCs) rather than CD4. In this study a novel biotinylated gp120 assay was used to determine whether CLR or CD4 were predominant receptors on MDDCs and ex vivo blood DCs. CLR bound more than 80% of gp120 on MDDCs, with residual binding attributable to CD4, reconfirming that CLRs were the major receptors for gp120 on MDDCs. However, in contrast to recent reports, gp120 binding to at least 3 CLRs was observed: DC-SIGN, mannose receptor, and unidentified trypsin resistant CLR(s). In marked contrast, freshly isolated and cultured CD11c(+ve) and CD11c(-ve) blood DCs only bound gp120 via CD4. In view of these marked differences between MDDCs and blood DCs, HIV capture by DCs and transfer mechanisms to T cells as well as potential antigenic processing pathways will need to be determined for each DC phenotype. (Blood. 2001;98:2482-2488) (C) 2001 by The American Society of Hematology.

Bitter-sweet symphony: defining the role of dendritic cell gp120 receptors in HIV infection

Background: Dendritic cells (DC) are believed to be one of the first cell types infected during HIV transmission. Recently a single C-type lectin receptor (CLR), DC-SIGN, has been reported to be the predominant receptor on monocyte derived DC (MDDC) rather than CD4. The role of other CLRs in HIV binding and HIV binding by CLRs on other types of DC in vivo is largely unknown. Objectives and study design: Review HIV binding to DC populations, both in vitro and in vivo, in light of the immense interest of a recently re-identified CLR called DC-SIGN. Results and conclusions: From recent work, it is clear that immature MDDC have a complex pattern of HIV gp120 binding. In contrast to other cell types gp120 has the potential to bind to several receptors on DC including CD4 and several types of C type lectin receptor, not just exclusively DC-SIGN. Given the diverse types of DC in vivo future work will need to focus on defining the receptors for HIV binding to these different cell types. Mucosal transmission of HIV in vivo targets immature sessile DCs, including Langerhans cells which lack DC-SIGN. The role of CLRs and DC-SIGN in such transmission remains to be defined. (C) 2001 Elsevier Science B.V. All rights reserved.