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Masashi Akiyama

Publications and source records attributed to Masashi Akiyama.

At least 19 recordsLinked to original sources
PubMed2007

Small-diameter porous poly (epsilon-caprolactone) films enhance adhesion and growth of human cultured epidermal keratinocyte and dermal fibroblast cells.

Autologous keratinocyte grafts provide clinical benefit by rapidly covering wounded areas, but they are fragile. We therefore developed biocompatible hexagonal-packed porous films with uniform, circular pore sizes to support human keratinocytes and fibroblasts. Cells were cultured on these porous poly (epsilon-calprolactone) films with pore sizes ranging from novel ultra-small 3 microm to 20 microm. These were compared with flat (pore-less) films. Cell growth rates, adhesion, migration, and ultrastructural morphology were examined. Human keratinocytes and fibroblasts attached to all films. Furthermore, small-pore (3-5 microm) films showed the highest levels of cell adhesion and survival and prevented migration into the pores and opposing film surface. Keratinocyte migration over small-pore film surface was inhibited. Keratinocytes optimally attached to 3-microm-pore films due to a combination of greater pore numbers (porosity), a greater circumference of the pore edge per unit surface area, and greater frequency of flat surface areas for attachment, allowing better cell-substrate and cell-cell attachment and growth. The 3-microm pore size allowed cell-cell communication, together with diffusion of soluble nutrients and factors from the culture medium or wound substrate. These characteristics are considered important in developing grafts for use in the treatment of human skin wounds.

Biocompatible Materials↗
PubMed2006

DNA-based prenatal diagnosis of harlequin ichthyosis and characterization of ABCA12 mutation consequences.

Until the identification of ABCA12 as the causative gene, prenatal diagnosis (PD) for harlequin ichthyosis (HI) had been performed by electron microscopic observation of fetal skin biopsy samples. We report the first case of HI DNA-based PD. Direct sequence analysis of ABCA12 revealed that the deceased proband was a compound heterozygote for two novel mutations. The maternal nonsense mutation p.Ser1249Term likely leads to nonsense-mediated messenger RNA decay. The paternal mutation c.7436G>A affects the last codon of exon 50 and was expected to be a splice site mutation. For their third pregnancy, the parents requested PD. Direct sequence analysis of fetal genomic DNA from amniotic fluid cells at 17 weeks gestation revealed the fetus was a compound heterozygote for both mutations. The parents requested the pregnancy to be terminated. Analysis of ABCA12 transcripts of cultured keratinocytes from the abortus showed the presence of six abnormally spliced products from the allele carrying the splice site mutation. Four of them lead to premature termination codons whereas the two others produced shortened proteins missing 21 and 31 amino acids from the second ATP-binding cassette. This report provides evidence for residual ABCA12 expression in HI, and demonstrates the efficiency of early DNA-based PD of HI.

ATP-Binding Cassette Transporters↗
PubMed2006

N-linked neutral oligosaccharides in the stratum corneum of normal and ichthyotic skin.

N-Glycan oligosaccharides are thought to play multiple, important roles in a variety of biological events. However, N-glycan profiles in the stratum corneum of human skin have not yet been studied in detail. To clarify the N-glycan profiles in the stratum corneum of normal and ichthyotic epidermis, N-glycan profiles were studied by high-performance liquid chromatography using normal human epidermal samples and scales from hyperkeratotic skin of ichthyosis patients. Chromatograms of patient scale samples showed unique alterations in three peaks eluted at 15.8, 18.8 and 26.9 min. The N-glycan profiles were significantly altered in ichthyotic hyperkeratotic skin compared with normal non-hyperkeratotic controls. These findings indicate the reduction of N-acetylglucosaminyltransferase II and fucosyltransferase 8 activities. Alteration of N-glycan structures in hyperkeratotic skin suggests the biological role of N-glycans in keratinization.

Chromatography, High Pressure Liquid↗
PubMed2006

Dietary glucosylceramide improves skin barrier function in hairless mice.

BACKGROUND: Sphingolipids are known to play an important role in both water retention and epidermal permeability barrier function in mammalian stratum corneum. However, little is known about the effects on epidermal function of orally administered sphingolipids. OBJECTIVE: We examined the effect of dietary glucosylceramide (GluCer) on the maintenance and recovery of epidermal barrier function. METHODS: Hairless mice were fed a particular diet (HR-AD) for 4 weeks to induce chronic skin perturbation. Subsequently, a normal diet supplemented with GluCer (from rice bran and germ) was provided for the next 4 weeks. Transepidermal water loss (TEWL) and stratum corneum flexibility were measured throughout this recovery phase. Additional hairless mice were fed a diet with or without a maize-extracted GluCer supplement for 5 weeks, then their skin was acutely perturbed with repeated tape-stripping, and the TEWL was measured. RESULTS: Although skin functions were generally lower following chronic perturbation, in GluCer-fed mice the TEWL was significantly reduced at 2 weeks and the stratum corneum flexibility was increased at 3 weeks compared to controls. Following acute barrier perturbation by tape-stripping, mice an HR-AD fed a GluCer diet exhibited enhanced recovery compared with the control diet group. CONCLUSION: These results demonstrate that in hairless mice skin barrier functions impaired by chronic or acute perturbations were improved by dietary GluCer. The oral administration of GluCer may be useful for the preservation and recovery of epidermal barrier functions an HR-AD.

Adhesives↗
PubMed2006

Targeted skipping of a single exon harboring a premature termination codon mutation: implications and potential for gene correction therapy for selective dystrophic epidermolysis bullosa patients.

This study examined the feasibility of antisense oligoribonucleotide (AON) therapy for dystrophic epidermolysis bullosa (DEB). AON was designed to induce skipping of a targeted exon containing a premature termination codon mutation, resulting in restoration of the open reading frame. We targeted exon 70 of COL7A1, as a recurrent mutation 5818delC in Japanese DEB patients was localized to exon 70. We found that one AON induced effective skipping of normal exon 70 containing 16 amino acids. Attachment and migration analyses showed that recombinant collagen without contribution of exon 70 was similar in effect to normal type VII collagen. Next, we synthesized mutation-specific AON by deleting cytosine at 5818. Introduction of this AON into DEB keratinocytes harboring 5818delC showed that the AON induced skipping of exon 70 in the abnormal 5818delC allele. Furthermore, 6.2% of DEB keratinocytes started to express type VII collagen in vitro after application of the mutation-specific AON. Injection of the AON into rat model grafted with DEB keratinocytes and fibroblasts induced a low amount of type VII collagen expression. We conclude that skipping of targeted exons using mutation-specific AON may show potential for future gene therapy for DEB patients.

Animals↗
PubMed2006

Compound heterozygous mutations including a de novo missense mutation in ABCA12 led to a case of harlequin ichthyosis with moderate clinical severity.

Harlequin ichthyosis (HI) is one of the most devastating genodermatoses. Recently, ABCA12 mutations were identified as the cause of HI. A newborn Japanese male demonstrated the typical features of HI. The patient was treated with oral etretinate and his general condition has been good (now aged 1.5 years). This patient with moderate clinical severity was compound heterozygous for a novel de novo missense mutation 1160G > A (S387N) in exon 10 and a maternal deletion mutation 4158_4160delTAC (T1387del) in exon 28 of ABCA12. T1387del was a deletion of a highly conserved threonine residue within the first adenosine 5' triphosphate-binding domain and is thought to seriously affect the function of the ABCA12 protein. Conversely, the residue 387 is located outside the known active sites of ABCA12 and S387N is predicted not to lead to a serious functional deficiency in ABCA12. Electron microscopy revealed abnormal lamellar granules in the granular layer cells and a moderate number of lipid vacuoles in the cornified cells. Disturbed glucosylceramide transport was confirmed in the cultured keratinocytes from the patient. No de novo mutation in ABCA12 has yet been reported either in HI or lamellar ichthyosis. The present case suggested that a de novo ABCA12 mutation might underlie HI.

ATP-Binding Cassette Transporters↗
PubMed2006

COL7A1 mutation G2037E causes epidermal retention of type VII collagen.

COL7A1 glycine substitution (GS) mutations result in dominant and recessive dystrophic epidermolysis bullosa (DDEB and RDEB). Here, we report a DDEB family in which retention of type VII collagen by epidermal keratinocytes was observed for a female proband. Mutational analysis detected a GS mutation, G2037E, in the proband and her affected father. To demonstrate direct association of G2037E and type VII collagen retention we introduced this mutated COL7A1 gene into cultured keratinocytes using retroviral methods. This mutation was dominant, so we transferred a 1:1 mixture of wild-type (unaffected) and G2037E-mutated COL7A1, together, in addition to the unaffected gene or the mutated gene alone. The increase in type VII collagen cytoplasmic staining in the G2037E/wild transfectant cell samples was compared with that for control/wild-type cells. Intracellular collagen VII staining in the G2037E (alone)-transfected cells was even stronger than for the G2037E/wild transfection sample. These results indicate that the G2037E COL7A1 mutation leads to increased epidermal retention of type VII collagen in vivo, and also suggests that homozygotes carrying this dominant GS mutation may have more severe phenotypes than heterozygotes. This study furthers our understanding of GS COL7A1 mutations in DEB.

Collagen Type VII↗
PubMed2006

Harlequin ichthyosis and other autosomal recessive congenital ichthyoses: the underlying genetic defects and pathomechanisms.

Autosomal recessive congenital ichthyoses (ARCI) include several severe subtypes including harlequin ichthyosis (HI), lamellar ichthyosis and non-bullous congenital ichthyosiform erythroderma. Patients with these severe types of ichthyoses frequently show severe hyperkeratosis and scales over a large part of the body surface form birth and their quality of life is often severely affected. Recently, research into the pathomechanisms of these severe congenital ichthyoses have advanced dramatically and led to the identification of several causative genes and molecules underlying the genetic defects. To date, seven loci have been identified that are associated with ARCI and, among them, five causative genes and molecules have been detected. The five genes are transglutaminase 1 gene (TGM1), ABCA12, two lipoxygenase genes, ALOXE3 and ALOX12B and ichthyin. One of these components, ABCA12, has recently been shown to be a keratinocyte lipid transporter associated with lipid transport in lamellar granules and loss of ABCA12 function leads to a defective lipid barrier in the stratum corneum, resulting in the HI phenotype. Transglutaminse 1 deficiency was reported to cause a malformed cornified cell envelope leading to a defect in the intercellular lipid layers in the stratum corneum and defective stratum corneum barrier function resulting in an ichthyosis phenotype. Thus, defective intercellular lipid layers are major findings in autosomal recessive congenital ichthyoses. Information concerning ARCI genetic defects and disease pathomechanisms are beneficial for providing better treatments and genetic counseling including prenatal diagnosis for families affect by ichthyoses.

ATP-Binding Cassette Transporters↗
PubMed2006

Pathomechanisms of harlequin ichthyosis and ABCA transporters in human diseases.

OBJECTIVES: To review recent advances in our understanding of the genetic pathomechanisms of harlequin ichthyosis (HI) (the most devastating subtype of congenital ichthyoses) and its prenatal diagnosis and to discuss the possibility of future gene therapy. DATA SOURCE: PubMed search for articles about HI, its causative protein adenosine triphosphate-binding cassette A12 (ABCA12), and related molecules. STUDY SELECTION: English-language studies were selected if they provided useful information about the pathomechanisms of HI and ABCA lipid transporters. DATA SYNTHESIS: This article describes ABCA12 as a causative molecule involved in defects in HI, summarizes the known genetic disorders caused by genetic defects in ABCA lipid transporters, and highlights the prospects of prenatal diagnosis and gene therapy for HI. CONCLUSIONS: Harlequin ichthyosis is caused by a serious functional deficiency of ABCA12. ABCA12 and ABCA3 are essential lipid transporters for human adaptation to a dry terrestrial environment. In clinical practice, information regarding the genetic defects and pathomechanisms underlying HI is important for precise diagnosis, genetic counseling, and prenatal diagnosis.

ATP-Binding Cassette Transporters↗
PubMed2006

Nail dystrophy and blisters as sole manifestations in myeloma-associated amyloidosis.

We report the case of a 61-year-old Japanese man with IgG lambda-type multiple myeloma, who presented with nail dystrophy as the initial manifestation of systemic amyloidosis. Subsequently he developed bullous amyloidosis. This report documents these two rare signs of systemic amyloidosis and demonstrates the precise location of cutaneous blister formation and amyloid deposition by fluorescence antigen mapping and electron microscopy.

Amyloidosis↗
PubMed2006

Fibroblasts show more potential as target cells than keratinocytes in COL7A1 gene therapy of dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-transferred cells can most efficiently express collagen VII in the skin. We demonstrated that gene transfer using a combination of G protein of vesicular stomatitis virus-pseudotyped retroviral vector and retronectin introduced COL7A1 into keratinocytes and fibroblasts from a DEB patient with the lack of COL7A1 expression. Real-time polymerase chain reaction analysis of the normal human skin demonstrated that the quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-transferred fibroblasts supplied higher amount of collagen VII to the new dermal-epidermal junction than the gene-transferred keratinocytes. An ultrastructural study revealed that collagen VII from gene-transferred cells formed proper anchoring fibrils. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes.

Animals↗
PubMed2006

Molecular cloning, mRNA expression, and immunocytochemical localization of a putative blue-light photoreceptor CRY4 in the chicken pineal gland.

In non-mammalian vertebrates, the pineal gland contains an endogenous circadian oscillator and serves as a photosensitive neuroendocrinal organ. To better understand the pineal phototransduction mechanism, we focused on the chicken putative blue-light photoreceptive molecule, Cryptochrome4 (cCRY4). Here we report the molecular cloning of pineal cCry4 cDNA, the in vivo expression of cCry4 mRNA, and the detection of cCRY4 protein. cCry4 is transcribed in a wide variety of chick tissues out of which the pineal gland and retina contain high levels of cCry4 mRNA. In the pineal gland, under 12 h light : 12 h dark cycles, the levels of both cCry4 mRNA and cCRY4 protein showed diurnal changes, and in cultured chick pineal cells, the cCry4 mRNA level was not only up-regulated by light but also controlled by circadian signals. Immunoblot analysis with a cCRY4-specific antibody detected cCRY4 in a soluble fraction of the pineal lysate. Immunocytochemistry revealed that cCRY4 was expressed in many parenchymal cells and a limited number of stromal cells. These cCRY4 features strikingly contrast with those of the chick pineal photoreceptor pinopsin, suggesting a possible temporal and/or spatial duplicity of the pineal photoreceptive system, the opsin- and CRY-based mechanisms.

Animals↗
PubMed2006

Expression of RNA-binding protein Musashi in hair follicle development and hair cycle progression.

Epithelial stem cells reside in the hair follicle (HF) bulge region and possess the ability to differentiate into a variety of cutaneous epithelial cells. The evolutionarily conserved Musashi family of RNA-binding proteins is associated with maintenance and/or asymmetric cell division of neural progenitor cells, and a mammalian Musashi protein is expressed in various epithelial stem/progenitor cells, including gut, stomach, and mammary gland. Thus, we hypothesized that Musashi might be expressed in stem cells and early progenitor cells of HF epithelium. Reverse transcriptase-polymerase chain reaction and immunoblotting identified Musashi-1 (Msi-1) and Musashi-2 (Msi-2) mRNA and protein in cultured mouse keratinocytes, but only Msi-1 was identified in human keratinocytes. In mice, immunohistochemical studies showed that Msi-1 and Msi-2 were expressed in the epidermis and HFs from E14.5 until adulthood. In the early anagen phase, Msi-1 and Msi-2 were expressed in the bulge and secondary germ cells and subsequently in inner root sheath (IRS) cells, especially the middle IRS cells, during the late anagen phase. In human skin, Msi-1 was detected in fetal HF cells but not in adult HFs. These observations suggest that Musashi functions not only in the asymmetric division of early progenitor cells but also in the differentiation of IRS cells during HF development and hair cycle progression.

Adult↗
PubMed2006

A novel N14Y mutation in Connexin26 in keratitis-ichthyosis-deafness syndrome: analyses of altered gap junctional communication and molecular structure of N terminus of mutated Connexin26.

Connexins (Cxs) are transmembranous proteins that connect adjacent cells via channels known as gap junctions. The N-terminal 21 amino acids of Cx26 are located at the cytoplasmic side of the channel pore and are thought to be essential for the regulation of channel selectivity. We have found a novel mutation, N14Y, in the N-terminal domain of Cx26 in a case of keratitis-ichthyosis-deafness syndrome. Reduced gap junctional intercellular communication was observed in the patient's keratinocytes by the dye transfer assay using scrape-loading methods. The effect of this mutation on molecular structure was investigated using synthetic N-terminal peptides from both wild-type and mutated Cx26. Two-dimensional (1)H nuclear magnetic resonance and circular dichroism measurements demonstrated that the secondary structures of these two model peptides are similar to each other. However, several novel nuclear Overhauser effect signals appeared in the N14Y mutant, and the secondary structure of the mutant peptide was more susceptible to induction of 2,2,2-trifluoroethanol than wild type. Thus, it is likely that the N14Y mutation induces a change in local structural flexibility of the N-terminal domain, which is important for exerting the activity of the channel function, resulting in impaired gap junctional intercellular communication.

3T3 Cells↗
PubMed2005

Colocalization of multiple laminin isoforms predominantly beneath hemidesmosomes in the upper lamina densa of the epidermal basement membrane.

Multiple laminin isoforms including laminins 5 (alpha3 beta3 gamma2), 6 (alpha3 beta1 gamma1), 10 (alpha5 beta1 gamma1), and possibly laminins 7 (alpha3 beta2 gamma1) and 11 (alpha5 beta2 gamma1) are present in the epidermal basement membrane. However, only the precise epidermal ultrastructural localization of laminin 5 (alpha3 beta3 gamma2) has been elucidated. We therefore determined the precise expression and ultrastructural localization of the alpha5, beta1, beta2, and gamma1 chains in the epidermis. The expression of laminin chains in skin samples was analyzed from patients with epidermolysis bullosa (EB, n=15) that harbor defects in specific hemidesmosome (HD)-associated components. The expression of the alpha5, beta1, and gamma1 chains (present in laminins 10/11) and beta2 chain (laminins 7/11) was unaffected in all intact (unseparated) skin of EB patients including Herlitz junctional EB with laminin-5 defects (n=6). In the basement membrane of human epidermis, the alpha5, beta1, beta2, and gamma1 chains were expressed but also localized to the dermal vessels. Immunogold electron microscopy of normal human epidermis localized the alpha5, beta1, beta2, and gamma1 chains to the upper lamina densa, with between 84% and 92% of labeling restricted to beneath the HDs, similar to laminin 5 (n> or =200 gold particles per sample, sample number n=4) but distinct from collagen IV labeling (with only 63% labeling beneath HDs, p<0.001). Taken together, the majority of the alpha5beta1/beta2gamma1 laminin chains are located beneath HDs. This suggests that laminin-10-associated chains have specific functions or molecular interactions beneath HDs in the epidermal basement membrane.

Adolescent↗