Lessons Learned from Contact Tracing COVID-19 cases in Dental Settings in East Scotland.

INTRODUCTION: Dental settings were considered high risk settings for COVID-19. A Dental Public Health Team in East Scotland worked to risk assess each situation timeously to break chains of transmission. AIM: To present learning from routine data collected from contact tracing COVID-19 cases in the dental setting. DESIGN: Retrospective analysis of a routine data set of COVID-19 cases associated with a dental setting reported via the national contact tracing system for two health board areas in the East of Scotland. METHODS: Descriptive statistics summarise the data collected over a 13-month period (Oct 2020-Dec 2021) during which all included COVID-19 cases were confirmed by PCR. A narrative presents output from contact tracing of all cases and includes themes identified during contact tracing that led to transmission within a dental setting. A case study illustrates impact of transmission. RESULTS: 752 cases are included. No evidence of staff to patient transmission or vice versa was found in this study. Staff to staff transmission occurred in non-clinical areas contributing to 33% of total staff cases with the remainder assessed to result from community transmission. CONCLUSION: Transmission of COVID-19 in a dental setting, in the context of this study, appears to be confined to non-clinical areas with the majority of staff cases resulting from community transmission. Future pandemic plans should include tools to aid with implementation of guidance in non-clinical areas.

Lessons Learned from Contact Tracing COVID-19 cases in Dental Settings in East Scotland.

INTRODUCTION: Dental settings were considered high risk settings for COVID-19. A Dental Public Health Team in East Scotland worked to risk assess each situation timeously to break chains of transmission. AIM: To present learning from routine data collected from contact tracing COVID-19 cases in the dental setting. DESIGN: Retrospective analysis of a routine data set of COVID-19 cases associated with a dental setting reported via the national contact tracing system for two health board areas in the East of Scotland. METHODS: Descriptive statistics summarise the data collected over a 13-month period (Oct 2020-Dec 2021) during which all included COVID-19 cases were confirmed by PCR. A narrative presents output from contact tracing of all cases and includes themes identified during contact tracing that led to transmission within a dental setting. A case study illustrates impact of transmission. RESULTS: 752 cases are included. No evidence of staff to patient transmission or vice versa was found in this study. Staff to staff transmission occurred in non-clinical areas contributing to 33% of total staff cases with the remainder assessed to result from community transmission. CONCLUSION: Transmission of COVID-19 in a dental setting, in the context of this study, appears to be confined to non-clinical areas with the majority of staff cases resulting from community transmission. Future pandemic plans should include tools to aid with implementation of guidance in non-clinical areas.

A Health Inequalities Impact Assessment of the surveillance of COVID-19 in asymptomatic patients attending dental settings in Scotland.

INTRODUCTION: A key aspect of the public health response to COVID-19 in Scotland was enhanced community surveillance, including testing in dental settings. Across Scotland, dental settings offered patients over 5-years-old the opportunity to participate in community surveillance of COVID-19. METHODS: A Health Inequalities Impact Assessment (HIIA) was conducted to understand the differential impacts the programme would have on the population and to improve the accessibility of the programme. HIIA is a tool to allow the assessment, understanding, and mitigation of impacts on people of a proposed policy or practice. It fulfils an organisational duty to meet the requirements of the Equality Act and Fairer Scotland Duty. The HIIA was conducted rapidly in parallel with the programme development. An action research approach included an online workshop, consultation, review of population data and a literature search. RESULTS: Adjustments were required to improve the programme's accessibility. Stakeholders, including dental teams from across Scotland were involved in the consultation and brought their front-line experience in different settings. Common issues identified included digital literacy and access, language and cultural barriers to participation, and issues relating to the implications of a positive COVID-19 result. Literature indicated limited evidence on the acceptability, accessibility, and equity of asymptomatic COVID-19 surveillance. CONCLUSION: This HIIA was conducted during the COVID-19 pandemic. As an example of good practice in tackling inequalities in access to programmes it should represent the benchmark for other similar initiatives.

Clinical and echocardiographic characteristics of cardioembolic stroke.

BACKGROUND AND PURPOSE: Ischaemic stroke frequently has a cardioembolic (CE) source. Clinical and echocardiographic parameters associated with CE stroke were evaluated. METHODS: In all, 93 consecutive ischaemic stroke patients who underwent a transthoracic echocardiogram were retrospectively analysed; strokes were classified by TOAST (Trial of Org 10172 in Acute Stroke Treatment) criteria. Echocardiographic parameters related to CE stroke, including left atrial volumes and function, were compared to 73 healthy controls. RESULTS: Of 93 patients (mean age 66.1 years, 56% male), nine (10%) had large artery atherosclerosis, 38 (41%) CE stroke, two (2%) small vessel disease, two (2%) other and 42 (45%) undetermined aetiology. Left atrial (LA) maximum volumes (LAVImax ) and minimum volumes (LAVImin ) were larger in the CE group than the non-CE group (45 vs. 32 ml/m2 , 32 vs. 13 ml/m2 , respectively, P < 0.001), whilst LA function indices including LA emptying fraction and LA function index (LAFI) were lower in the CE group (34% vs. 55%, and 0.12 vs. 0.35, respectively, P < 0.001). Adjusting for clinical characteristics, LAFI ≤0.3 was an independent predictor of CE stroke (adjusted odds ratio 5.3, P = 0.001). Additionally, LAVImax and LAVImin were larger (61 vs. 44 and 32 vs. 24 ml/m2 respectively, P < 0.01) and LAFI significantly lower (0.34 vs. 0.52, P < 0.001) in the undetermined aetiology group versus healthy controls. CONCLUSIONS: Left atrial enlargement with reduced LA function was associated with CE stroke and LAFI was the best independent predictor. LA parameters were also altered in the undetermined aetiology group, suggesting an underlying LA myopathy in this subset.

Placebo-controlled pilot trial testing dose titration and intravenous, intramuscular and subcutaneous routes for ketamine in depression.

OBJECTIVE: This pilot study assessed the feasibility, efficacy and safety of an individual dose-titration approach, and of the intravenous (IV), intramuscular (IM) and subcutaneous (SC) routes for treating depression with ketamine. METHOD: Fifteen treatment-refractory depressed participants received ketamine or midazolam (control treatment) in a multiple crossover, double-blind study. Ketamine was administered by IV (n = 4), IM (n = 5) or SC (n = 6) injection. Dose titration commenced at 0.1 mg/kg, increasing by 0.1 mg/kg up to 0.5 mg/kg, given in separate treatment sessions separated by ≥1 week, with one placebo control treatment randomly inserted. Mood, psychotomimetic and hemodynamic effects were assessed and plasma ketamine concentrations assayed. RESULTS: Twelve participants achieved response and remission criteria, achieved at doses as low as 0.1 mg/kg. All three routes of administration resulted in comparable antidepressant effects. Fewest adverse effects were noted with the SC route. Antidepressant response, adverse effects and ketamine concentrations were dose-related. CONCLUSION: Antidepressant response occurred at a range of doses and at <0.5 mg/kg. The dose-titration approach is a practical method for optimizing the efficacy - side-effects trade-off on an individual patient basis. This pilot study provides preliminary evidence for SC injection as a practical, feasible and efficacious treatment approach.

SARS-CoV-2 Positivity in Asymptomatic-Screened Dental Patients.

Enhanced community surveillance is a key pillar of the public health response to coronavirus disease 2019 (COVID-19). Asymptomatic carriage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a potentially significant source of transmission, yet remains relatively poorly understood. Disruption of dental services continues with significantly reduced capacity. Ongoing precautions include preappointment and/or at appointment COVID-19 symptom screening and use of enhanced personal protective equipment (PPE). This study aimed to investigate SARS-CoV-2 infection in dental patients to inform community surveillance and improve understanding of risks in the dental setting. Thirty-one dental care centers across Scotland invited asymptomatic-screened patients aged over 5 y to participate. Following verbal consent and completion of sociodemographic and symptom history questionnaire, trained dental teams took a combined oropharyngeal and nasal swab sample using standardized Viral Transport Medium-containing test kits. Samples were processed by the Lighthouse Lab and patients informed of their results by SMS/email with appropriate self-isolation guidance in the event of a positive test. All positive cases were successfully followed up by the national contact tracing program. Over a 13-wk period (from August 3, 2020, to October 31, 2020), 4,032 patients, largely representative of the population, were tested. Of these, 22 (0.5%; 95% CI, 0.5%-0.8%) tested positive for SARS-CoV-2. The positivity rate increased over the period, commensurate with uptick in community prevalence identified across all national testing monitoring data streams. To our knowledge, this is the first report of a COVID-19 testing survey in asymptomatic-screened patients presenting in a dental setting. The positivity rate in this patient group reflects the underlying prevalence in community at the time. These data are a salient reminder, particularly when community infection levels are rising, of the importance of appropriate ongoing infection prevention control and PPE vigilance, which is relevant as health care team fatigue increases as the pandemic continues. Dental settings are a valuable location for public health surveillance.

Adducin: Ca++-dependent association with sites of cell-cell contact.

Adducin is a protein recently purified from erythrocytes and brain that has properties in in vitro assays suggesting a role in assembly of a spectrin-actin lattice. This report describes the localization of adducin to plasma membranes of a variety of tissues and the discovery that adducin is concentrated at sites of cell-cell contact in the epithelial tissues where it is expressed. Adducin in tissues and cultured cells always was observed in association with spectrin and actin, although spectrin and actin were evident in the absence of adducin. In sections of intestinal epithelial cells spectrin was present on all plasma membrane surfaces while adducin was restricted to the lateral cell borders. Adducin also was not detected in association with actin stress fibers in cultured cells. The presence of adducin at cell-cell contact sites of cultured epithelial cells requires extracellular Ca++ and occurs within 15 min of addition of 0.3 mM Ca++. Redistribution of adducin after addition of extracellular Ca++ is independent of formation of desmosomal and adherens junctions since assembly of adducin at contact sites requires lower concentrations of Ca++ and occurs more rapidly than redistribution of desmoplakin or vinculin. Treatment of keratinocytes and MDCK cells with nanomolar concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) induces redistribution of adducin away from contact sites. The effect of TPA may be a direct consequence of phosphorylation of adducin, since adducin is phosphorylated in TPA-treated cells and the phosphorylation of adducin occurs before disassembly of adducin from sites of cell-cell contact. Spectrin and adducin are both present in a detergent-insoluble form at cell-cell contact sites of cultured cells. These observations are consistent with the idea that adducin recognizes and associates with specific "receptors" localized at regions of cell-cell contact and promotes assembly of spectrin into a more stable structure, perhaps analogous to the highly organized spectrin-actin network of erythrocyte membranes.

Restricted tissue distribution of a 37-kD possible adherens junction protein.

A major polypeptide of M(r) 37,000 was purified from a desmosome-enriched citric acid-insoluble pellet of pig tongue epithelium. The polypeptide was solubilized from the 4-M urea-insoluble pellet with 9 M urea, and extracts were separated by carboxymethyl cellulose and gel filtration chromatography. The 37-kD protein was obtained in milligram quantities as a single band on two-dimensional gels in 30% yield after 21-fold purification from the citric acid-insoluble fraction. The protein is not glycosylated and has a pI of approximately 8.7. Although isolated from a fraction rich in desmosomes, the 37-kD protein is not a desmosomal protein. Indirect immunofluorescence analysis of frozen sections of tongue and other tissues demonstrated that antibodies raised to the 37-kD protein bound only to suprabasal cell layers at punctate regions of the periphery of the cell and was absent from most regions of epidermis, whereas antibodies to desmoplakins I and II, desmosomal proteins, bound similarly but in all epidermal layers. Immunoelectron microscopy localized the 37-kD protein to the cell periphery in regions between, but never in, desmosomes. By immunofluorescence, the 37-kD protein colocalized with actin as well as with vinculin and uvomorulin in oral tissues. Like the 37-kD protein, vinculin and uvomorulin were absent from the basal layer. Based on its appearance, localization, and solubility properties, the 37-kD protein is probably a component of adherens junctions; its restriction to suprabasal cells and exclusion from the epidermis are unique.

Calcium-induced assembly of adherens junctions in keratinocytes.

Extracellular calcium concentration has been shown to control the stratification of cultured keratinocytes, presumably by regulation of formation of desmosomes. Previous studies have shown that keratinocytes cultured in medium containing 0.1 mM Ca++ form loose colonies without desmosomes. If the Ca++ is raised to 1 mM, desmosomes are assembled and the distribution of keratin filaments is altered. We have examined the disposition of vinculin and actin in keratinocytes under similar conditions. Using immunofluorescence microscopy we show that raising [Ca++] in the medium dramatically alters the distribution of vinculin and actin and results in the formation of adherens-type junctions within 15 min after switching to high calcium medium. Borders of cells at the edge of colonies, which are not proximal to other cells, are not affected, while cells in the interior of the colony form junctions around their periphery. Attachment plaques in keratinocytes grown in low calcium medium are located at the ventral plane of the cell, but junctions formed after switching to high calcium are not, as demonstrated by interference reflection microscopy. In cells colabeled with antibodies against vinculin and desmoplakin, vinculin-containing adherens junctions were visible before desmosomal junctions when cells were switched to high calcium. Although newly formed vinculin-containing structures in high calcium cells, like desmosomes, colocalize with phase-dense structures, superimposition of video fluorescence images using digitized fluorescence microscopy indicates that adherens junctions and desmosomes are discrete structures. Adherens junctions, like desmosomes, may play an essential role in controlling stratification of keratinocytes.

Epidermal growth factor receptor number decreases during rat liver regeneration.

The potential role of epidermal growth factor (EGF) in the regulation of rat liver regeneration was examined by assessing the binding of 125I-EGF to hepatic membranes isolated at various times after partial hepatectomy. The results demonstrated a fall in 125I-EGF binding detectable as early as 8 h after partial hepatectomy. The nadir in EGF binding, less than 40% of that observed in sham-operated control rats, was seen 36 and 48 h after partial hepatectomy. Scatchard analysis showed that the decrease in binding capacity was due to a fall in receptor number. The specificity of the observed loss of EGF receptors was substantiated in parallel studies of 125I-insulin and 125I-wheat germ lectin binding; the binding of these ligands did not decrease appreciably during liver regeneration. The data are consistent with the hypothesis that EGF or a similar substance is one component of the complex humoral signal that regulates liver regeneration.

Specific affinity between fibronectin and the epidermolysis bullosa acquisita antigen.

Autoantibodies in the skin and sera of patients with epidermolysis bullosa acquisita bind to a large matrix molecule within the lamina densa region of skin basement membrane. At the site of these immune complexes, the epidermis separates from the dermis, which creates a subepidermal blister just below the lamina densa. The target molecule for the autoantibodies is in close apposition to fibronectin, a major extracellular matrix molecule that is abundant in the upper dermis of skin. In this report, we show specific affinity between fibronectin and the 290,000-D chain of the epidermolysis bullosa acquisita antigen, and that this affinity is mediated by the gelatin/collagen-binding domain of fibronectin (Mr = 60,000). Since blistering in epidermolysis bullosa acquisita often occurs in the absence of clinical and histological inflammation, a direct interruption in the fibronectin-epidermolysis bullosa acquisita antigen bond may be involved in the pathogenesis of epidermal-dermal disadherence that occurs in this bullous disease.

Human autoantibodies against desmoplakins in paraneoplastic pemphigus.

Recently, a previously unrecognized autoantibody mediated blistering disease, paraneoplastic pemphigus has been described. Paraneoplastic pemphigus is associated with lymphoid malignancies, thymomas, and poorly differentiated sarcomas. Serum of affected patients contain pathogenic autoantibodies that immunoprecipitate from normal keratinocytes a characteristic complex of four polypeptides with M(r) of 250, 230, 210, and 190 kD. As our preliminary studies indicated that the 250-kD and the 210-kD antigens comigrated with desmoplakins I and II, we investigated the possibility that autoantibodies against the desmoplakins were a component of this autoimmune syndrome. 11 sera from affected patients were tested by indirect immunofluorescence against desmosome containing tissues, immunoprecipitation of metabolically labeled keratinocytes, and Western immunoblotting of desmoplakins I and II that had been purified to homogeneity from pig tongue epithelium. By indirect immunofluorescence, 9 of 11 sera showed strong binding to epithelial and nonepithelial desmosomes, and 2 were weakly reactive. All 11 immunoprecipitated 250- and 210-kD bands of variable intensity that comigrated with bands identified by a murine monoclonal antidesmoplakin antibody, and immunoblotting confirmed binding of the serum autoantibodies to purified desmoplakins. This demonstrates that paraneoplastic pemphigus is the first human autoimmune syndrome in which autoantibodies against the desmoplakins are a prominent component of the humoral autoimmune response.

Minoxidil: inhibition of proliferation of keratinocytes in vitro.

Previous studies have suggested that minoxidil stimulates growth of keratinocytes, possibly in a manner similar to the action of epidermal growth factor. Using both a short-term assay, thymidine incorporation, and a longer term assay, cell counting, to assess proliferative growth, we tested the activity of minoxidil in human keratinocyte cultures grown in 0.1 mM Ca(++). Minoxidil failed to stimulate growth in these assays. At concentrations of 5-10 micrograms per ml, minoxidil showed half-maximal inhibition of both EGF- and placental extract-stimulated thymidine incorporation. Minoxidil also inhibited proliferative growth in the presence or absence of placental extract. Direct measurement of the ability of minoxidil to compete for binding to the EGF receptor indicated that minoxidil probably does not bind to the EGF receptor. Minoxidil was not toxic, as keratinocytes continued to survive and grow, although at a slower rate, in the presence of minoxidil.

Stimulation of thymidine incorporation in keratinocytes by insulin, epidermal growth factor, and placental extract: comparison with cell number to assess growth.

The results of a thymidine incorporation assay were compared with direct measurement of cell number in assessment of proliferative growth of human keratinocytes in monolayer culture. Keratinocytes were cultured in supplemented MCDB 153 medium in 0.1 mM Ca2+, and plated in 24-well trays. The ability of insulin, placental extract, and epidermal growth factor to enhance growth and thymidine incorporation were compared. Autoradiography was performed to determine the percentage of cells with labeled nuclei. Epidermal growth factor increased thymidine incorporation under the conditions of the assay, and placental extract increased incorporation by up to 50-fold, since the control cells plated in the absence of epidermal growth factor and other growth factors survived but proliferated minimally. Both cell number and thymidine incorporation showed similar concentration dependence upon insulin and placental extract. If placental extract was added to cells plated 28 h earlier, incorporation was maximal after 17 h in the presence of the extract. If cells were plated in the presence of the extract, 85% of nuclei were shown by autoradiography to be labeled after 23 h, but only 24% of nuclei were labeled in the absence of the extract. A plating density of 10(4) cells/2-cm2 well was optimal. The assay permits rapid identification of growth-promoting fractions without prolonged growth periods, and is a valid indicator of these agents in keratinocyte cultures.

Modulation of the epidermal growth factor receptor of human keratinocytes by calcium ion.

Human keratinocytes grown in medium containing reduced calcium concentrations (0.07 mM) have been found to show altered morphology and decreased differentiation in comparison with cells grown in medium with physiologic calcium concentrations (1-2 mM). Since such alterations could be mediated by growth factors, we measured binding of [125I]epidermal growth factor (EGF). Neonatal keratinocytes were subcultured without feeder layers and grown to confluence in 1.1 mM calcium. Medium was changed and cells incubated for various periods in reduced calcium prior to binding assays. Scatchard plots of binding data showed a 5-fold increase in receptor number with no change in affinity (3 X 10(-9) M) after 4-24 h at 37 degrees C. Maximal binding occurred at 0.02-0.04 mM calcium and decreased sharply with increasing calcium concentrations. The increase could be prevented by calcium added soon after binding began to increase but was altered less after substantial elevation had occurred, although morphologic changes at reduced calcium concentrations were reversed within several hours. Substantial increases in binding of [125I]somatomedin C and [125I]concanavalin A were detected, but binding of [125I]pindalol, a beta-receptor ligand, was changed little. Keratinocytes at reduced calcium concentrations responded to added EGF by decreasing surface EGF receptors briskly in a time- and temperature-dependent fashion. The data suggest that keratinocytes show enhanced binding of EGF and some other cell surface ligands under conditions in which differentiation is retarded.

Epidermal growth factor receptor in human epidermal cells: direct demonstration in cultured cells.

The interaction of [125I] labeled murine epidermal growth factor (EGF)( with cultured human keratinocytes has been studied. Epidermal cells from neonatal foreskins were propagated to confluence in 24-well culture trays and incubated with [125I] EGF for binding assays. Association reached equilibrium within 2-4 hr at 4 degrees and slightly earlier at 37 degrees. EGF bound at 37 degrees dissociates very slowly from cells, since it can be shown to enter cells and is degraded to trichloracetic-acid-soluble material. Cells exposed to chloroquine, an inhibitor of lysosomal enzymes, fail to degrade internalized [125I] EGF. Scatchard plots of the binding data yield a dissociation constant of 1 X 10(-9) m and show that epidermal cells bind approximately 3-4 X 10(4) molecules of EGF. Cells exposed to EGF alter their ability to bind EGF by decreasing the number of binding sites in a time- and concentration-dependent manner. Differentiation of epidermal cells in culture poses a problem in assessment of binding of EGF and possibly of other biologically active ligands, since cells lose the ability to bind EGF as they differentiate. These findings indicate that isolated epidermal cells possess a functional receptor for EGF which binds and responds to EGF in a manner similar to that described for other cells.

Stimulation of growth of keratinocytes by basic fibroblast growth factor.

Numerous heparin-binding growth factors active in different types of cells have recently been shown to belong to the family of fibroblast growth factors. Because these factors are active in some types of epithelial cells, we tested the activity of basic fibroblast growth factor (bFGF) from bovine brain in human keratinocyte cultures. bFGF stimulated thymidine incorporation and cellular proliferation in these cultures with half-maximal activity at approximately 60 pg/ml (4 X 10(-12) M). Stimulation of thymidine incorporation was associated with increased nuclear labeling after 22 h in the presence of bFGF under the same conditions used in the thymidine incorporation assay. bFGF was nearly as effective as epidermal growth factor (EGF) in stimulating keratinocyte growth and substantially less effective than crude placental extract, and was not additive with EGF in stimulating thymidine incorporation or proliferation of cells. The findings indicate that bFGF is a potent growth factor for keratinocytes.

Type IV collagen and fibronectin enhance human keratinocyte thymidine incorporation and spreading in the absence of soluble growth factors.

In various cell culture systems, extracellular matrix components have been demonstrated to be mitogenic and, in some cases, to substitute for growth factors. In order to study the effects of various matrices on keratinocyte growth, we assessed the incorporation of tritiated thymidine and cell number on short-term cultures of human keratinocytes plated on different substrata. For determination of whether thymidine incorporation by keratinocytes was related to the ability of the cells to attach and spread on the substratum, experiments to determine the percentage of attached and spread cells on each matrix surface were performed. High levels of attachment and incorporation of thymidine with no preferential attachment to a given matrix were evident when the cells were cultured in the presence of growth factors. When growth factors were absent, keratinocytes likewise showed no preferential attachment to a given matrix component, but demonstrated enhanced thymidine incorporation when apposed to type IV collagen or fibronectin in comparison with tissue culture plastic or laminin. In the absence of epidermal growth factor (EGF) and bovine pituitary extract (BPE), increased spreading on type IV collagen and fibronectin was associated with enhanced incorporation of thymidine. In agreement with the thymidine incorporation results, when keratinocytes were cultured for 7 d, cell numbers were increased in cultures plated on type IV collagen only if growth factors were excluded from the medium. When attachment of cells to substrata with or without growth factors was compared, either EGF or BPE enhanced attachment to all of the substrata tested. It is concluded that under suboptimal growth conditions extracellular matrix components can modulate keratinocyte growth. Also, under these conditions, spreading, but not attachment, correlates with growth potential.

Trichohyalin: presence in the granular layer and stratum corneum of normal human epidermis.

Trichohyalin, a protein contained in granules in the cells of the hair-follicle inner root sheath and in the medulla of the hair shaft, has been purified previously from sheep hair bulbs and is also a major protein of filiform papillae of tongue epithelium. Polyclonal affinity-purified antibodies and a monoclonal antibody raised to purified pig tongue trichohyalin both stained the inner root sheath of hair follicles and the medulla of hair fibers and identified human trichohyalin as a single 220-kDa band on immunoblots of human hair bulb proteins. These antibodies were used to examine human epidermis by immunofluorescence and immunoblotting. The antibodies decorate granules in cells in the granular layer and stratum corneum of non-hair-bearing human skin, and immunoblots identify a protein in epidermis comigrating with trichohyalin from human hair and human tongue epithelium. Absorption of antibody to trichohyalin on a trichohyalin affinity column abrogated staining of the epidermis and the bands on the immunoblots. Trypsin-separated epidermis contained 220 and 160 kDa bands identified as trichohyalin, but epidermis shaved from skin and quickly frozen showed only a single 220-kDa band, indicating that the 160-kDa protein was generated by proteolysis. Double immunofluorescence for trichohyalin and filaggrin showed that some cells containing filaggrin also contain trichohyalin. These studies show that trichohyalin is not limited to hair and tongue but is present in isolated cells in the granular layer and stratum corneum of normal epidermis.

Tyrosine phosphorylation of human keratinocyte beta-catenin and plakoglobin reversibly regulates their binding to E-cadherin and alpha-catenin.

We show that tyrosine phosphorylation, produced by incubation of normal human keratinocytes with the tyrosine phosphatase inhibitor peroxovanadate, directly and reversibly regulates the association of beta-catenin and plakoglobin with E-cadherin and alpha-catenin. Prior studies have demonstrated a correlative, but not causal, association between increased tyrosine phosphorylation and decreased adherens junction mediated cell-cell adhesion. We observed that (i) binding of tyrosine phosphorylated beta-catenin and plakoglobin to E-cadherin and to alpha-catenin was substantially reduced, but could be restored in vitro by removal of phosphate from beta-catenin and plakoglobin with added tyrosine phosphatase, and (ii) tyrosine phosphorylation of beta-catenin and plakoglobin was associated with decreased cell-cell adhesion. These findings support a direct and causal role for tyrosine phosphorylation of beta-catenin and plakoglobin in regulating adherens junction mediated cell-cell adhesion. We propose that tyrosine phosphorylation of specific and probably different residues is responsible for regulating the binding of beta-catenin or plakoglobin to (i) E-cadherin and (ii) alpha-catenin. Additionally, because beta-catenin and plakoglobin have both structural and regulatory functions, the data raise the possibility that beta-catenin or plakoglobin released from the adherens junctions by tyrosine phosphorylation may transduce a signal to the nucleus regarding the adhesive state of the cell.