Lipocortin I (Annexin I) Is Preferentially Localized on the Plasma Membrane in Keratinocytes of Psoriatic Lesional Epidermis as Shown by Immunofluorescence Microscopy

Lopocortin I (LPC-I, also called annexin I) is a 35-kD protein that binds phospholidpids and actin ina a Ca++-dependetn manner. It is also a major substrate for EGF recepto/kinase and rotein kinase C,. and a putative inhibito of phospholipase A2, which produces chemical mediators to cause inflammation. Psoriasos (PS) is an inflammatory skin disease characterized by a rapid turnover of keratinocytes and a defect in keratinization with increased activities of phospholipase C and A2, and EGF receptor. To understand the mechanism of the PS lesion formation and the function of LPC-I, its didtribution was studied in the epiedermis of PS, subacure eczema and normal skin, and in tumor, cells of seborheic keratosis and Bowen's disease. This study involved immunofluorescence and immunoblotting using affinity-purified polyclonal and monclonal antibodies specific to LPC-I and to its Ca++- bound form. In normal, nonlesional PS and subacute eczema epidermis, LPC-I was detected , mainly in the cytoplasm of the suprabasal cells, although it was on the inner aspects of the plasma membrane in some parts of the granular layer. In lesional epidermis of PS it was localized mainly on the inner aspects of the plasma membrane, but not in the cytoplasm of the whole suprabasal cells as the Ca++-bond form, indicating a preferential localization of the plasma membrane. This membrane-binding of LPC-I was also observed in seborrheic keratosis, but not in Bowen's disease. These results suggest that the binding of LPC-I to the plasma membrane occurs actually in living cells, plays a role not necessarily disease specific, in the PS lesion formation, and has some relevance to normal or abnormal differentiation of keratinocytes

Mice Transgenic for KitV620A: Recapitulation of Piebaldism but not Progressive Depigmentation Seen in Humans with this Mutation

Piebaldism is an autosomal dominant genetic pigmentary disorder, characterized by congenital white hair and patches located on the forehead, anterior trunk, and extremities. Most piebald patients have a mutation of the KIT gene, which encodes a tyrosine kinase receptor involved in pigment cell development. The white hair and patches of such patients are already completely formed at birth and do not usually expand thereafter. This stability of pigmented spots also applies to KitW and KitlSl mutant mice. However, two novel cases of piebaldism were reported in 2001, in which both mother and daughter having a novel Val620Ala mutation in their KIT gene showed progressive depigmentation. To prepare an animal model of this mutation, to explore undefined functions of KIT signaling for maintaining pigmented melanocytes in the skin or more specifically the integrity of the melanocyte stem cell system in the postnatal skin, we produced transgenic mice expressing Val620Ala Kit. These mice well mimicked the white spotting pattern of patients; however, no change in this pattern was observed after birth, even after increasing the transgene expression by various means. Here, we report the unexpectedly extremely stable maintenance of the melanocyte stem cell system under stringent conditions for KIT signaling

Expression of activin A in human keratinocytes at early stages of cultivation

AbstractActivins are members of the TGF-β superfamily and are classified into 3 types: activin A, which consists of a homodimer of βA, activin B, which consists of a homodimer of βB, and activin AB, which consists of a heterodimer of βAβB. We studied the expression of activin mRNAs by RT-PCR in normal human epidermis, cultured keratinocytes, and DJM-1 cells (a squamous cell carcinoma line). We could detect only activin A mRNA (βA) in normal human epidermis. In cultured keratinocyte and DJM-1 cells, activin βA mRNA was observed at 4 h but not at 96 h after plating. Activin A activity was detected in the conditioned medium of DJM-1 cells within 48 h. In addition, although follistatin mRNA was not observed in human epidermis in situ, it was transiently expressed in cultured cells at 4 h after plating. These findings suggest that the expression of these molecules in keratinocytes is associated with cell proliferation. In an in vitro tissue injury model, activin A was observed at the wound edge, where cell migration and proliferation may be activated. In DJM-1 cells cultured for 92 h, βA mRNA was observed 4 h after injury treatment. These findings suggest that activin A acts as a potent inducer of proliferation in vitro, at least in keratinocytes

Bullous Pemphigoid IgG Induces BP180 Internalization via a Macropinocytic Pathway

Bullous pemphigoid (BP) is an autoimmune blistering skin disease induced by pathogenic autoantibodies against a type II transmembrane protein (BP180, collagen type XVII, or BPAG2). In animal models, BP180 autoantibody-antigen interaction appears insufficient to develop blisters, but involvement of complement and neutrophils is required. However, cultured keratinocytes treated with BP-IgG exhibit a reduction in the adhesive strength and a loss of expression of BP180, suggesting that the autoantibodies directly affect epidermal cell–extracellular matrix integrity. In this study, we explored the consequences of two distinct epithelial cells treated with BP-IgG, particularly the fate of BP180. First, we followed the distribution of green fluorescent protein–tagged BP180 in an epithelial cell line, 804G, and normal human epidermal keratinocytes after autoantibody clustering. After BP-IgG treatment, the adhesive strength of the cells to their substrate was decreased, and BP180 was internalized in both cell types, together with the early endosomal antigen-1. By using various endocytosis inhibitors and a fluid-uptake assay, we demonstrated that BP-IgG–induced BP180 internalization is mediated via a macropinocytic pathway. Moreover, a macropinocytosis inhibitor rescued a BP-IgG–induced reduction in the adhesive strength of the cells from their substrate. The results of this study suggest that BP180 internalization induced by BP-IgG plays an important role in the initiation of disease pathogenesis

Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration

Regulation of gene expression requires selective incorporation of histone H3 variant H3.3 into chromatin. Histone H3.3 has several subsidiary variants but their functions are unclear. Here we characterize the function of histone H3.3 sub-variant, H3mm7, which is expressed in skeletal muscle satellite cells. H3mm7 knockout mice demonstrate an essential role of H3mm7 in skeletal muscle regeneration. Chromatin analysis reveals that H3mm7 facilitates transcription by forming an open chromatin structure around promoter regions including those of myogenic genes. The crystal structure of the nucleosome containing H3mm7 reveals that, unlike the S57 residue of other H3 proteins, the H3mm7-specific A57 residue cannot form a hydrogen bond with the R40 residue of the cognate H4 molecule. Consequently, the H3mm7 nucleosome is unstable in vitro and exhibited higher mobility in vivo compared with the H3.3 nucleosome. We conclude that the unstable H3mm7 nucleosome may be required for proper skeletal muscle differentiation

Single Event Tolerance of X-ray SOI Pixel Sensors

We evaluate the single event tolerance of the X-ray silicon-on-insulator (SOI) pixel sensor named XRPIX, developed for the future X-ray astronomical satellite FORCE. In this work, we measure the cross-section of single event upset (SEU) of the shift register on XRPIX by irradiating heavy ion beams with linear energy transfer (LET) ranging from 0.022 MeV/(mg/cm2) to 68 MeV/(mg/cm2). From the SEU cross-section curve, the saturation cross-section and threshold LET are successfully obtained to be $3.4^{+2.9}_{-0.9}\times 10^{-10}~{\rm cm^2/bit}$ and $7.3^{+1.9}_{-3.5}~{\rm MeV/(mg/cm^2)}$, respectively. Using these values, the SEU rate in orbit is estimated to be $\lesssim$ 0.1 event/year primarily due to the secondary particles induced by cosmic-ray protons. This SEU rate of the shift register on XRPIX is negligible in the FORCE orbit.Comment: 9 pages, 5 figures, accepted for publication in JATI

X-ray Radiation Damage Effects on Double-SOI Pixel Detectors for the Future Astronomical Satellite "FORCE"

We have been developing the monolithic active pixel detector "XRPIX" onboard the future X-ray astronomical satellite "FORCE". XRPIX is composed of CMOS pixel circuits, SiO2 insulator, and Si sensor by utilizing the silicon-on-insulator (SOI) technology. When the semiconductor detector is operated in orbit, it suffers from radiation damage due to X-rays emitted from the celestial objects as well as cosmic rays. From previous studies, positive charges trapped in the SiO2 insulator are known to cause the degradation of the detector performance. To improve the radiation hardness, we developed XRPIX equipped with Double-SOI (D-SOI) structure, introducing an additional silicon layer in the SiO2 insulator. This structure is aimed at compensating for the effect of the trapped positive charges. Although the radiation hardness to cosmic rays of the D-SOI detectors has been evaluated, the radiation effect due to the X-ray irradiation has not been evaluated. Then, we conduct an X-ray irradiation experiment using an X-ray generator with a total dose of 10 krad at the SiO2 insulator, equivalent to 7 years in orbit. As a result of this experiment, the energy resolution in full-width half maximum for the 5.9 keV X-ray degrades by 17.8 $\pm$ 2.8% and the dark current increases by 89 $\pm$ 13%. We also investigate the physical mechanism of the increase in the dark current due to X-ray irradiation using TCAD simulation. It is found that the increase in the dark current can be explained by the increase in the interface state density at the Si/SiO2 interface.Comment: 15 pages, 12 figures, accepted for publication in Journal of Astronomical Telescopes, Instruments, and System