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Background: Epidermolytic palmoplantar keratoderma (EPPK) is characterized by diffuse hyperkeratosis affecting palms and soles with suprabasal epidermolysis or vacuolar degeneration histopathologically. The disorder is caused by heterozygous mutations in KRT9 or KRT1. Dominant-negative mutations in KRT1 could also result in epidermolytic ichthyosis with EPPK, a more severe entity affecting the entire body., Objective: To investigate the genetic basis and pathogenesis of two unrelated patients with EPPK and knuckle pads, both of whom were born to consanguineous parents of Chinese origin., Methods: Next-generation sequencing was applied to the two patients using genomic DNA extracted from peripheral blood. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining and Western blot (WB) were employed to evaluate mRNA and protein expression level. Ultrastructural changes of skin lesion were analysed using transmission electron microscopy., Results: Two novel homozygous mutations, c.457C>T (p.Gln153*) and c.33C>G (p.Tyr11*) in KRT1, were identified in patients 1 and 2 respectively. The nonsense mutations were predicted to result in nonsense-mediated mRNA decay and absence of keratin 1, which was confirmed in the skin lesions from patient 1. Upregulated keratin 2 was detected both in the affected and unaffected skin samples from patient 1, while the protein abundance and distribution pattern of keratin 10 remained unchanged. An aberrant and clumped staining pattern of keratin 9 was noted in the palmar skin of patient 1., Conclusions: Homozygous 'knockout' mutations in KRT1 resulted in EPPK with knuckle pads rather than epidermolytic ichthyosis. We speculated that sparing of non-acral skin might be due to compensatory effect of keratin 2 upregulation by forming heterodimer with keratin 10., (© 2022 European Academy of Dermatology and Venereology.)

Wool is composed primarily of proteins belonging to the keratin family. These include the keratins and keratin-associated proteins (KAPs) that are responsible for the structural and mechanical properties of wool fibre. Although all human keratin and KAP genes have been annotated, many of their ovine counterparts remain unknown and even less is known about their genomic organisation. The aim of this study was to use a combinatory approach including comprehensive cDNA and de novo genomic sequencing to identify ovine keratin and KAP genes and their genomic organisation and to validate the keratins and KAPs involved in wool production using ovine expressed sequence tag (EST) libraries and proteomics. The number of genes and their genomic organisation are generally conserved between sheep, cattle and human, despite some unique features in the sheep. Validation by protein mass spectrometry identified multiple keratins (types I and II), epithelial keratins and KAPs. However, 15 EST-derived genes, including one type II keratin and 14 KAPs, were identified in the sheep genome that were not present in the NCBI gene set, providing a significant increase in the number of keratin genes mapped on the sheep genome., (© 2018 Stichting International Foundation for Animal Genetics.)

The term palmoplantar keratoderma (PPK) indicates any form of persistent thickening of the epidermis of palms and soles and includes genetic as well as acquired conditions. We review the nosology of hereditary PPKs that comprise an increasing number of entities with different prognoses, and a multitude of associated cutaneous and extracutaneous features. On the basis of the phenotypic consequences of the underlying genetic defect, hereditary PPKs may be divided into the following: (i) non-syndromic, isolated PPKs, which are characterized by a unique or predominant palmoplantar involvement; (ii) non-syndromic PPKs with additional distinctive cutaneous and adnexal manifestations, here named complex PPKs; (iii) syndromic PPKs, in which PPK is associated with specific extracutaneous manifestations. To date, the diagnosis of the different hereditary PPKs is based mainly on clinical history and features combined with histopathological findings. In recent years, the exponentially increasing use of next-generation sequencing technologies has led to the identification of several novel disease genes, and thus substantially contributed to elucidate the molecular basis of such a heterogeneous group of disorders. Here, we focus on hereditary non-syndromic isolated and complex PPKs. Syndromic PPKs are reviewed in the second part of this 2-part article, where other well-defined genetic diseases, which may present PPK among their phenotypic manifestations, are also listed and diagnostic and therapeutic approaches for PPKs are summarized., (© 2018 European Academy of Dermatology and Venereology.)

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial-to-mesenchymal transition (EMT) is a key contributor in the metastatic process. In this study, we found that miR-655 was down-regulated in TNBC, and its expression levels were associated with molecular-based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR-655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR-655 not only induced the up-regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal-like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR-655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR-655 significantly suppressed Prrx1, as demonstrated by Prrx1 3'-untranslated region luciferase report assay. Our study demonstrated that miR-655 inhibits the acquisition of the EMT phenotype in TNBC by down-regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Chronic rhinosinusitis (CRS) defines a group of disorders characterized by persistent inflammation of the sinonasal tract. Epithelial changes and structural remodelling are present, but whether epithelial differentiation is altered remains uncertain., Methods: To evaluate the differentiation state of the sinonasal epithelium in CRS, sinonasal biopsies from patients with CRS with nasal polyps (CRSwNP) or CRS without nasal polyps (CRSsNP), or with allergic rhinitis (AR), as compared to controls, were processed by immunohistochemistry and RT-qPCR for terminal differentiation (E-cadherin, high molecular weight cytokeratins (Hmw CK) and CK5, vimentin) and lineage differentiation (ß-tubulin IV+ ciliated cells, MUC5AC+ goblet cells, p63 + basal cells). Findings were correlated with subepithelial fibrosis and clinical CT score., Results: Expression of E-cadherin was decreased at protein and mRNA levels in CRSwNP and CRSsNP, as compared to controls. Staining for Hmw CKs was also reduced in CRSwNP and CRSsNP, and CK5 mRNA was decreased in CRSwNP. These features were not due to changes in lineage specification, but associated with increases in vimentin-expressing epithelial cells. In addition, vimentin expression correlated with the basement membrane thickening and with CT score, as well as with tissue eosinophils., Conclusion: Features of epithelial dedifferentiation towards a mesenchymal phenotype are observed in CRSwNP and CRSsNP and correlate with airway fibrosis and inflammation., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In higher plants, the MADS-box genes encode a large family of transcription factors (TFs) involved in key developmental processes, most notably plant reproduction, flowering and floral organ development. SEPALLATA 3 (SEP3) is a member of the MADS TF family and plays a role in the development of the floral organs through the formation of multiprotein complexes with other MADS-family TFs. SEP3 is divided into four domains: the M (MADS) domain, involved in DNA binding and dimerization, the I (intervening) domain, a short domain involved in dimerization, the K (keratin-like) domain important for multimeric MADS complex formation and the C (C-terminal) domain, a largely unstructured region putatively important for higher-order complex formation. The entire K domain along with a portion of the I and C domains of SEP3 was crystallized using high-throughput robotic screening followed by optimization. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 123.44, b = 143.07, c = 49.83 Å, and a complete data set was collected to 2.53 Å resolution.

Hereditary conditions are traditionally classified based either on physical/physiological attributes or using the names of the individuals credited with identifying the condition. For the 170 plus conditions classified as ectodermal dysplasias (EDs), both of these nosological systems are used, at times interchangeably. Over the past decade our knowledge of the human genome and the molecular basis of the EDs have greatly expanded providing the impetus to consider alternative classification systems. The incorporation of the molecular basis of hereditary conditions adds important information allowing effective transfer of objective genetic information that can be lacking from traditional classification systems. Molecular information can be added to the nosological system for the EDs through a hierarchical- and domain-based approach that encompasses the condition's name, mode of inheritance, molecular pathway affected, and specific molecular change. As new molecular information becomes available it can be effectively incorporated using this classification approach. Integrating molecular information into the ED classification system, while retaining well-recognized traditional syndrome names, facilitates communication at and between different groups of people including patients, families, health care providers, and researchers., ((c) 2009 Wiley-Liss, Inc.)

Propionibacterium acnes is one of the most significant pathogenic factors of acne vulgaris. This bacteria relates to acne by various pathways. It has also been reported that P. acnes influences pro-inflammatory cytokine production in keratinocytes in vitro. However, the influence on the differentiation of keratinocytes by P. acnes has not been studied extensively. We analyzed the expression of keratinocyte differentiation-specific markers, keratins, and pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEK) exposed to P. acnes in vitro. All P. acnes strains used in this study increased transglutaminase (TGase), keratin 17 (K17) and interleukin (IL) mRNA expression levels in NHEK, and decreased K1 and K10 expression levels. Some P. acnes strains increased involucrin and K6 mRNA expression levels in NHEK and decreased filaggrin, K6 and K16 expression levels in vitro. This experiment clarified that P. acnes influences the differentiation of NHEK in vitro. As a result, P. acnes influenced the expression of not only pro-inflammatory cytokines but also some keratinocyte differentiation-specific markers and keratins in NHEK. Our results suggest that P. acnes relates to acne pathogenesis by not only the induction of inflammation but also in the differentiation of keratinocytes. Moreover, it was considered that the reaction of NHEK to P. acnes may be different depending on the type of bacteria.

This article presents a short summary of our current knowledge of cicatricial alopecia disease pathogenesis and the hypothetical disease mechanisms that may be involved in scarring alopecia development. Several forms of scarring alopecia likely involve targeted cytotoxic action against hair follicle cells mediated by a folliculocentric inflammation. However, the specific nature of the inflammatory interference in hair follicle growth is open to question. A popular hypothesis of lymphocyte-mediated scarring alopecia development involves autoimmune targeting of hair follicle-specific self-antigens, although there is no direct evidence in support of such a view. Alternative hypotheses focus on defects in sebaceous gland function, destruction of hair follicle stem cells, and interference in the communication between hair follicle mesenchyme and epithelium. Many questions arise from these hypotheses, and addressing them with a systematic research approach may enable significant advances in understanding cicatricial alopecia etiology.

Oral submucous fibrosis (OSF) is characterized by abnormal collagen metabolism in the submucosal connective tissue. Its influence on the overlying epithelium is not known but about 14% of OSF cases undergo malignant transformation to squamous cell carcinoma indicating association with abnormality of the epithelium. Here, we have defined the keratin expression profile, by immunohistochemistry and quantitative image analysis, using a panel of 22 anti-keratin monoclonal antibodies on 28 OSF samples. We observed an increase of K1 and K10 in the suprabasal layers, induction of K6 in the basal layer and complete loss of K19 in the epithelium. Furthermore, there was increased K17 expression in the suprabasal layers, which correlated with disease severity. In a subset of the most severe OSF cases (14%), K17 expression was completely lost in the basal layer which might define them to be at most risk to undergo malignant transformation. There was no detectable expression of K8, K18, K7 and K9 and the expression of K4, K13, K14, K15 and K16 did not change in OSF. We propose that the altered keratin profiles could be useful as histological diagnostic markers and provide important insights into the pathogenesis of the disease and its predisposition to malignancy.

Background: Ductopenia is observed in end-stage human cholestatic diseases. The limited capability of cholangiocytes for proliferation is suggested to be the principal reason. Recently, bone marrow cells (BMCs) have been reported to behave as hepatic stem cells; however, their capability to differentiate into cholangiocytes in cholestasis remains unclear., Methods: Normal mice were lethally irradiated to suppress the proliferation of self-BMCs; thereafter, the BMCs from enhanced green fluorescent protein (EGFP)-transgenic mice were transferred to recipients. Chronic cholestasis was induced by 0.1%alpha-naphtylisothiocyanate (ANIT) feeding. The proliferation of cholangiocytes and oval cells was assessed morphologically and immunohistchemically (cytokeratin-7 (CK-7), A6). Proliferative activity (proliferating cell nuclear antigen (PCNA) protein expression), hepatic growth factor (HGF) receptor (c-Met), stem cell factor receptor (c-kit), Notch2 and Hes1 expression were also evaluated., Results: Marked cholangiocyte proliferation was observed in ANIT-fed mice. However, no EGFP/CK-7 double positive cells were identified in any of the liver specimens after BMCs transfer (Tx). In hepatic parenchyma, there were scattered EGFP-positive cells, although none of them were positive for CK-7., Conclusions: In spite of the significant ductular proliferations after ANIT feeding, no EGFP-positive cholangiocytes were confirmed by any other means in this chronic cholestasis model. Thus, different from hepatocytes, BMCs Tx seems not to contribute to the differentiation of cholangiocytes. Future studies are feasible to clarify the origin of proliferative cholangiocytes observed in this chronic cholestatic ductular hyperplasia model.

Pachyonychia congenita type 2 (PC-2) is an autosomal dominant disorder characterized by hypertrophic nail dystrophy, focal keratoderma, multiple pilosebaceous cysts, and other features of ectodermal dysplasia. It has been demonstrated that PC-2 is caused by mutations in the keratin 17 and keratin 6b genes. In this report, we describe a missense mutation in the keratin 17 gene, M88T, in a Korean patient whose phenotype included early onset steatocystoma multiplex and Hutchinson-like tooth deformities along with other typical features of PC-2 such as hypertrophic nails, natal teeth and follicular hyperkeratosis.

Bullous congenital ichthyosiform erythroderma (BCIE) is an autosomally dominant inherited disorder characterized by erythematous, erosive, and bullous skin lesions over the entire body at birth and abnormal hyperkeratosis on the palmoplantar sufaces as the patient grows older. BCIE is caused by a mutation in the keratin 1 (K1) and/or keratin 10 (K10) genes, and most pathogenic mutations are found within the helix initiation and termination motifs of the central helical rod domain (K1 and K10) or the upstream H1 homology domain (K10). In addition to inherited cases, sporadic cases due to a new mutation account for approximately half the total cases of BCIE. We report herein a typical sporadic case of BCIE with erythroderma, erosion, and blisters on the entire body surface at birth and palmoplantar and flexuaral areas of hyperkeratosis in the later stage. We found in this case a novel mutation, 559C to T, at amino acid position 187, which resulted in a leucine to phenylalanine substitution within the helix initiation motif of K1.

Objective: Angiogenesis plays an important role in psoriasis, but its role in atopic dermatitis is unknown. The authors examined the dermal microvasculature of an IL-4 transgenic mouse model of atopic dermatitis to determine whether angiogenesis was present., Methods: Transmission and scanning electron microscopy and confocal microscopy studies were performed., Results: Transmission electron microscopy showed sprouting, transcapillary pillars of intussusception, thickened endothelial cells with large nuclei, and increased interendothelial junctional cleft number and length. Compared to nontransgenic littermates, there was a significant increase in the lengths and numbers of the interendothelial junctional clefts, along with a decrease in the length ratios of tight junction to interendothelial junctional clefts in both the early and late disease stages. In the early and late skin lesions, scanning electron microscopy of vascular corrosion casts showed disorganization of the capillary network hierarchy with increased density of capillary sprouts. Confocal microscopy of the animals with early and late skin lesions showed significant reduction in tight junction protein claudin-5., Conclusions: Angiogenesis is the major pathologic feature in this model of atopic dermatitis. The chronic skin inflammation is intertwined with and may cause the angiogenesis, but the angiogenesis itself is likely to be important in this disease process.

Diseases caused by mutations in gene encoding keratin intermediate filaments (IF) are characterized by a loss of structural integrity in the cells expressing those keratins in vivo. This is manifested as cell fragility, compensatory epidermal hyperkeratosis, and keratin filament aggregation in some affected tissues. Keratin disorders are a novel molecular category including quite different phenotypes such as epidermolysis bullosa simplex (EBS), bullous congenital ichthyosiform erthroderma (BCIE), pachyonychia congenital (PC), steatocystoma multiplex, ichthyosis bullosa of Siemens (IBS), and white sponge nevus (WSN) of the orogenital mucosa., ((c) 2004 Wiley-Liss, Inc.)

The ichthyoses are a large, clinically, genetically, and etiologically heterogeneous group of disorders of cornification due to abnormal differentiation and desquamation of the epidermis. Although they differ in clinical features, inheritance, and structural and biochemical abnormalities of the epidermis, they often pose a diagnostic challenge. For each of the 12 ichthyoses and related disorders described here, the major disease genes have been identified and genotype-phenotype correlation have begun to emerge. The molecular findings reveal the functional importance and interactions of many different epidermal proteins and metabolic pathways, including major structural proteins (keratins, loricrin), enzymes involved in lipid metabolism (transglutaminase 1, lipoxygenases, fatty aldehyde dehydrogenase, steroid sulfatase, glucocerebrosidase, Delta8-Delta7 sterol isomerase, 3beta-hydroxysteroid dehydrogenase), and protein catabolism (LEKTI), peroxisomal transport and processing (Peroxin 7 receptor, Phytanoyl-CoA hydroxylase) and DNA repair (proteins of the transcription repair complex). This review highlights the spectacular advances in the molecular genetics and biology of heritable ichthyoses over the past decade. It illustrates the power of molecular diagnostics for refining disease classification, providing prenatal diagnosis, improving genetic counseling, and clinical management., ((c) 2004 Wiley-Liss, Inc.)

Objective and Background: Our previous studies suggest that little adverse effect on the growth of the periodontal ligament would be expected, if tetracycline, minocycline, ofloxacin, roxithromycin, clarithromycin, and azithromycin were topically administered to the periodontal pocket at their MIC90 doses required to inhibit the growth of 90% of periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. In the present study, we investigated the cytocidal effects of eight antibacterial agents on the human gingival epithelial cell line NDUSD-1. We also used NDUSD-1 cells to examine the effects of these agents on the mRNA and protein expressions of genes associated with the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment., Methods: The cytocidal effect of the test agents was measured as a decrease in cell survival. To obtain a quantitative measure of the cytocidal effect, the LD50, i.e. the concentration which results in a 50% decrease in cell survival relative to the controls, was extrapolated from the concentration-response curves. The effects of the agents on the mRNA and protein expressions in NDUSD-1 cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively., Results: The cytocidal effect increased in a concentration-dependent manner as the concentration of each of the eight test agents increased. The order of the agents according to their cytocidal effects (LD50) was minocycline > tetracycline > enoxacin > clarithromycin > roxythromycin approximately ofloxacin > azithromycin > erythromycin. The cytocidal effects of minocycline, tetracycline, enoxacin, clarithromycin, roxythromycin, ofloxacin, and azithromycin ranged from 1.2 to 23.2 times greater than that of erythromycin. The maximum non-cytocidal concentrations (MNCCs) of these agents for NDUSD-1 cells were: 0.3 microm for minocycline, 1 microm for tetracycline, 3 microm for ofloxacin and erythromycin, 10 microm for enoxacin, clarithromycin, and azithromycin, and 100 microm for roxythromycin. The MNCCs of ofloxacin, azithromycin, clarithromycin, and roxythromycin were greater than their MIC90 concentrations for periodontopathic bacteria described above. The effects on the mRNA and protein expressions of epithelial-cell- or cell-adhesion-related genes were examined in NDUSD-1 cells exposed to clarithromycin, roxythromycin, ofloxacin, and azithromycin at their MNCCs. None of the agents affected the mRNA expressions of five genes: keratinocyte growth factor receptor, keratin 18, integrin beta1, integrin beta4, and laminin 5gamma2. Clarithromycin and ofloxacin slightly decreased the protein expression of integrin beta4. Roxythromycin markedly decreased the protein expressions of integrin beta4 and laminin 5gamma2. Azithromycin had little inhibitory effects on the protein expressions of any of the five genes., Conclusions: These results suggest that little, if any, adverse effects on growth, differentiation, and adhesion of basal epithelial cells would be expected with topical administration of clarithromycin, ofloxacin or azithromycin to the periodontal pocket at a dose equivalent to the MIC90. It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominantly inherited disease. We studied a family from Shandong, China, having patients suffering from EPPK with a unique symptom-knuckle pads. We noticed that both the hyperkeratosis and knuckle pads in the Chinese family were friction-related. Candidate gene analysis was carried out using linkage analysis and direct sequencing. A novel L160F mutation in keratin 9 was found, and its effects on the secondary structure of keratin 9 were studied. We predict that the L160F mutation is also responsible for the knuckle pads in the family. Our study provides a new clue for the study of the function of keratin 9., (Copyright 2003 Wiley-Liss, Inc.)

We described a 5-year-old Japanese girl with epidermolytic palmoplantar keratoderma and examined her for a keratin 9 gene mutation. Physical examination disclosed diffuse yellowish hyperkeratosis with an erythematous border limited strictly to the palms and soles. Histological examination revealed hyperkeratosis with vacuolar degeneration in the spinous and granular layers of the epidermis. Sequence analysis demonstrated an A to G transition at the middle position of codon 160 in the 1A domain of the keratin 9 gene. The amino acid at codon 160 was deduced to have changed from asparagine (Asn) to serine (Ser). This is the first case with an Asn160Ser mutation in a Japanese. The substitution of Ser for Asn at codon 160 of the keratin 9 gene is assumed to be fatal for keratin filament assembly regardless of race or ethnicity.

The epidermis of skin and the oral mucosa are highly specialized stratified epithelia that function to protect the body from physical and chemical damage, infection, dehydration, and heat loss. To maintain this critical barrier, epithelial tissues undergo constant renewal and repair. Epithelial cells (keratinocytes) undergo a program of terminal differentiation, expressing a set of structural proteins, keratins, which assemble into filaments and function to maintain cell and tissue integrity. Two types of cell adhesion structures, desmosomes and hemidesmosomes, function to glue keratinocytes to one another and to the basement membrane, and connect the keratin cytoskeleton to the cell surface. Keratinizing epithelia such as the epidermis and oral gingiva that have to withstand severe physical and chemical forces produce a toughened structure, the cornified cell envelope. This envelope is a major component of the epithelial barrier at the tissue surface. This article summarizes our current understanding of the structure and function of these different cellular components and discusses various genetic and acquired diseases that alter tissue integrity and barrier function. We also highlight recent work demonstrating how loss or attenuation of certain proteases can lead to early onset periodontitis and tooth loss as well as other epithelial abnormalities.

Epidermolysis bullosa simplex (EBS) is classified into three main types and is caused, in most cases, by missense mutations in the genes encoding keratin (K) 5 and K14. In this study, we clinically, ultrastructurally, immunohistochemically, and molecularly studied a patient with a dominant EBS, Köbner type. Using sequence analysis of genomic DNA, a novel K14 nonsense mutation was identified. A heterozygous mutation G1231T of KRT14 was found to be associated with the disease in the patient. The mutation created a premature stop codon (amino acid codon 411, residue 106 of the 2B helix) in the K14 molecule. This residue lies in a highly conserved region and was recently found to be absolutely required for molecular stability and intermediate filament assembly in K5 and K14. The E411X (E106X) heterozygous ablation, missing the last 16 amino acid residues of the 2B and the entire tail domain of K14, led to disease but did not result in clumping of keratin filaments. It is the first premature stop codon mutation of K14 found in dominant EBS.

Background: Epidermolytic palmoplantar keratoderma is an autosomal dominant inherited disorder of keratinization., Methods: We studied five members of a Jewish family with epidermolytic palmoplantar keratoderma. Genomic DNA was extracted from leucocytes, and exon 1 of the keratin 9 gene was amplified using polymerase chain reaction techniques., Results: The mutation was found in exon 1 of the keratin 9 gene in codon 160., Conclusions: Like most of the other families with clinical features of epidermolytic palmoplantar keratoderma the mutation is found in exon 1 of the keratin 9 gene.

We described a 19-year old Japanese female with bullous congenital ichthyosiform erythroderma (BCIE) and examined the keratin gene mutation. Physical examination disclosed generalized erythema, ichthyosiform skin with scales, and erosions without palmoplantar keratoderma. Histological examination revealed hyperkeratosis with vacuolar degeneration in the granular layer of the epidermis. Sequence analysis demonstrated a C to G transition at the first position of codon 156 in the keratin 10 gene. The amino acid at codon 156 was deduced to have changed from arginine to cystine. Substitution from arginine to cysteine at codon 156 of the K 10 gene is assumed to be fatal for keratin filament assembly regardless of racial or ethnic difference.

Epidermolysis bullosa simplex (EBS) is a blistering disorder affecting the basal layer of the epidermis usually inherited in an autosomal dominant fashion. Most cases are caused by mutations in the genes encoding keratin 5 (K5) and keratin 14 (K14) and are characterized by cytolysis within the basal layer of the epidermis. We report a patient manifesting the Dowling-Meara variant of EBS in whom we characterized a cytosine to thymine transition at codon 125 (R125C) in K14. This missense mutation is located at the amino terminus of the helical rod domain of the keratin 14 molecule, resulting in defective pairing with K5, thereby disrupting keratin tonofibril integrity.

Gene expression for the differentiation-specific keratins (K4, K13, K1 and K10) was analyzed in oral non-dysplastic keratoses, oral lichen planus (OLP) and lichenoid reactions (LR) by comparative in situ hybridization (ISH) and immunohistochemistry (IHC) to investigate molecular changes in the altered differentiation pattern from non- to para- or orthokeratinization. At the protein level, K4 and K13 were detected homogeneously in the suprabasal compartment of parakeratotic epithelium but showed reduced expression in orthokeratoses, particularly in the presence of lymphocytes. Corresponding transcripts were restricted to basal and lower prickle cells. Synthesis of K1 and K10 was upregulated and more pronounced in orthokeratotic epithelia. The study showed an alteration in the pattern of differentiation-specific keratins, although involvement of the lymphocytic infiltrate in OLP and LR resulted in further gene modulation. In both diseases, K1 and K10 showed transcriptional control, proteins having the same distribution as their transcripts. This represented a change from post-transcriptional regulation in normal buccal epithelium, in which mRNAs for K1 and K10 are more widely expressed than their proteins. Thus, the pattern of keratin gene expression may be altered in response to frictional/smoking stimuli or immune-mediated mechanisms.

Recently, mutations of keratin 1 gene (KRT1) and keratin 10 gene (KRT10) have been reported in various patients with bullous congenital ichthyosiform erythroderma (BCIE). The substitution of arginine (R) to histidine (H) at amino acid residue 156 (R156H) of coiled 1A region is one of the most frequent mutations of KRT10. In this study, we searched for a mutation in KRT1 and KRT10 in a Japanese family with BCIE and detected mutation R156H in KRT10. Our search led to the detection of R156H. This mutation was not detected in 50 normal individuals. These results confirmed that codon 156 is a frequently mutated site, and that R156H in KRT10 is likely also to be a mutation hotspot in Japanese patients with BCIE.

Objectives/hypothesis: To discover unique genes specific for squamous cell carcinoma of the head and neck for eventual development as tumor markers and vaccine candidates., Study Design: Molecular biological analysis of fresh-frozen head and neck squamous cell cancer (HNSCC)., Methods: A subtractive library was made from two HNSCC and six normal tissues using a polymerase chain reaction (PCR)-based approach. Genes from this library were PCR amplified and placed on a microarray glass slide. RNA was prepared or obtained from 16 fresh-frozen HNSCC and 22 normal tissue sources. Fluorescent probes were made from the polyA+ RNA derived from the tumor and normal tissues. The probes were hybridized to the glass slides and excited by a tuneable laser. One hundred seven of the genes showing the highest differential fluorescence value between tumor and normal tissue were identified by sequence analysis., Results: Thirteen independent genes were found to be overexpressed in tumor tissues. Of these, nine were previously known: keratins K6 and K16, laminin-5, plakophilin-1, matrix metalloproteinase-2 (MMP), vascular endothelial growth factor, connexin 26, 14-3-3 sigma, and CaN19. The level of polyA+ RNA of these genes in the tumors was significantly different from the levels in normal tissue (P < .05). Four previously unidentified genes were also discovered to have increased expression in tumor tissue. Comparing the total tumor group (n = 16) to the normal group (n = 22), only one of these genes showed significant overexpression., Conclusion: We report the identification of nine known genes that are significantly overexpressed in HNSCC as compared to normal tissue using subtractive and microarray technology. In addition, we present four previously unidentified genes that are overexpressed in a subset of tumors. These genes will be developed as tumor markers and vaccine candidates.

Cytokeratin (CK) is a filament which plays a central role in epithelial tissue and, like the polypeptides of intermediate filaments in general, shows a high degree of tissue specificity. The CK expression patterns of odontogenic epithelia are still poorly described. We studied the distribution of individual CK polypeptides in the human enamel organ at bell stage and in remnants of the dental lamina. Our immunohistochemical study showed that epithelial cells stained for CKs 7, 13, 14 and 19 with slight changes in their pattern during the differentiation phase of odontogenesis. There was negative staining for all other CK polypeptides tested (CKs 8, 10, 16, 17 and 18). Most of the CKs in the enamel organ epithelia did not show differences related to the stage-specific state of differentiation, except for CKs 14 and 19 at the inner enamel epithelium. A strong label for CK 14 was present at the inner dental epithelium at early bell stage, and this was substituted by CK 19 at the late bell stage when the ameloblasts were fully differentiated.

Epidermolytic palmoplantar keratoderma appears to be due to defects in keratin 9, the palmoplantar specific type 1 keratin. We report a case of spontaneous mutation, a C to T transition at codon 162, resulting in an arginine to tryptophan substitution in the 1 A region of the alpha helical rod domain of keratin 9. This provides further evidence that this codon is an important spot for mutation in keratin 9.

We have identified the gene coding for the canine ortholog of the human keratin 9 protein using the inverse-polymerase chain reaction (PCR) strategy. Sequence comparison and structure analysis of the gene show marked similarity with the human gene. This gene spans about 7 kb and spreads over eight exons. In the dog gene, the reading frame is extended by 20 codons, the first in-frame stop codon being in exon 8 in the dog rather than in exon 7 as in humans. Alignment of human and dog predicted amino acid sequences confirms the high analogy, reaching 75% identity and 95% similarity in the rod domain. Interestingly, the glycine-loop motif number in the C-terminal V2 variable subdomain of the protein increases from 19 in human to 43 in dog, generating a size difference of 12 kDa between the two proteins. Due to its restricted expression pattern in mammalian epidermis, dog keratin 9 gene was a good candidate gene for the genetic palmoplantar hyperkeratosis observed in the Dogue de Bordeaux. However, no polymorphism associated with the pathology was detected within an affected Dogue de Bordeaux pedigree ruling out this hypothesis.

Ameloblastomas are slowly growing, locally invasive neoplasms with a potentially destructive behaviour. The molecular mechanisms that regulate the cell growth and invasion of ameloblastoma cells are unknown. Because ameloblastoma cells placed in culture have a very limited lifespan, the establishment of immortalized clones of ameloblastoma cells would aid its study. We produced an immortalized ameloblastoma cell line (AM-1) using human papillomavirus type-16. This cell line maintains epithelial cell morphology and expresses cytokeratins K8, K14, K18, K19. Furthermore, bcl-2 protein, which prevents apoptosis, is expressed. We investigated the behaviour of these cells on a collagen matrix in vitro. These cells grew in a monolayer over foci of collagen degradation and could invade the collagen gel at such sites. Since the behavior of cell line AM-1 mimics the behavior of ameloblastoma in vivo, it may be a valuable model for the study of these neoplasms.

Mutations in keratin genes account for a number of inherited keratodermas in humans. The groups of basic and acidic keratin genes are clustered on human chromosomes 12 and 17, respectively. The present authors have assigned the two putative keratin gene clusters to canine chromosomes using canine cosmid clones. Successful fluorescence in situ hybridization mapped the putative cluster of canine acidic genes to dog chromosome 20 and the putative cluster of basic keratin genes to a small autosome not yet included in the partial canine standard karyotype.

Cytokeratins (CKs) are now considered to be reliable markers for following the development and differentiation of epithelial tissue. We have investigated the pathway of differentiation in human developing junctional epithelium using monoclonal antibodies and two-dimensional gel electrophoresis of microdissected tissue to identify CK 19, CK 16, CK 14, CK 13, CK 6, CK 5, CK 4 in the junctional epithelium (JE) over partially erupted human teeth. The CK profile was similar to that of developing oral epithelia, suggesting that the junctional epithelium in teeth during eruption is of odontogenic origin. The present study used in situ hybridization to determine the distribution of the mRNAs of CKs 19, 16, 13 and 4 in human developing junctional epithelium and to examine the correlation between mRNAs and their encoded proteins. CK 19 mRNA was abundant in the basal cell layers of the primary junctional epithelium (PJE) but less concentrated in the suprabasal layers. CK16, 13 and 4 mRNAs were abundant in the basal cell layers of the PJE. The parabasal cell layers reacted intensely to the cRNA probe complementary to CK16 mRNA, as were the reactions in the suprabasal cell layers of the PJE for the CK 13 and 4 probes. Our results demonstrate that the PJE express the genes encoding for CKs 16 and 4 that have been revealed previously only by electrophoresis. They therefore confirm that the PJE is a well-differentiated stratified epithelium with a complex unique phenotype that produces CKs specific for basal cells (CK 19), CKs associated with hyperproliferation (CK 16), and finally those associated with stratification (CKs 4 and 13). Only synthesis of CK 19 protein and mRNA are strictly parallel. CKs 4 and 13 mRNAs are present in basal and suprasal cells, while their encoded proteins were not, except for CK 13 in suprabasal cell layers of PJE, where the amount of its mRNAs was coincident with the expression of the protein.

Cytokeratin (CK) expression in untreated, paraffin-treated or dimethylbenzanthracene (DMBA)-treated hamster cheek pouch epithelium was investigated utilizing monoclonal antibodies AE1 or AE3, which react with type I or type II CKs, respectively, and by in situ hybridization utilizing type I CK-specific probes. The latter were isolated from a cDNA library of hamster cheek pouch mRNA and designated CK 13 and CK 10 based on their respective homologies (> 95% amino acids) with murine CK 13 and human CK 10. Treatment of hamster cheek pouch epithelium with DMBA resulted in increased expression of type I CK, detected immunohistochemically with monoclonal AE1, but decreased expression of type II CKs detected with AE3. Despite an overall increase in type I CKs, in situ hybridization demonstrated differential expression of type I CKs with altered distribution of CK 13 mRNA and reduced expression of CK 10 mRNA, providing additional sensitive markers for DMBA-associated changes in CKs. These changes were constant at 2 to 22 weeks in the pre-neoplastic and neoplastic epithelium following the initial application of DMBA.

The junctional (JE) and oral gingival (OGE) epithelium show distinct morphological phenotypes and express different cell surface and keratin markers. Transforming growth factor-beta (TGF-beta) has been shown to stimulate extracellular matrix formation and inhibit proteolytic matrix degradation in periodontal wound healing. To elucidate potential roles of TGF-beta in gingival epithelial regeneration and reattachment, the present study examined the effects of TGF-beta on JE and OGE cell growth and determined the patterns of expression of mRNAs for the TGF-beta isotypes beta 1, beta 2 and beta 3 and TGF-beta receptor types I, II and III. Primary cell cultures were initiated from JE and OGE and the cell phenotypes confirmed using monoclonal antibodies to specific keratins. TGF-beta induced a significant growth inhibition in OGE cells derived from 6 different patients with a mean inhibition of 46% and a range of 16-70% (p = 0.031). Although responses varied between patients, in general maximum inhibition occurred at 10 ng/ml TGF-beta. JE cells from 5 patients showed no significant growth inhibition by TGF-beta (p = 0.125). Greater expression of TGF-beta 2 and receptor type I mRNA was found in OGE than JE cells and thus appeared to be associated with differentiating epithelial cells. JE cells expressed more TGF-beta type II receptor specific mRNA than did OGE cells, but TGF-beta 1 mRNA expression was similar in JE and OGE cells. JE or OGE cultures derived from 2 of 3 patients showed expression of mRNA for the TGF-beta type III receptor. TGF-beta 3 mRNA was not detected in any of the JE or OGE samples examined. The greater sensitivity of OGE than JE to the growth inhibiting effects of TGF-beta correlated with higher expression of receptor type I mRNA which, together with the type II receptor, is required for sensitivity to growth inhibition by TGF-beta. The results suggest that, in addition to structural differences, the development of functional differences in the responses of JE and OGE to TGF-beta may be associated with the formation of JE from OGE cells and the reformation of attachment after periodontal surgery.

To characterize further the nature of calcifying odontogenic cyst (COC), we studied histologically and immunohistochemically an extraosseous and two intraosseous lesions. The extraosseous COC was in continuity with the stratified squamous epithelium of the alveolar mucosa. Immunostaining with monoclonal antibodies showed reactivity of both low- and high-molecular-weight cytokeratins, the degree of coexpression decreasing with the increasing morphological diversity of the cyst/tumour epithelium. Staining for the matrix glycoprotein tenascin-C was seen not only in the connective tissue, where its distribution patterns corresponded to the stage of hard tissue formation, but also in epithelial elements. The staining patterns were analogous to those described during normal tooth formation. Both the morphological characteristics and expression patterns of the various cytokeratin types and tenascin-C implied that COC represents a pathological counterpart of normal odontogenesis. In the case of the extraosseous COC, the correspondence could be traced back to early stages of tooth development.

To establish a three-dimensional in vitro test system mimicking the physiological situation of the oral cavity, organotypic co-cultures consisting of primary gingival cells on a collagen matrix with fibroblasts were generated. The histomorphological development after 7 and 14 d revealed close similarity with the non-keratinized gingiva epithelium. Furthermore, as epithelial specific markers synthesis and localization of keratins as well as the deposition of basement membrane components were assessed on frozen sections by immunofluorescence and keratin expression by in situ hybridization. Primary keratinocytes in conventional culture strained positive for keratin K14 and the mucosal differentiation-specific keratins K4 and K13, while primary fibroblasts, isolated from the same tissue source, and also some keratinocytes, were positive for vimentin. In organotypic co-cultures the keratinocytes formed a multilayered epithelium within 14 d containing basal cells and flattened cells in the uppermost layers. Comparable to native non-keratinized gingiva keratin 14 gene expression was clearly detectable in the basal cell compartment but showed extending immunolocalization. In addition, particularly at the early stage (7 d), basally located keratinocytes were also vimentin positive. According to morphological differentiation K4 and K13 were detectable in suprabasal position a the RNA and protein level. The major basement membrane constituents collagen type IV and laminin increased with time revealing first an interrupted and later a fully extended staining underneath the basal cells. Maintenance of basal cell function was further demonstrated by cell proliferation (BrdU incorporation) which was initially high (7 d) but declined towards the later stages (14-21 d). The results demonstrate i) that this co-culture system leads to a stratified surface epithelium with morphological and biochemical characteristics of the non-keratinized gingiva epithelium and ii) that a state of physiological tissue balance was reached, thus rendering a suitable model for tissue compatibility studies.