Basket-weave structure in the stratum corneum is an important factor for maintaining the physiological properties of human skin as studied using reconstructed human epidermis and tape stripping of human cheek skin.

BACKGROUND: The normal stratum corneum (SC) has an upper basket-weave (BW) pattern layer and a lower compact layer. The transition from compact to BW SC is well associated with a transition from diffuse to peripheral distributions of corneodesmosomes (CDs). The loss of transition from compact SC to BW SC appears to cause structural and barrier-function impairments. OBJECTIVES: To show the involvement of the BW SC in maintaining the physiological properties of the skin. METHODS: Reconstructed human epidermis (RHE) with a complete BW structure was created by treatment with prepared emulsion-A, an oil-in-water emulsion. The RHE tissues were subjected to histological analysis, and the distribution of CDs on the SC with or without BW SC was analysed by anti-desmoglein (Dsg)1 antibody immunofluorescence and ultrastructural and Western blotting analyses. Ultrastructural analysis of intercellular lipids was performed. The mechanical properties of the RHE were evaluated. RESULTS: Emulsion-A successfully generated the BW SC in the RHE in which the degradation of CDs was promoted. The intercellular space of the BW SC generated by emulsion-A was filled with multilamellar lipid sheets. The softness of the SC with a BW structure formed with emulsion-A was higher than that of the compact SC in RHE. The outermost SC Dsg1 degradation (formation of the BW SC as determined with Dsg1 pixels) was correlated with water-barrier functions and the SC softness of healthy human cheek, which varied widely. CONCLUSIONS: Emulsion-A successfully generated the BW SC in RHE for the first time. This method is suggested to be a useful tool for investigating the physiological significance of the BW SC in vitro. Determination of Dsg1 content in the SC obtained by tape stripping from human skin allows study of the effects of external stimulants, such as creams and ointments, including cosmetics, on the completeness of the BW SC in situ without biopsy. What's already known about this topic? The normal stratum corneum (SC) has two layers, an upper basket-weave (BW) pattern layer and a lower compact layer. Epidermal diseases such as ichthyosis vulgaris and X-linked ichthyosis have an incomplete or no BW SC and impaired SC barrier functions, in which corneodesmosome (CD) degradation in a peripheral distribution is impaired. The roles of the BW SC in the physiological properties of human skin have not been clearly elucidated. What does this study add? Reconstructed human epidermis (RHE) with a complete BW structure was generated for the first time by treatment with oil-in-water emulsion-A. The formation of the BW SC was associated with a decrease in Dsg1 content, which represents the CD number in the SC. The intercellular space of the BW SC generated by emulsion-A, but not compact SC, was filled with multilamellar lipid sheets. The softness of the SC with a BW structure formed by emulsion-A treatment was higher than that of the compact SC in RHE. What is the translational message? RHE with a complete BW SC generated by emulsion-A treatment is suggested to be a useful tool for investigating effects on the physiological functions of the BW SC, as in treatments with creams and ointments including cosmetics. Determination of desmoglein 1 content in the SC obtained by tape stripping from human skin can make it possible to study the effects of external stimulants, such as creams and ointments, including cosmetics, on the completeness of the BW SC in situ without biopsy.

The efficacy of adjunct tacrolimus treatment in pregnancy outcomes in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) involves multiple organ systems and primarily affects women during their reproductive years. Pregnancy in a woman with SLE may lead to higher rates of disease flares. Little is known regarding which medications are safe to maintain remission and/or treat flares throughout such pregnancies. Here we retrospectively analyzed the efficacy of tacrolimus (TAC) in the pregnancy outcomes of SLE patients. We studied the 54 deliveries of 40 SLE patients over an eight-year period from 2008 to 2016. We used analyses of covariance with adjustments for the propensity score and inverse probability of treatment weights to compare the patient backgrounds between the TAC users and non-TAC users. TAC was administered to the patient in 15 of the 54 (27.8%) pregnancies, and these patients had a significantly higher dose of prednisolone, hypocomplementemia, lower estimated glomerular filtration rate, past history of lupus nephritis, and complication with antiphospholipid syndrome. In the adjusted background of the TAC deliveries, the risks of decreased fetal body weight, low birth weight infant, non-reassuring fetal status (NRFS), and preterm birth were not increased compared to the non-TAC deliveries. Thrombocytopenia and hypertension during the pregnancy were extracted as independent predictive risk factors for decreased fetal body weight and NRFS, respectively. We had anticipated that the maternal and fetal outcomes in the TAC-use deliveries would be poor before the analysis; however, the TAC-use group showed no significant difference in risks contributing to outcomes compared to the non-TAC group, suggesting that adjunct TAC treatment corrected various risk factors during the lupus pregnancies.

Binding of pemphigus vulgaris IgG to antigens in desmosome core domains excludes immune complexes rather than directly splitting desmosomes.

BACKGROUND: Pemphigus vulgaris (PV) is characterized by autoantibodies against desmoglein (Dsg) 3 or both Dsg1 and Dsg3, i.e. desmosomal adhesion molecules. OBJECTIVES: We examined whether or not PV IgG binding to Dsg3 directly impairs the adhesion of desmosomes. METHODS: For immunofluorescence microscopy, keratinocytes were first incubated with PV IgG for 30 min in low Ca(2+) medium, in which no desmosomes were formed, and then for 1 h in high Ca(2+) medium to generate desmosomes. For immunoelectron microscopy, after a 30-min incubation with PV IgG in low Ca(2+) medium, cells were incubated with antihuman IgG with 5-nm gold particles for 5 min; after washing, cells were further incubated in high Ca(2+) medium for 1 h. For tracing of PV IgG/Dsg3 immune complexes formed in the desmosomal core domain, cells were first incubated with PV IgG for 5 min to allow PV IgG to bind the desmosomal core domain and were further incubated with PV IgG-free medium for different times. RESULTS: Immunofluorescence microscopy revealed that PV IgG bound in a random-punctate pattern on the cell surface in low Ca(2+) medium was translocated to the cell-cell contacts forming a dotted-linear distribution, suggesting desmosome generation even in the presence of PV IgG. Immunoelectron microscopy revealed that half-desmosome-like structures decorated with gold particles in low Ca(2+) keratinocytes coupled to form desmosomes and gold particles were sandwiched in the desmosomal core domain after Ca(2+) switch, even though their surfaces were covered with PV IgG/antihuman IgG 5-nm gold particles. In the tracing experiments, although PV IgG demonstrated a dotted-linear distribution along the cell-cell contacts colocalized with desmoplakin (DPK) after a 30-min tracing, it disappeared from cell-cell contacts after a 5-h tracing, leaving DPK and desmocollin 3. CONCLUSIONS: These results suggest that the PV IgG/Dsg3 immune complexes are excluded from the desmosomal core domain rather than directly splitting the desmosome.

Tetrahymena strives to maintain the fluidity interrelationships of all its membranes constant. Electron microscope evidence.

When cells of Tetrahymena pyriformis, strain NT-1, were chilled from their growth temperature of 39.5 degrees C to lower temperatures, the plasma membrane, outer alveolar, nuclear, outer mitochondrial, food vacuolar, and endoplasmic reticulum membranes each responded in a fashion quite characteristic of the membrane type. In most cases a distinctive rearrangement of intramembrane particles, as discerned by freeze-fracture electron microscopy, began abruptly at a definitive temperature. By comparing the freeze-fracture patterns of membranes in cells grown at 39.5, 27, and 15 degrees C, it was shown that the initial particle rearrangement in a given membrane always occurred at a fixed number of degrees below the growth temperature of the cell. Gradual chilling of a cell grown at constant temperature induced these membrane changes first in the outer alveolar membrane, then, in order of decreasing response to temperature, in the endoplasmic reticulum, outer mitochondrial membrane, nuclear envelope, and vacuolar membrane. The normally stable relationships between the physical properties of the several membrane types could in some cases be reversed, but only temporarily, by fatty acid supplementation or during the initial phases of acclimation to growth at a different temperature. The system provides a unique opportunity to study the effects of environmental change upon the physical properties of several functionally distinct but metabolically interrelated membranes within a single cell.

Differentiation of food vacuolar membranes during endocytosis in Tetrahymena.

The origin and differentiation of Tetrahymena pyriformis food vacuolar membranes has been studied by freeze-fracture electron microscopy. By measuring the temperature needed to induce the onset of lipid phase separation (as inferred by the appearance of particle-free regions in replicas) and calculating the changes in average intramembrane particle distribution, a distinct modification of the vacuolar membrane could be observed from the time of its formation from disk-shaped vesicles to its maturation before egestion of its indigestible contents. Whereas the nascent vacuolar membrane first showed signs of phase separation at 9 degrees C, this temperature rose to 14 degrees C in the completed vacuole and then, after lysosomal fusion, eventually declined to 12 degrees C. The average membrane particle density on the PF face increased from 761 +/- 219 to 1,625 +/- 350 per micron 2 during membrane differentiation. Like other membranes of the cell, the vacuolar membrane underwent adaptive changes in its physical properties in cells maintained for several hours at low temperature. This exposure to low temperature caused an equal effect in vacuoles formed before, during, or after the temperature shift-down. Normal changes in the properties of the vacuolar membrane may have some bearing on its programmed sequence of fusion reactions.

Methylation-induced silencing of ASC and the effect of expressed ASC on p53-mediated chemosensitivity in colorectal cancer.

Tumor suppressor p53 is known to play a crucial role in chemosensitivity in colorectal cancer. We previously demonstrated that an apoptosis-associated speck-like protein, ASC, is a p53-target gene which regulates p53-Bax mitochondrial apoptotic pathway. ASC is also known to be a target of methylation-induced gene silencing. An inactivation of ASC might thus cause resistance to chemotherapy, and if this is the case, then the expression of ASC would restore the chemosensitivity. The aim of this study was to clarify this hypothesis. ASC was methylated in 25% of all resected specimens in patients with colorectal cancer; however, ASC methylation did not always correspond to a lack of ASC protein. When expressed in colon cancer cells, in which ASC is absent due to methylation, ASC was found to enhance the chemosensitivity in a p53-dependent manner. In p53-null cells, ASC increased the p53-mediated cell death induced by p53-expressing adenovirus infection. Our data suggest that the methylation-induced silencing of ASC might cause resistance to p53-mediated chemosensitivity in colorectal cancer. The gene introduction of ASC may thus restore such chemosensitivity, and this modality may therefore be a useful new treatment strategy for colorectal cancer.

Activation of the hypoxia-inducible factor pathway induced by prolyl hydroxylase domain 2 deficiency enhances the effect of running training in mice.

AIMS: Hypoxic response mediated by hypoxia-inducible factor (HIF) seems to contribute to the benefit of endurance training. To verify the direct contribution of HIF activation to running training without exposure to atmospheric hypoxia, we used prolyl hydroxylase domain 2 (PHD2) conditional knockout mice (cKO), which exhibit HIF activation independent of oxygen concentration, and we examined their maximal exercise capacity before and after 4 weeks of treadmill exercise training. METHODS: Phd2f/f mice (n = 26) and Phd2 cKO mice (n = 24) were randomly divided into two groups, trained and untrained, and were subjected to maximal running test before and after a 4-week treadmill-training regimen. RESULTS: Prolyl hydroxylase domain 2 deficiency resulted in HIF-α protein accumulation. Phd2 cKO mice exhibited marked increases in haematocrit values and haemoglobin concentrations, as well as an increase in the capillary number in the skeletal muscle. The 4-week training elicited an increase in the capillary-to-fibre (C/F) ratio and succinyl dehydrogenase activity of the skeletal muscle. Importantly, trained Phd2 cKO mice showed a significantly greater improvement in running time than trained control mice (P < 0.05). Collectively, these data suggest that the combination of training and the activation of the HIF pathway are important for maximizing the effect of running training. CONCLUSION: We conclude that the activation of the HIF pathway induced by PHD2 deficiency enhances the effect of running training.

Elevation of both cyclooxygenase-2 and prostaglandin E2 receptor EP3 expressions in rat placenta after uterine artery ischemia-reperfusion.

Intrauterine growth restriction (IUGR) has a multifactorial pathogenesis and is an important cause of perinatal mortality. The relationship between fetal weight and placental blood flow in an animal model of IUGR has been investigated, showing that fetal growth is regulated by placental blood flow. The aim of the present study was to determine whether ischemia-reperfusion (I/R) injury stimulates the prostaglandin E2 (PGE2) system or the vascular endothelial growth factor (VEGF) system in the placenta of a rat IUGR model. COX-2 is reported to be involved in ischemic damage in many organs. There are 4 types of PGE2 receptor (EP1, EP2, EP3 and EP4). It is well known that EP1 and EP3 is associated with vasoconstriction. In the present study, vessels were occluded in the right uterine horn on day 17 of pregnancy in rats, and the clamps were removed after 30 min of ischemia. At 24h, 48 h, and 5 days after I/R injury, the live fetuses and placentas were obtained by cesarean section. This study revealed that I/R injury caused IUGR 5 days after the treatment. COX-2 expression and EP3 receptor expression were significantly elevated at 24h after I/R injury, but VEGF mRNA expression was not altered in the placenta from the ischemic horn compared with the non-ischemic horn. These results suggested that induction of the COX-2-EP3 system in the placenta may be one of the causes of IUGR induced by uterine ischemia, because the EP3 receptor and PGE2 are well known to mediate vasoconstriction in many organs.

Hypoxia accelerates cancer invasion of hepatoma cells by upregulating MMP expression in an HIF-1alpha-independent manner.

Tumor hypoxia has been reported to induce tumor progression in several carcinomas. Current studies have shown that hypoxia inducible factor-1alpha (HIF-1alpha) is stabilized under hypoxic conditions and transactivates various genes related to cancer aggressiveness. In the present study, we examined whether hypoxia affects cancer invasion in hepatocellular carcinoma. We aimed to solve the molecular mechanism of tumor invasion under the hypoxic condition. We showed that tumor hypoxia accelerated cancer invasion in two hepatoma cell lines. Using Western blot and RT-PCR analyses we demonstrated striking evidence that the expression of HIF-1alpha, ETS-1, MMP-7 and MT1-MMP was strongly upregulated by hypoxic stimulation. To examine whether these invasion-related genes are regulated by HIF-1alpha, we treated hepatoma cells with TX-402, which was reported to repress HIF-1alpha expression. HIF-1alpha expression was strongly repressed by the TX-402 treatment. In contrast, the expression of ETS-1, MMP-7 and MT1-MMP mRNA was not affected by TX-402 treatment. We further established stable transfectants in which HIF-1alpha dominant negative vector was introduced into Hep3B cells (pHIF-1alphaDN). In the pHIF-1alphaDN cells, the expression of ETS-1, MMP-7 and MT1-MMP was not repressed. Moreover, the invasion activity of pHIF-1alphaDN was not altered, compared with that of the mock. In hepatoma cells, we provided evidence that hypoxic stress accelerates cancer invasion by upregulating ETS-1 and the MMP family by an HIF-1alpha-independent pathway.

Biphasic effects of 12-O-tetradecanoylphorbol-13-acetate on the cell morphology of low calcium-grown human epidermal carcinoma cells: involvement of translocation and down regulation of protein kinase C.

The present study was performed to investigate involvement of protein kinase C in the biphasic effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell morphology in low calcium (0.07 mM)-grown cells of a human epidermal squamous cell carcinoma cell line. The low calcium-grown cells formed no desmosomal cell-cell contact and showed roughly circular arrangements of keratin intermediate filaments around the nucleus. Treatment with 10 ng/ml of TPA induced a rapid formation (within 15 min) of cell-cell contact and reorganization of keratin intermediate filaments from a circular organization to a radial arrangement in these low calcium-grown cells. These structural phenomena were associated with a transient increase in membrane-bound protein kinase C activity. However, the prolonged treatment longer than 24 h led to a prominent decrease in the number of cell-cell contacts, that had been once formed, and caused fibroblastic changes of cell morphology in association with a decrease in the membrane-bound protein kinase C activity. Addition of 20 microM 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a potential inhibitor of protein kinase C, to the medium with TPA blocked the formation of cell-cell contact. Addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride alone to normal calcium-grown cell cultures exhibiting cell-cell contact resulted in a decrease in the number of cell-cell contacts and in the fibroblastic morphological changes after 24-h incubation. These results suggest that the effects of TPA are biphasic as follows: the initial stage, inducing cell-cell contact formation associated with the translocation of protein kinase C activity from the cytosol to the membrane; and the late stage, exhibiting a fibroblastic morphological change with a decrease in the number of cell-cell contacts associated with the down regulation of this enzyme activity by TPA.

Isolation, ultrastructure and chemical composition of the outermost layer ("exo-layer") of the Epidermophyton floccosum cell wall.

The outer-most layer ("exo-layer") of the wall was isolated from cell walls of Epidermophyton floccosum. The pure cell walls, obtained by disruption in a Ribi cell fractionator, sonication and centrifugation, were digested with snail enzyme for 12 h. Thereafter, the exo-layer preparation was obtained as the fraction resistant to the snail enzyme. Electron microscopy showed that the exo-layer is a thin, stranded network structure 10-20 nm thick. Chemical analysis of the exo-layer showed that the main components are protein (63 percent), mannose (10 percent) and glucosamine (17 percent). Sodium dodecyl sulfate polyacrylamide gel electrophoresis has revealed that the main band is a glycoprotein containing mannose.

Effects of lipid-phase separation on the filipin action on membranes of ergosterol-replaced Tetrahymena cells, as studied by freeze-fracture electron microscopy.

The effects of lipid-phase separation on the filipin action on pellicle membranes of ergosterol-replaced Tetrahymena pyriformis cells were studied by freeze-fracture electron microscopy. The pellicle membranes with phase separations induced by chilling from 34 degrees C (growth temperature) to lower temperatures (30, 22 and 15 degrees C) were treated with filipin. This produced filipin-induced lesions ("pits") only in the particulated (liquid) regions along the margin between solid and liquid domains, while they were produced in the particle-free (solid) areas when membranes were chilled to 15 degrees C. The pellicle membranes with lesions induced by filipin at 34 degrees C were chilled to 22 degrees C. This chilling raised larger particle-free areas and more condensed particle-aggregations on the membranes than on the membranes without the filipin treatment. These results suggest that the membrane fluidity affects induction and development of the ergosterol-filipin complex in the membrane.

Freeze-fracture ultrastructural alterations induced by filipin, pimaricin, nystatin and amphotericin B in the plasmia membranes of Epidermophyton, Saccharomyces and red complex-induced membrane lesions.

The effects of chemically different polyenes on fungal membranes (Epidermaphyton floccosum, a human pathogenic fungus, and Saccharomyces cerevisiae) and human red blood cell membranes were studied by freeze-fracture electron microscopy in order to elucidate the interaction of these antibiotics with ergosterol. Each type of neutral, small amphoteric and large amphoteric polyenes produces a distinct morphoneutral, small amphoteric and large amphoteric polyenes produces a distinct morphological effect on the fungal membranes: (1) Pit formation type. Filipin, a neutral polyene, produces 250-300 A diameter "pits" or "invagination" both in ergosterol-containing fungal plasma membranes and cholesterol-containing red blood cell ghost membranes. (2) Network particle aggregation type. The small amphoteric polyene, pimaricin, produces a network of membrane particle aggregation which encloses 1000 A diameter particle-free areas in fungal membranes. These areas are slightly elevated toward the outside of the cell. (3) Random particle aggregation type. The large amphoteric polyenes, amphotericin B and nystatin, cause a random segregation of the fungal plasma membrane and the red blood cell ghost membranes into particle-free and aggregated areas. It is concluded that these morphological differences are due to different mechanisms of polyene-sterol interactions in which the different size of the mocrolide ring in the antibiotic structure may be involved. Since all of these antibiotics, except filipin, cause no alterations on whole red blood cells detectable by negative staining and freeze-fracture electron microscopy, it is possible that they have a higher affinity to ergosterol than cholesterol in membranes.

Self-regulation of membrane fluidity. The effect of saturated normal and methoxy fatty acid supplementation on Tetrahymena membrane physical properties and lipid composition.

Tetrahymena cells elongated and desaturated massive supplements of palmitic or lauric acid at nearly twice the rates employed by unfed cells, thereby maintaining constant the physical properties of their membrane lipids. However, when a mixture of the 9- and 10-monomethoxy derivatives of stearic acid was administered, these compounds were incorporated without further metabolism. The marked fluidizing effect of the phospholipid-bound methoxy-fatty acids elicited an immediate reduction in fatty acid desaturase activity, the pattern of change being very similar to that induced by supplements of polyunsaturated fatty acids. The modulation of fatty acid desaturase activity by methoxy-acids clearly seems to be governed by membrane fluidity rather than by some form of end product inhibition of the type which might have been postulated to explain the similar effect caused by polyunsaturated fatty acids.

Changes in membrane lipid composition during temperature adaptation by a thermotolerant strain of Tetrahymena pyriformis.

Experiments on temperature adaptation have been conducted using a thermotolerant clone of Tetrahymena pyriformis designated as strain NT-1. The strain was able to grow well at 39.5 and 15 degrees C and could adapt quickly when transferred from one of these temperatures to the other. Cells grown at the extreme temperatures differed markedly in their membrane lipid composition, particularly in the phospholipid polar head groups and hydrocarbon chains. The levels of fatty acid unsaturation increased at the lower temperature (e.g. 15 degrees C cells contained 31% gamma-linolenic acid vs. 25% at 39.5 degrees C) as did the content of alkyl glyceryl ether derivatives. Ethanolamine phosphoglycerides decreased by more than 10 mol % of the lipid phosphorus with the drop in temperature, the decrease being offset by a concomitant rise in 2-aminoethylphosphonolipid. These temperature-induced changes were noted in certain purified membrane preparations as well as in whole cells. Experiments with [14C]palmitic acid and sodium[14C]acetate showed that fatty acids are first incorporated into phospholipids predominantly in a saturated form. The membranes served as a reservoir of fatty acid substrate for desaturase activity. Tetrahymena pyriformis, strain NT-1, was proposed as a useful model system for studying the temperature adaptation process in eukaryotic cells.

Molecular cloning of a gene encoding phospholipase D from the pathogenic and dimorphic fungus, Candida albicans.

A phospholipase D gene (CaPLD) has been cloned from the Candida albicans genomic DNA library. The CaPLD is a member of a highly conserved gene family of PLD and has the highest homology to Saccharomyces cerevisiae PLD (SPO14) with an overall homology of 42%. Phylogenetic analysis indicated that fungus PLDs including CaPLD composed one of the three clusters of PLD genes.

Purification and characterization of lysophospholipase-transacylase (h-LPTA) from a highly virulent strain of Candida albicans.

A lysophospholipase-transacylase (h-LPTA) was purified to homogeneity from a clinical isolate of Candida albicans (C. albicans) that had high extracellular phospholipase activity (strain 16240). The purified enzyme was a glycoprotein with molecular mass of 84 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of the enzyme were 117 mumol/min per mg protein for fatty acid release and 459 mumol/min per mg protein for phosphatidylcholine (PC) formation. An apparent Km of the hydrolase activity of the enzyme for 1-palmitoyl-sn-glycero-3-phosphocholine (1-palmitoyl-lyso-PC) was 60.6 microM. The enzyme had a pH optimum at 6.0. Transacylase activity of the enzyme was partially inhibited by palmitoylcarnitine (35% inhibition) and N-ethylmaleimide. In contrast, the hydrolase activity of the enzyme was stimulated by palmitoylcarnitine but was partially inhibited by N-ethylmaleimide. The enzyme exhibited broad specificity to lyso-phospholipids. The h-LPTA activity was not dependent on divalent cations (Ca2+ and Mg2+) and was not inhibited by addition of EDTA or EGTA. These results show that C. albicans strain 16240 with high extracellular phospholipase activity produced h-LPTA in large amount. This enzyme is biochemically distinct from the LPTA enzyme previously isolated from C. albicans 3125.

Alteration in the arrangement of the keratin-type intermediate filaments during mitosis in cultured human keratinocytes.

The behavior of the keratin-type intermediate filaments (KIFs) during mitosis was characterized in cultured human keratinocytes by immunofluorescence microscopy using polyclonal antibodies to keratin. The structural relationship of KIFs with microtubules (MTs) was also studied at the same time using a monoclonal antibody to alpha-tubulin. The KIFs and MTs showed similar but different cytoskeletal networks and underwent structural rearrangements independently during the cell cycle. KIFs in keratinocytes formed two different arrangements during meta- and anaphase: a global aggregation of filaments around the spindle and a fibrous array radiating from the central, global aggregation of filaments to the cell periphery where they were connected with those of the adjacent cells at desmosomal sites. These radiating fibrous portions of KIFs appeared to play a role in retaining the cell in its correct relationship to the surrounding cells during mitosis. This behavior of KIFs in normal keratinocytes was different from the KIF-alterations which had been previously described in SV40-transformed keratinocytes and other cells which expressed two different IFs (keratin and vimentin).

Pemphigus vulgaris-IgG causes a rapid depletion of desmoglein 3 (Dsg3) from the Triton X-100 soluble pools, leading to the formation of Dsg3-depleted desmosomes in a human squamous carcinoma cell line, DJM-1 cells.

In this study, we examined desmoglein (Dsg) 3 and other desmosomal molecules after pemphigus vulgaris (PV)-immunoglobulin G (IgG) binding to the Dsg3 on the cell surface in DJM-1 cells, a human squamous cell carcinoma cell line. After cells were incubated with PV-IgG for various time periods (0, 5, 10, 20, 30, 60 min, or 30 h), cells were fractionated into phosphate-buffered saline soluble (cytosol), phosphate-buffered saline insoluble-Triton X-100 soluble (membrane), and Triton X-100 insoluble (cytoskeleton) fractions, and subjected to immunoblotting and immunofluorescence microscopy using antibodies against Dsgl, Dsg3, plakoglobin, desmoplakin 1, and cytokeratins. Immunoblot analysis with PV-IgG revealed that Dsg3 was already dramatically depleted from the membrane fraction 20 min after PV-IgG treatment, whereas no reduction of Dsg3 was detected in the cytoskeleton fraction as examined by immunoblotting. A 30 h incubation with PV-IgG, however, caused a marked disappearance of Dsg3, but not other desmosomal molecules, from cytoskeleton fractions. Furthermore, double-staining immunofluorescence microscopy revealed that Dsg3 was depleted from the desmosomes whereas Dsg1, desmoplakin 1, plakoglobin, and keratin filaments were bound to desmosomes. These results provide a novel interpretation for a better understanding of mechanisms for blistering in PV; i.e., a possibility that PV-IgG generates the formation of aberrant desmosomes, which are lacking in Dsg3, but not other desmosomal constituents.