Simple mucin-type carbohydrates in oral stratified squamous and salivary gland epithelia

Simple mucin-type carbohydrate antigens, T, Tn, and sialosyl-Tn, have been found to be good markers of malignant transformation in several epithelial tissues as a result of incomplete synthesis with precursor accumulation. The T, Tn, and sialosyl-Tn antigens represent the initial, most immature glycosylation of serine and threonine amino acids of proteins. In normal adult cells these structures are generally masked by addition of further saccharides to form more complex structures. We analyzed simple mucin-type carbohydrates in human labial stratified squamous and minor salivary gland epithelia in order to define the glycosylation pattern in normal cells in relation to epithelial differentiation and maturation. A panel of monoclonal antibodies with well-characterized specificity for T, Tn, sialosyl-Tn and the histo-blood group H and A variants hereof were used in immunohistology of sections from 30 individuals with known ABO, Lewis, and secretor status. In stratified epithelium the sialylated T structure was confined to cell membranes of immature basal cells, whereas the H and A variants were observed on cell membranes of more mature parabasal and spinous cell layers. Furthermore, superficial spinous cells produced a fine granular cytoplasmic staining for Tn and sialosyl-Tn antigens. In minor salivary glands mucous cells expressed Tn and sialosyl-Tn as well as the H and A variants in the area of the nucleus, whereas T and the H variant were found in duct cells and unsubstituted T antigen in myoepithelial cells. These results indicate that incomplete synthesis, i.e., deletion of sialyltransferases and/or histo-blood group ABH transferases, may result in accumulation of T, Tn, and sialosyl-Tn antigens in oral epithelia, thus offering a baseline for further studies of changes in premalignant and malignant oral epithelia.     

Delineation of diversified desmoglein distribution in stratified squamous epithelia: implications in diseases

Desmogleins play critical roles in cell adhesion and skin blistering diseases, as they are the target antigens of autoimmune antibodies and bacterial toxins. We recently cloned several novel members of the desmoglein gene family, bringing the number of desmogleins to four in the rat and human genomes and six in the mouse. Here, we have produced a monoclonal antibody to a cytoplasmic epitope of Dsg4, assessed its specificity and compared it to several existing Dsg1-3 antibodies. We also demonstrated cross-reactivity of commercially available and often used Dsg1 antibodies. Using these tools, we delineated the unique expression patterns of each desmoglein isoform in various human and mouse stratified squamous epithelia, including skin, hair, palm, and oral mucosa. Interestingly, in the epidermis, the expression of each desmoglein correlates with their gene arrangement in the cadherin locus. In human, Dsg4 was detected primarily in the granular and cornified cell layers of the epidermis, while present throughout all differentiated layers of the oral mucosa and palm, and in the matrix cells of anagen hair bulb. Similar pattern of expression for Dsg4 was observed in mouse, with the exception that it was expressed at significantly lower levels in the mouse epidermis. These results demonstrate the complexity of desmoglein gene expression and provide additional insights into the correlation between tissue expression patterns and disease phenotypes.     

鼻疽诺卡菌基因组DNA及细胞脂肪酸组成分析

目的：比较鼻疽诺卡菌与其它三种常见诺卡菌随机扩增DNA片段及脂肪酸组成的异同，并初步探讨其分类学意义。方法：(1)采用随机扩增DNA多态性(RAPD)方法，分析鼻疽诺卡菌、星形诺卡菌、巴西诺卡菌、豚鼠诺卡菌基因组DNA片段、(2)采用盐酸甲醇法提取上述菌株脂肪酸，以气相色谱法检测，并以计算机分析其组成。结果：四种诺卡菌DNA片段图谱各异，虽然可见少数一致性片段，但总体差异明显；脂肪酸组成成份有一定相似性，但在饱和／不饱和脂肪酸比值、饱和脂肪酸棕榈酸(C16:0)／油酸(C18:1)比值、不饱和脂肪酸棕榈酸(C16:1)／硬脂酸(C18:0)比值及是否含有C15:0和C17:0脂肪酸方面存在较大差异。结论:采用RAPD分析及气相色谱法脂肪酸组成分析，发现四种诺卡菌间既有遗传共性又存在种间差异。     

Alterations in skin and stratified epithelia by constitutively activated PPARalpha

Peroxisome proliferator-activated receptor (PPAR)alpha is a pleiotropic regulator in many cell types and has recently been implicated in skin homeostasis. To determine the role of PPARalpha in skin physiology, transgenic mice were generated using the tetracycline Tet-off regulatory system to target constitutively activated PPARalpha to the epidermis and other stratified epithelia by the bovine keratin K5 promoter. Expression of the transgene during early development resulted in postnatal lethality within 2 days after birth. A thin epidermis, few hair follicles, and abnormal development of the tongue were observed in neonatal transgenic mice. Early mortality was not observed when transgenic PPARalpha expression was diminished by administration of doxycycline (dox) to the mothers. The alterations noted in neonatal mice were not observed in adult mice upon re-expression of the PPARalpha transgene by withdrawing dox. Attenuated hyperplasia of interfollicular epidermis after topical application of the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was observed in adult mice expressing the PPARalpha transgene. In addition, expression of the PPARalpha transgene in mammary gland during pregnancy resulted in abnormal development of this organ and impaired lactation. Further investigations using primary keratinocytes revealed that expression of the transgene in keratinocytes resulted in increased differentiation and decreased proliferation, which may contribute to the observed phenotype in the transgenic mice. Thus, these results indicate that PPARalpha plays an important role in the development of stratified epithelia including skin, tongue, and mammary gland.     

Keratin 15 expression in stratified epithelia: downregulation in activated keratinocytes

Keratin 15 (K15) is a type I keratin without a defined type II partner whose expression in epidermal diseases has not been investigated. In this study we have used LHK15, a monoclonal antibody raised against the last 17 amino acids of the K15 polypeptide, to show that K15 is expressed primarily in the basal keratinocytes of stratified tissues, including the fetal epidermis and fetal nail. Although K15 in normal hair follicles was virtually absent from hair bulbs, it was expressed by a subset of keratinocytes in the outer root sheath. By comparison, K14 expression was found throughout the outer root sheath of hair follicles; however, when both K14 alleles were naturally ablated, the expression of K15 was also observed throughout the outer root sheath of the follicles. Expression of K15 mRNA was assessed by in situ hybridization and corroborated the data from immunostaining. An increase in K15 mRNA and protein expression in hair follicles from the K14 ablated epidermis suggested an upregulation of the K15 gene in the absence of the K14 protein. In organotypical cultures where differentiating keratinocytes expressed markers of activated phenotype, i.e., K6 and K16, expression of K15 was undetectable. The expression of K15 mRNA and protein was also downregulated in two hyperproliferating situations, psoriasis and hypertrophic scars. Because keratinocytes in psoriasis and hypertrophic scars are activated, we conclude that K15 expression is not compatible with keratinocyte activation and the K15 gene is downregulated to maintain the activated phenotype.     

Variation in sebum fatty acid composition among adult humans

Quartz capillary gas chromatography was used to analyze the wax ester fatty acids of 4 sebum samples collected at 2-week intervals from each of 10 adult human subjects. Marked differences in wax ester fatty acid composition between subjects were apparent. The greatest variation was present in the even-carbon-numbered iso-branched acids, which ranged from 1-22% of the monounsaturated acids and from 1-13% of the saturated acids. The anteiso chain structures formed 3-7.5% of the unsaturated acids and 3-13.5% of the saturated acids. Fatty acids bearing one or more methyl branches at other positions in the chain made up 12-22% of the saturated acids, but were not present in the unsaturated fatty acid fraction. This and other features of the composition of the unsaturated fatty acids indicate that the delta 6-desaturase that produces the monounsaturated fatty acids of human sebum requires a substrate having a straight chain of at least 12 carbon atoms extending from the carboxyl group. The differences in fatty acid composition between subjects and the constancy of composition for each of the subjects over the 2-month period indicate that the synthesis of each of the types of chain structure is under genetic control.     

Localization of cathepsin H and its inhibitor in the skin and other stratified epithelia

Summary The rat-skin-derived cysteine proteinase, so-called BANA-hydrolase, which is capable of hydrolysing benzoylarginine naphthylamide and leucine naphthylamide was shown to be immunologically identical to cathepsin H purified from rat liver. The enzyme was immunocytochemically localized in the basal cell layer of rat epidermis. A natural inhibitor of cathepsin H with a molecular weight of about 13,000 was mainly localized in the keratinizing cell layers and showed only a weak reaction in the basal cells. Thus, cathepsin H appears to be a characteristic feature of the proliferating cell layer, whereas the cysteine-proteinase inhibitor is a characteristic feature of keratinizing cells.     

Expression pattern of REIC/Dkk-3 in mouse squamous epithelia

The REIC/Dkk (reduced expression in immortalized cells/Dickkopf-3) gene was originally identified as a tumour-suppressor gene with reduced expression in immortalized cells, cancer-cell lines and tumour tissues. Of the four members of the Dkk family, the REIC/Dkk-3 protein is unique in terms of DNA sequence, expression profiles and biological functions. In this study, we investigated and compared the expression patterns of the REIC/Dkk-3 protein in mouse squamous epithelia. Expression of REIC/Dkk-3 in the back skin was localized to the upper layer of the interfollicular epidermis, and the inner root sheath of hair follicles. Expression of REIC/Dkk-3 was detected in the ear skin, oral mucosa, tongue, oesophagus, uterine cervix, footpad and tail skin, but not in the cornea. Interestingly, expression was localized to the upper layers of these epithelial tissues. The physiological function of REIC/Dkk-3 is still unclear, but our detailed observation highlight its unique expression pattern in squamous epithelia.     

气相色谱法检测申克孢子丝菌细胞脂肪酸

目的：探讨不同临床表现型和不同颜色菌落的申克孢子丝菌细胞的脂肪酸组成成分之间的相关性。方法：采用盐酸甲醇法提取8株申克孢子丝菌细胞的脂肪酸，包括固定型5株，淋巴管型3株，其中菌落为白色5株，褐色3株，以气相色谱法检测，配以计算机分析各种菌株的饱和与不饱和脂肪酸含量、成分及主要饱和与不饱和脂肪酸比值。结果：申克孢子丝菌的饱和脂肪酸棕榈酸（C16．0）和不饱和脂肪酸油酸（C18：1）、亚油酸（C18：2）的含量较高，而不饱和脂肪酸棕榈油酸（C16：1）、亚麻酸（C18：3）、硬脂酸（C18：0）、扁油酸（廿二烷酸：C22：0）则含量很少，部分菌株含有少量豆蔻酸（十四烷酸：C14：0），但所有菌株皆不含花生酸（C22：0）。不同临床型和不同菌落颜色的申克孢子丝菌的饱和与不饱和脂肪酸比值无明显差异。结论：气相色谱法方法简便、快速，可用于孢子丝菌的脂肪酸检测，但不同临床型和不同颜色的菌株间脂肪酸组成和含量未见明显区别。     

Human essential fatty acid deficiency: treatment by topical application of linoleic acid.

An essential fatty acid (EFA) deficiency developed in a 19-year-old man who was being maintained on a long-term regimen of fat-free, intravenous hyperalimentation fluids. The EFA deficiency was reversed after 21 days by daily, topical application of linoleic acid to the patient's skin. The ratio of eicosatrienoic acid (20:3, n-9) to eicosatetraenoic acid (20:4, n-6) decreased to normal levels in the skin and serum with clinical improvement of the EFA deficiency syndrome. The cutaneous manifestations (scalp dermatitis, alopecia, and depigmentation of hair) were reversed with continued, topical application of safflower oil, which contains 60% to 70% linoleic acid.     

Postnatal lethality of P-cadherin/desmoglein 3 double knockout mice: demonstration of a cooperative effect of these cell adhesion molecules in tissue homeostasis of stratified squamous epithelia

To investigate the cooperativity of different cell adhesion molecules in maintaining the structural integrity of the epidermis, we have generated mice deficient for both a classical cadherin, P-cadherin, and a desmosomal cadherin, desmoglein 3. In epithelial cells, P-cadherin is localized to the adherens junction, whereas desmoglein 3 is found in desmosomes. Previous studies have shown that these two junctional complexes are important for keratinocyte cell-cell adhesion. Both P-cadherin and desmoglein 3 expression are restricted to the basal and most immediate suprabasal cells of the epidermis, whereas both proteins are found throughout the oral mucosal epithelium. Although P-cadherin mutant mice have no apparent defect in epithelial cell adhesion, the desmoglein 3 mutant phenotype resembles that of patients with the autoimmune disease pemphigus vulgaris, in that the mice develop spontaneous mucous membrane blisters and trauma-induced skin blisters. The oral lesions in DSG3-/- mice reduce their food intake, resulting in a runted phenotype; however, most animals recover and live past weaning age. In contrast, animals mutant for both P-cadherin and desmoglein 3 die before weaning. The majority of the double mutant animals die around 1 wk after birth, apparently due to malnutrition. These studies suggest that loss of P-cadherin leads to a more severe desmoglein 3 mutant phenotype in the double knockout mice. This is the first in vivo evidence of possible synergism between a classical and desmosomal cadherin.     

The requirement for perp in postnatal viability and epithelial integrity reflects an intrinsic role in stratified epithelia

Mice lacking the desmosome protein Perp exhibit blistering in their stratified epithelia and display postnatal lethality. However, it is unclear if these phenotypes are strictly related to Perp function in stratified epithelia, as Perp expression is not restricted to these tissues during embryogenesis, and certain desmosomal blistering diseases such as pemphigus vulgaris and pemphigus foliaceus have non-cell-intrinsic bases. Furthermore, we show here that Perp is expressed in the heart, raising the possibility that defects in heart function could account for lethality in the Perp-deficient mice. To determine conclusively if Perp function in stratified epithelia is crucial for postnatal survival and epithelial adhesion, we specifically ablated Perp in stratified epithelia by breeding conditional Perp knockout mice to keratin 5 (K5)-Cre transgenic mice. We found that the majority of mice lacking Perp in stratified epithelia die within 10 days after birth, accompanied by blistering and hyperproliferation in the epithelia, similar to the constitutive Perp null mice. Together, these findings indicate that Perp's requirement for both viability and epithelial integrity reflects a role in the stratified epithelial compartment.     

Reduced neutrophil LTB4 release in atopic dermatitis patients despite normal fatty acid composition.

Propositions about an abnormal fatty acid metabolism in atopic dermatitis patients prompted us to compare the phospholipid fatty acid composition and LTB4 release of neutrophils from 15 atopic dermatitis patients, as well as the adipose tissue triglyceride fatty acid composition, to that of 15 healthy controls matched by age, gender, and smoking habits. We found no differences in the tissue fatty acid composition between the two groups. The release of leukotriene B4 from Ca-ionophore-stimulated neutrophils in patients was on the average only 42% (p less than 0.001) of that measured in the control group, despite the very similar arachidonic acid contents of these cells. Our study does not support the assumption of an abnormal fatty acid desaturation in atopic dermatitis patients. Rather, the capacity to release and/or convert arachidonic acid into leukotrienes in neutrophils appears to be affected by this disease.     

Analysis of epidermal-type transglutaminase (transglutaminase 3) in human stratified epithelia and cultured keratinocytes using monoclonal antibodies

BACKGROUND: Epidermal-type transglutaminase (TGase 3) is involved in the cross-linking of structural proteins in the epidermis, which results in the formation of the cornified envelope. TGase 3 is activated by limited proteolysis of a 77 kDa zymogen during keratinocyte differentiation. OBJECTIVE: To characterize the expression of TGase 3 in human epidermis and cultured keratinocytes, we established specific monoclonal antibodies against the TGase 3. METHODS: Recombinant proteins for human TGase 3 produced in bacteria and baculovirus-infected insect cells were purified as an antigen. Hybridomas are established and used for characterization of expression in epidermis and keratinocytes. RESULTS: Four antibodies were generated against recombinant human TGase 3, which reacted with the 77 kDa zymogen and in some cases either the 47 or 30 kDa active proteolytic fragments. In human epidermis and cultured keratinocytes, only the zymogen form of TGase 3 was detected. Immunohistochemical analysis of the skin revealed that the enzyme is present in the cells of the granular and cornified layers consistent with its role in cornified envelope formation. In cultured keratinocytes, TGase 3 was expressed in differentiating cells coincident with profilaggrin and keratin 10 expressions. CONCLUSION: Using monoclonal antibody against human TGase 3, we showed the expression of TGase 3 in upper layers of epidermis. TGase 3 displayed a diffuse cytoplasmic distribution in vitro consistent with its proposed role in the early phase of cornified cell envelope assembly in the cytoplasm.     

Changes in proliferative activity as cells move along undulating basement membranes in stratified squamous epithelium

The filiform papillae of mouse tongue provide evidence for a flow of interphase basal cells along the basement membrane. This implies that they are aligned on that structure according to age. In addition there is a decrease in proliferative potential as the basal cells age. This is probably due at least in part to a fall in growth fraction. Such a method of basal cell replacement may exist in other stratified squamous epithelia characterized by undulating basement membranes.     

Apoptosis and cellular proliferation in human epidermal squamous cell neoplasia

We examined cell loss (apoptosis) and proliferation in a histopathological spectrum of epidermal squamous cell neoplasia, including 11 cases of solar keratosis (SK), 18 Bowen's diseases (BD) and 19 invasive squamous cell carcinomas (SCC). Apoptotic and proliferative cells were determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) and by the detection of nuclear antigen Ki-67, respectively. Few apoptotic cells were observed in normal epidermis, while TUNEL index (TI; percentage of TUNEL-positive cells) was highest for SCCs, followed by BDs and SKs, in the order given. Although the mean Ki-67 index did not differ between SCCs and BDs. both disease types showed a significantly higher index than the SKs. Of SCCs, both TI and Ki-67 index values were significantly higher in poorly than in well differentiated carcinomas. TI was significantly higher in SCCs without P53 immunohistochemical expression than in SCCs with P53 expression, while TI and Ki-67 indices did not correlate with P53 expression in the SKs and BDs. These results suggest that apoptosis reflects not only cell loss, but also proliferative activity in the epidermal neoplastic lesions.     

A morphometric study of alterations in rough endoplasmic reticulum during differentiation in stratified squamous epithelium

Summary Stereological techniques were applied to investigate several structural parameters characterising the rough endoplasmic reticulum (RER) during differentiation in hamster cheek-pouch epithelium. Mucosal samples from five Syrian golden hamsters were obtained and processed for electron microscopy. Following a strict sampling regime, micrographs were obtained from defined basal, spinous and granular layers, and subjected to stereological point and intersection counting procedures. This enabled volume and surface densities, and volume-to-surface ratios of RER to be determined for each cellular layer. From previous estimaes of the mean cytoplasmic volume of the average basal, spinous and granular cell in this tissue, it was possible to calculate the absolute volume and surface area of RER present in these average cells. Both volume and surface densities of RER decreased between basal and granular layers, whereas the total volume and surface area present in the average spinous and granular cell were both higher than in the average basal cell. These data suggest that RER is being synthesised during epithelial differentiation. In view of the role of the RER in the production of exportable proteins, it is possible that increased amounts of this organelle are required to synthesise the enzymes and glycoproteins found in membrane-coating granules, since these are also seen with increasing frequency in successively higher strata.