The morphology of the denuded epidermal basal cell layer of the hairless mouse after different preparation methods. A scanning and transmission electron microscopical study

The denuded basal cell layer of the hairless mouse epidermis is described in the present scanning (SEM) and transmission electron microscopical (TEM) study. The suprabasal layers were removed mechanically after trypsinization or by extracellular calcium depletion. Trypsinization before removal of the suprabasal cells caused the basal cells to shrink. Characteristic surface plication and hemi-desmosomal attachment to the basement membrane were generally preserved. SEM revealed partly maintained intercellular bridging, whereas by TEM such contacts were absent because half desmosomes were internalized. Total calcium depletion induced more serious damage to the basal cell surface, which was smooth with apparent perforations. However, cell bridges, and occasional desmosomes were present. The cell interior demonstrated important cellular injury. If the calcium deprived explants were allowed to recover in calcium-containing medium, the cells acquired an activated "regenerative" morphology, without junctions, similar to that observed in wound healing. Epidermal non-keratinocytes were seen only after trypsinization. Control experiments revealed that they adapted poorly to organ culture conditions. By TEM, we observed several interesting aspects of the differences, between dark and clear basal keratinocytes. This was unexpected because fixation studies had shown, that with the present fixation method, typical dark and clear cells do not occur in untreated epidermis. We believe that membrane injury through mechanical stripping of partly adhering epidermal layers induced "clear cells", whereby the neighboring cells appeared darker. This provides additional evidence as to the origin of the two sub-populations, dark and clear basal cells. The clear cells may be injured cells, caused by cell damage, and not by processes of cellular differentiation. The results of the present investigation supports the view that basal keratinocytes have a polygonal shape with numerous free surface extensions and they are anchored to the basement membrane with "foot pads". Our study also shows that SEM of the epidermal basal layer might be feasible. Various artifacts, however, must be considered, depending on the denudation method used. We prefer trypsinization to calcium depletion because it is less time-consuming and results in a cell morphology which in TEM is comparable to that of basal cells in untreated whole epidermis. Extra-cellular calcium depletion, however, might be useful as a method to prepare single cell suspensions for flow cytometry. Restoration of a normal calcium concentration after stripping, provides an opportunity to mimic wound healing in situ, as an alternative t     

Changes in cytokeratin expression in epidermal keratinocytes during wound healing

In order to investigate the re-epithelialization process during wound healing, the hair on the back of guinea pigs was shaved and then excisional wounds were made through the entire thickness of the skin. Histological changes were observed and changes in the expression of different cytokeratin polypeptides were examined using an immunohistochemical technique. Immunohisto chemical study revealed that the proliferating and migrating keratinocytes expressed the same cytokeratins as the basal cells of normal epidermis. In addition, the entire epidermis of fairly remote areas from the edges of the wound where no thickening was observed showed a temporarily abnormal staining pattern. The suprabasal cells in the regenerating epidermis temporarily expressed cytokeratins not only specific for suprabasal cells but also specific for basal cells. The cytokeratins expressed in normal basal keratinocytes were also present in the thickened granular layers. These data indicate that the expression of cytokeratins in the epidermal keratinocytes (even in fairly remote areas from the wound edges) changes during wound healing, that the origin of the migrating keratinocytes from the remaining epidermis seems to be the basal cells in the epidermis, and that the appearance of keratohyalin granules is not related to changes in cytokeratin expression.     

Impact of glutaraldehyde versus glutaraldehyde-formaldehyde fixative on cell organization in fish corneal epithelium

The purpose of this study was to examine the impact of a low osmolality glutaraldehyde fixative and a high osmolality glutaraldehyde-formaldehyde fixative on the structural organization of a tissue that could be exposed to low and high osmolality environments. The corneas of freshwater trout were prepared for transmission and scanning electron microscopy using either a fixative of 2% glutaraldehyde in 60 mM cacodylate buffer (pH 7.8, 260 mOsm/l) or a fixative prepared by adding 2.5% glutaraldehyde to a solution of 1% formaldehyde and buffering the solution with 0.1 M cacodylate (pH 7.6, 850 mOsm/l; Karnovsky-type fixative). The corneal epithelial cell layer thickness was greater after glutaraldehyde compared to glutaraldehyde-formaldehyde fixation (67 vs 55 mum), as was the thickness of the superficial cells (5.1 vs 3.4 mum) and basal cells (43 vs 38 mum). The intermediate (wing) cells of the epithelium were, however, less thick after glutaraldehyde fixation (15 vs 18 mum). The width of the squamous, intermediate and basal cells was greater following glutaraldehyde fixation with the effect being greatest in the superficial layers and insignificant at the level of the basal cells. The results show that chemical fixatives with extremes of osmolality cannot only produce different cell sizes in a tissue but also determine the overall organization of the cells in a positional-dependent fashion.     

Immunocytochemical and electrophoretic distribution of cytokeratins in the resting stage epidermis of the lizard Podarcis sicula

The distribution of three anti-cytokeratin (alpha-keratin) antibodies (AE1, AE2, AE3) in the epidermis of a lizard has been studied by immunocytochemistry at light and electron microscope and by immunoblot analysis. This study shows the expression of different keratins in the resting stage epidermis of the lizard Podarcis sicula. In this stage the epidermis has an external beta-layer, an underlying alpha-layer, some layers of living suprabasal cells and a basal stratum germinativum. The AE1 antibody is localized in the basal and suprabasal cells only in the outer scale surface, but is absent from the inner surface, the hinge region and from the keratinized beta- and alpha-layers. The AE2 antibody is mainly localized at the level of the hinge region and of the alpha-layer and gives a lower reaction in the beta-layer. The AE3 antibody is mainly localized in basal and suprabasal cells, lower in the alpha-layer, and absent from the beta-layer. The electron microscope shows that all the three antibodies immunolabel cytoplasmic fibrillar structures in the deep alpha-layers and that AE2 and AE3 antibodies label small electron-dense areas in the external dense beta-layer within the electron-lucid matrix. Immunoblot analysis of the keratins extracted and separated by gel electrophoresis demonstrates the presence of a band of high molecular weight (67-68 kDa) positive to all three antibodies. In addition AE1 antibody recognizes a 44-45 kDa band and a 57-58 kDa band, AE2 recognizes a 60-61 kDa band, and AE3 recognizes a 47 kDa and a 56-57 kDa band. The localization of the keratins identified by immunoblot analysis in the epithelial layers is discussed taking in account the immunolabeling at light and electron microscope. The present study suggests that also in the normal epidermis of this reptiles, in both the alpha- and the beta-layer, the molecular masses of keratins increase from the basal to the keratinized layers, a phenomenon which is generalized to adult and embryonic amniotes epidermis.     

Direct evidence for spatial and temporal regulation of transforming growth factor beta 1 expression during cutaneous wound healing

The expression of transforming growth factor (TGF beta 1) protein in human and porcine skin has been analyzed by immunohistochemistry with two polyclonal antibodies (anti-CC and anti-LC) following cutaneous injury. The anti-LC antibody binds intracellular TGF beta 1 constitutively expressed in the nonproliferating, differentiated suprabasal keratinocytes in the epidermis of normal human skin, while the anti-CC antibody does not react with the form of TGF beta 1 present in normal skin as previously shown. TGF beta 1 may play a role in wound healing as suggested by its effect on multiple cell types in vitro and its acceleration of wound repair in animals. We have evaluated the natural expression and localization of TGF beta 1 protein in situ during initiation, progression, and resolution of the wound healing response in two models of cutaneous injury: the human suction blister and the dermatome excision of partial thickness procine skin. Anti-CC reactive TGF beta 1 in the epidermis is rapidly induced within 5 minutes following injury and progresses outward from the site of injury. The induction reflects a structural or conformational change in TGF beta 1 protein and can be blocked by the protease inhibitor leupeptin or by EDTA, suggesting a change in TGF beta 1 activity. One day post-injury anti-CC reactive TGF beta 1 is present in all epidermal keratinocytes adjacent to the wound including the basal cells. This corresponds temporally to the transient block of the basal keratinocyte mitotic burst following epithelial injury. Three to 4 days post-injury anti-CC reactive TGF beta 1 is localized around the suprabasal keratinocytes, in blood vessels, and in the papillary dermis in cellular infiltrates. The exclusion of TGF beta 1 from the rapidly proliferating basal cells and its extracellular association with suprabasal keratinocytes may represent physiological compartmentation of TGF beta 1 activity. Anti-CC staining is strong in the leading edge of the migrating epithelial sheet. The constitutive anti-LC reactivity with suprabasal keratinocytes seen in normal epidermis is neither relocalized nor abolished adjacent to the injury, but anti-LC staining is absent in the keratinocytes migrating within the wound. As the wound healing response resolves and the skin returns to normal, anti-CC reactive TGF beta 1 disappears while constitutive anti-LC reactive TGF beta 1 persists. Thus, changes in the structure or conformation of TGF beta 1, its localization, and perhaps its activity vary in a spatial and temporal manner following cutaneous injury and correlate with physiological changes during wound healing.     

The shift from aquatic to terrestrial phenotype in Lissotriton italicus: larval and adult remodelling of the skin

Morphology and ultrastructure of the skin of Lissotriton italicus (previously named Triturus italicus) have been described in different phases of its biological cycle: larval stage, metamorphic stage and adult stage with emphasis on modifications occurring between aquatic and terrestrial adults. In the present study, light microscopy and both scanning and transmission electron microscopy were employed to analyze the histological and cytological remodelling that occurs in the skin of L. italicus during metamorphosis. The ultrastructure of the larval epidermis is arranged into three principal layers comprising an external layer of pavement cells, a basal layer and 1-3 intermediate layers consisting of Leydig cells along with accessory cells and mitochondria-rich cells. By the onset of metamorphosis, morphological changes of the skin include stratification and flattening of epidermal layers and disappearance of typical larval cells. In both aquatic and terrestrial adult phases the thin, cornified epidermis shows the same general arrangement as found in other vertebrates with an external stratum corneum and a variable number of intermediate cell layers. During the terrestrial adult phase, the skin is characterized by the presence of numerous tubercles; moreover, the lower epithelium is thicker than in the aquatic phase. Ultrastructural analysis revealed no substantial differences in the cellular composition of the skin between aquatic and terrestrial phases.     

Proliferation in murine epidermis after minor mechanical stimulation. Part 1. Sustained increase in keratinocyte production and migration

It was our objective to obtain an insight into the details and dynamics of the cell proliferative changes following minor barrier disruption, the mechanisms of recovery, and their regulation. Hair of the dorsal area of DBA2-mice was removed and the epidermis was tape stripped. Tritiated thymidine was injected into groups of mice at daily intervals thereafter. Labelling and nuclear densities were measured at several time intervals later in the various epidermal strata to characterize cell production and cell fluxes through the tissue. A dramatic proliferative response was observed at 24 h when the labelling density increased more than sixfold in the basal layer. Labelled cells rapidly appeared in suprabasal layers within a few hours in large quantities while this process took over 2 days in normal skin. Some cycling cells were also found in the suprabasal layer (pulse labelling at 24 h) in contrast with the controls. The cellular flux through the suprabasal layers was drastically (20-fold) increased and the transit time was shortened. Although the nuclear density in the basal layer showed only moderate changes it increased four-fold in the suprabasal layer within 5 days. A kinetic model analysis suggested that the cell cycle time of proliferative cells dropped from a normal value of about 200 h to less than 12 h post tape strip. After 7 days, the proliferative activation still persisted, even though at 3 days post tape strip the stratum corneum had been re-established. Hence, a mild mechanical alteration with removal of some parts of the cornified layer in mouse backskin epidermis triggers a huge proliferative response with massive overproduction of cells that lasts at least 7 days. Our findings suggest that the re-establishment of the cornified layer does not immediately shut down cell proliferation and that more complex, slower (long-term) regulatory processes are involved.     

Proliferation of human embryonic and fetal epidermal cells in organ culture

The morphology of human embryonic and fetal skin growth in organ culture at the air-medium interface was examined, and the labeling indices of the epidermal cells in such cultures were determined. The two-layered epidermis of embryonic specimens increased to five or six cell layers after 21 days in culture, and the periderm in such cultures changed from a flat cell type to one with many blebs. The organelles in the epidermal cells remained unchanged. Fetal epidermis, however, differentiated when grown in this organ culture system from three layers (basal, intermediate, and periderm) to an adult-type epidermis with basal, spinous, granular, and cornified cell layers. Keratohyalin granules, lamellar granules, and bundles of keratin filaments, organelles associated with epidermal cell differentiation, were observed in the suprabasal cells of such cultures. The periderm in these fetal cultures formed blebs early but was sloughed with the stratum corneum in older cultures. The rate of differentiation of the fetal epidermis in organ culture was related to the initial age of the specimen cultured, with the older specimens differentiating at a faster rate than the younger specimens. Labeling indices (LIs) of embryonic and fetal epidermis and periderm were determined. The LI for embryonic basal cells was 8.5% and for periderm was 8%. The fetal LIs were 7% for basal cells, 1% for intermediate cells, and 3% for periderm. The ability to maintain viable pieces of skin in organ culture affords a model for studying normal and abnormal human epidermal differentiation from fetal biopsies and for investigating proliferative diseases.     

Differential expression of CD44s and CD4v10 proteins and syndecan in normal and irradiated mouse epidermis

 The role of the CD44s adhesion molecule, its epithelial isoforms and its relationship to epidermal proteoglycans such as syndecan was studied in normal and irradiated mouse skin. In normal mouse skin, only 10% of basal cells are strongly CD44s-immunopositive, with a cytoplasmic expression pattern. Double-label experiments with the basal cell marker keratin 14 confirmed the epithelial nature of the strongly CD44s-positive cell type in the basal layer. Some spinous keratinocytes and the majority of the remaining basal cells exhibited a weak membranous staining pattern. In contrast, the epithelial isoform, CD44v10, was strongly present in all basal and suprabasal epithelial cells of the epidermis, with a membranous staining pattern. Syndecan was found in the granular layer of the normal epidermis only. After 1 week of daily irradiation, the entire basal cell layer of the epidermis expressed CD44s in the membrane, but with a varying degree of staining intensity. This reactivity spread to the upper spinous layer after 3 weeks of treatment. In hyperproliferative epidermis, there was no difference in the staining patterns between CD44s and CD44v10. The expression of syndecan switched from the granular layer to the basal and lower spinous layers after 2 weeks of daily irradiation. Immunoreactivity for syndecan was also strongly enhanced in the dermis of irradiated samples. The results suggest an important role for syndecan and CD44 in proliferative processes during radiation-induced accelerated repopulation. Accepted: 30 September 1996     

Osmolarity of osmium tetroxide and glutaraldehyde fixatives

Synopsis The evidence available to date for the importance of fixative osmolarity is considered together with some observations on the volume changes of crab axons after fixation by osmium tetroxide and glutaraldehyde. The results obtained are compared with those obtained from crab axons and from amphioxus skin cells which had been processed and examined with the electron microscope after initial fixation in fixatives of different composition. It is concluded that the osmolarity of the fixative vehicle is of considerable importance when the fixing agent is glutaraldehyde but is of less importance when the fixing agent is osmium tetroxide or a mixture of the two agents.Preliminary observations upon crab axons fixed with glutaraldehyde in a vehicle approximating to the internal composition of the cells suggest that this approach to the design of fixative vehicles may be useful.     

Transforming growth factor-beta 1 localization in normal and psoriatic epidermal keratinocytes in situ.

Transforming growth factor-beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGF beta 1-specific polyclonal antibodies (anti-LC and anti-CC) to determine the presence of TGF beta 1 peptide in keratinocytes in sections of normal human skin in situ and in both plaque and nonplaque skin from individuals with psoriasis. In contrast to the differentiation phenotype expressed by keratinocytes in normal epidermis, keratinocytes in the psoriatic plaque exhibit a hyperproliferative/regenerative differentiation phenotype. Anti-TGF beta 1 staining was observed primarily in the epidermis. Anti-LC TGF beta 1 antibody stained nonproliferating, differentiated suprabasal keratinocytes intracellularly in normal skin but did not stain psoriatic plaques from five of seven patients. In contrast, anti-CC TGF beta 1 antibody stained suprabasal keratinocytes extracellularly in psoriatic plaques, but did not stain normal skin. Both anti-LC and anti-CC stained suprabasal keratinocytes intracellularly in nonplaque psoriatic skin. Thus, the conformation or structure of TGF beta 1 and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGF beta 1 is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGF beta 1 with nonproliferating keratinocytes in the suprabasal layers of the epidermis and its exclusion from the proliferating keratinocytes in the basal layer suggest that it may be a physiological regulator of keratinocyte proliferation. In addition, the intracellular localization of TGF beta 1 peptide in both normal and psoriatic keratinocytes suggests that it is constitutively synthesized by epidermal keratinocytes in vivo.     

Correlation between tissue growth kinetics and modulation of mouse skin tumorigenesis by phorbol esters.

Previous studies on the influence of phorbol esters on mouse skin tumorigenesis have shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances development of malignant epithelial and mesenchymal skin tumors by a completely carcinogenic dose of 3-methylcholanthrene (MCA), while its congener phorbol-12, 13-diacetate (PDA) exerts an inhibitory effect. Differential effects of these two agents were analysed by histology, morphometry and cell kinetic techniques including autoradiography and estimation of labelled precursor incorporation into DNA by liquid scintillation counting. Epidermal hyperplasia induced on exposure of S/RV Cri mouse skin to a single or multiple TPA application after MCA injection was associated with a significant increase in the thickness of nucleated cell layers, stratum granulosum, number of suprabasal cells and dark basal cells. Enhancing effect of TPA on MCA-induced neoplastic development correlated well with an increase in mitotic activity, number of cells in S-phase and increased rate of DNA synthesis in the epidermis, dermis and subcutis as also mast cell number. In contrast, treatment of MCA-injected preneoplastic mouse skin with PDA resulted in epidermal hypoplasia and cellular damage evident as cytoplasmic vacuolation and nuclear pyknosis. Multiple PDA exposure also reduced the thickness, mitotic index and number of cells in S-phase in epidermis, dermis and subcutis. Thus, cellular toxicity and inability to recruit cells in DNA-synthetic phase may account for inhibition of progression of preneoplastic epithelial and mesenchymal cells into overt tumors by PDA.     

The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes. A morphometric study of so-called "dark cells" and their putative role in epidermal carcinogenesis

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered "clear cells" dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Co-localization of COX-2, CYP4F8, and mPGES-1 in epidermis with prominent expression of CYP4F8 mRNA in psoriatic lesions

Cyclooxygenase-2 (COX-2), cytochrome P450 4F8 (CYP4F8), and microsomal PGE synthase-1 (mPGES-1) form PGE and 19-hydroxy-PGE in human seminal vesicles. We have examined COX-2, CYP4F8, and mPGES-1 in normal skin and in psoriasis. All three enzymes were detected in epidermis by immunofluorescence and co-localized in the suprabasal cell layers. In lesional psoriasis the enzymes were also co-localized in the basal cell layers. Real-time RT-PCR analysis suggested that CYP4F8 mRNA was induced 15-fold in lesional compared to non-lesional epidermis. mRNA of all enzymes were present in cultured HEK and HaCaT cells, but the prominent induction of CYP4F8 mRNA in psoriasis could not be mimicked by treatment of these keratinocytes with a mixture of inflammatory cytokines or with phorbol 12-myristate-13-acetate. The function of CYP4F8 in epidermis might be related to lipid oxidation and keratinocyte proliferation.     

The bovine desmocollin family: a new gene and expression patterns reflecting epithelial cell proliferation and differentiation

We have discovered a third bovine desmocollin gene, DSC3, and studied expression of all three desmocollin genes, DSC1, 2, and 3, by Northern blotting, RT-PCR and in situ hybridization. DSC1 is strongly expressed in epidermis and tongue papillae, showing a "skin"-type pattern resembling that previously described for keratins 1 and 10. Expression is absent from the epidermal basal layer but appears in the immediate suprabasal layers and continues uniformly to the lower granular layer. In tongue epithelium, expression is suprabasal and strictly localized to papillae, being absent from interpapillary regions. In other epithelial low level DSC1 expression is detectable only by RT-PCR. The distribution of Dsc1 glycoproteins, detected by an isoform-specific monoclonal antibody, closely reflects mRNA distribution in epidermis and tongue. DSC2 is ubiquitously expressed in epithelia and cardiac muscle. In stratified epithelia, expression appears immediately suprabasal, continuing weakly to the lower granular layer in epidermis and to just above half epithelial thickness in interpapillary tongue, oesophageal, and rumenal epithelia. DSC3 expression is restricted to the basal and immediately suprabasal layers in stratified epithelia. In deep rete ridges DSC expression strikingly resembles the distribution of stem, transit-amplifying, and terminally differentiating cells described by others. DSC3 expression is strongly basal, DSC2 is strong in 5-10 suprabasal layers, and then weakens to be superseded by strong DSC1. These results suggest that desmocollin isoform expression has important functional consequences in epithelial proliferation, stratification, and differentiation. The data also provide a standard for nomenclature of the desmocollins.     

Immunohistochemical analysis of DNA mismatch repair enzyme hMSH-2 in normal human skin and basal cell carcinomas

We have analysed the expression and distribution of the DNA mismatch repair enzyme hMSH-2 in normal skin and basal cell carcinomas. hMSH-2 protein was investigated immunohistochemically (normal human skin: n=10; basal cell carcinomas: n=16) on frozen sections using a highly sensitive streptavidin–peroxidase technique and a specific mouse monoclonal antibody (clone FE11). In normal human skin, we found nuclear immunoreactivity for hMSH-2 in epidermal keratinocytes of the basal and first 1–3 suprabasal cell layers. All basal cell carcinomas analysed revealed strong nuclear imunoreactivity that was pronounced in peripheral tumour cells and cells of the palisade. Expression of hMSH-2 protein was consistently and strongly upregulated in tumour cells of the carcinomas as compared to adjacent unaffected epidermis or epidermis of normal human skin. Twelve of the sixteen carcinomas analysed revealed no visual correlation in comparing the labelling patterns for hMSH-2 with the labelling pattern for the proliferation marker Ki-67. Our findings indicate that (a) hMSH-2 is expressed in human epidermal keratinocytes, predominantly in lower cell layers of the viable epidermis; (b) expression of hMSH-2 protein is strongly upregulated in basal cell carcinomas as compared to unaffected epidermis; (c) the level of hMSH-2 proteins in the carcinomas is not exclusively regulated by the proliferative activity of these tumour cells; (d) inactivating mutations of the hMSH-2 gene may in the carcinomas not be involved in the carcinogenesis or microsatellite instability secondary to replication errors; (e) expression of hMSH-2 may be of importance for the genetic stability of basal cell carcinomas in vivo.     

松江鲈鱼皮肤的显微和亚显微结构

采用光学显微镜、扫描电镜和透射电镜,对松江鲈鱼(Trachidermus fasciatus)成体皮肤的显微和亚显微结构进行了观察。结果表明,松江鲈鱼体表不同部位皮肤的厚薄不一,但基本结构相似。皮肤由表皮和真皮层构成。松江鲈鱼的皮肤裸露无鳞,表皮层较薄,由约4～8层细胞构成,主要由复层上皮细胞和黏液细胞及基底细胞组成。表层细胞呈扁平、多边形,细胞之间主要靠桥粒紧密连接,连接处形成增厚的边缘嵴状突起。表皮细胞游离面向内凹陷,表面形成指纹状微嵴。黏液细胞呈圆形或卵圆形,散布在上皮细胞之间。黏液细胞内的黏原颗粒具有椭圆颗粒状、均匀致密的块状和疏松丝状3种不同形态。真皮通过基膜与表皮相连,由稀疏层和致密层构成。真皮结缔组织在腹部较厚而在其他部位较薄。表皮与真皮连接处有色素层,头部、背部、尾柄和体侧皮肤色素细胞分布多,色素层明显,而腹部和颏部皮肤缺少色素。松江鲈鱼黄河群体真皮层中有角质棘状突起,而滦河群体则无。头部、体侧和尾柄处皮肤上还分布有侧线孔和表面神经丘等感觉器官。     

Immunolocalization of the Tumor-Sensitive Calmodulin-Like Protein CALML3 in Normal Human Skin and Hyperproliferative Skin Disorders

Background and Objective

Calmodulin-like protein CALML3 is an epithelial-specific protein regulated during keratinocyte differentiation in vitro. CALML3 expression is downregulated in breast cancers and transformed cell lines making it an attractive marker for tumor formation. The objective of this study was to survey CALML3 localization in normal epidermis and in hyperproliferative skin diseases including actinic keratosis, squamous and basal cell carcinoma as well as verruca and psoriasis and to compare CALML3 immunoreactivity with the proliferation marker Ki-67.

Methods

Paraffin-embedded tissue sections from normal human skin and hyperproliferative skin disorders were examined by immunohistochemistry and analyzed for localization and expression of CALML3 and Ki-67.

Results

CALML3 was strongly expressed in differentiating layers of normal skin, staining the periphery in suprabasal cells and exhibiting nuclear localization in the stratum granulosum. CALML3 nuclear localization was inversely correlated to Ki-67 staining in each disease, indicating that CALML3 nuclear presence is related to terminal cell differentiation and postmitotic state.

Conclusions

Increased CALML3 expression in suprabasal layers is characteristic for differentiating keratinocytes in normal epidermis, and nuclear expression of CALML3 inversely correlates with expression of the proliferation marker Ki-67. This suggests that CALML3 is a useful marker for normal and benign hyperplastic epidermal development, whereas the loss of nuclear CALML3 indicates progression to a proliferative and potentially malignant phenotype.