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Biomedical subjects

M Mizoguchi

Publications and source records attributed to M Mizoguchi.

At least 55 records · Page 3Linked to original sources
PubMed1999

Distinct pattern of PCR-SSCP analysis of p53 mutations in human astrocytomas.

Clarification of somatic mutations during the progression of human astrocytomas is important in order to understand the mechanisms underlying the development of these tumors. We analyzed surgical specimens of human astrocytomas for mutations in the p53 gene using single-strand conformation polymorphism analysis of polymerase chain reaction product (PCR-SSCP analysis) at a low pH. Klenow fragment treatment after PCR amplification was an effective means to get rid of some extra bands on the SSCP gel. Five mutations in three of 24 astrocytomas were identified by this improved SSCP method. The frequency of p53 gene mutations in astrocytomas examined was 12.5%. Further examination by direct sequencing showed that all five mutants had single-base substitutions resulting in missense mutations. The present studies revealed a loss of heterozygosity and two point mutations on the remaining allele in one of the fibrillary astrocytomas. Finally, the improvement of PCR-SSCP analysis using Klenow treatment and low pH showed a distinct electrophoresis gel pattern and could be relevant for the prognosis of human astrocytomas.

Astrocytoma↗
PubMed1999

Derivation of melanocytes from embryonic stem cells in culture.

We report that embryonic stem (ES) cells were efficiently induced to differentiate to melanocytes in vitro. When undifferentiated ES cells were cocultured with a bone marrow-derived stromal cell line, a very small but significant number of melanocytes were reproducibly generated. This process was greatly enhanced by addition of dexamethasone to the culture and strictly depended on steel factor, the ligand for the c-Kit receptor tyrosine kinase. Expression of c-Kit on the precursor cells was confirmed by using SCL/tal-1-/- ES cells, which are defective for producing hematopoietic cells, which were thus ruled out as possible sources of nonmelanogenic c-Kit-expressing cells. The morphology, reactivity to growth factors, and expression of melanogenic markers of the cells generated all indicated unequivocally that these cells were melanocytes. This culture system may provide a potent tool for the study of development and function of melanocytes.

Animals↗
PubMed1999

Skin manifestations of a patient with mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes (MELAS syndrome).

Skin abnormalities in a patient with newly diagnosed MELAS syndrome are reported. He presented with scaly, pruritic, diffuse erythema with reticular pigmentation on his entire body. Moderate hypertrichosis was also noted on the lower extremities. Thickened arrector muscles with longitudinal and cross sections were seen in the dermis. Analysis of mitochondrial DNA in a biopsy specimen from the biceps brachii disclosed the presence of an A-to-G point mutation at nucleotide pair 3243. Electron microscopy revealed marked increase in the number of morphologically abnormal mitochondria in the arrector muscles of the involved skin. This is the first report of a MELAS syndrome patient with skin manifestations and evidence of morphologically abnormal mitochondria.

Adult↗
PubMed1999

Molecular confirmation of a Trichophyton violaceum isolate from human black-dot ringworm.

A clinical isolate from a black-dot ringworm lesion of a 28-year-old female Japanese was investigated by morphological and biochemical analyses as well as molecular analyses. The isolate grew well on thiamine enriched agar and did not produce violet pigment, macro-conidia or micro-conidia on Sabouraud's dextrose agar. Approximately 620-bp genomic DNA fragments of the CHS1 gene were amplified from Trichophyton mentagrophytes, T. rubrum, T. tonsurans and T. violaceum by polymerase chain reaction (PCR) and sequenced. The chitin synthase 1 (CHS1) nucleotide sequences of the clinical isolate showed more than 97% similarity to that of T. violaceum and less than 96% similarity to that of T. mentagrophytes, T. rubrum and T. tonsurans. The phylogenetic analysis of their sequences revealed that the clinical isolate was genetically close to T. violaceum and distinct from T. mentagrophytes, T. rubrum and T. tonsurans. Therefore, the isolate was confirmed as T. violaceum by mycological examination and molecular analyses.

Adult↗
PubMed1999

Keratin subunit expression in human cultured melanocytes and mouse neural crest cells without formation of filamentous structures.

The synthesis of keratin is considered to occur in epithelial and epidermal cells. Previous studies have not reported on keratin synthesis within melanocytes that derive from neural crest cells. Epithelial and neural crest cells originally develop from ectodermal tissue. We previously reported that the expression of keratin is a universal phenomenon seen in cultured melanoma cell lines, as demonstrated by two-dimensional polyacrylamide gel electrophoresis, western blot, and electron microscopy analyses. To further investigate the specificity of keratin function in melanocytic cells, we first examined the presence of keratin proteins in cultured human melanocytes, and unexpectedly found keratin subunits in melanocytes by the above-mentioned procedures. The keratin (K) subunits were composed of K1, K5, K8, K10, K14, K16, and K18, together with vimentin. Neural crest cells, which contain immature embryonic melanocytes developing from ectoderm, already expressed keratins; however, under electron microscopy, the expressed keratin did not form filamentous structures. Although the ATP synthase alpha-chain, which is expressed universally in cultured epidermal tumor cell lines, was also expressed in cultured melanocytes and neural crest cells, a novel malignant melanoma-related protein (MMRP) was absent in melanocytes and neural crest cells. We concluded that keratin subunits are present in both cells, but do not construct keratin filaments.

Animals↗
PubMed1999

Removal of stem cell factor or addition of monoclonal anti-c-KIT antibody induces apoptosis in murine melanocyte precursors.

Previous findings indicate that the protein c-KIT and its ligand, stem cell factor (SCF) play a crucial role in the development of melanocytes from their precursors in the embryonic neural crest cells. Using a monoclonal anti-c-KIT antibody, ACK2, which is an antagonistic blocker of c-KIT function, we and colleagues demonstrated that mouse melanocytes disappeared with the injection of ACK2 during certain periods of embryonic and postnatal life. The precise mechanisms of this disappearance, however, remain unclear. Because melanocytes disappeared without any inflammation in these in vivo studies, we suspect that apoptosis was a main cause of their disappearance. In this study, to clarify the underlying mechanism, we studied whether ACK2 induces apoptosis in c-KIT-positive melanoblasts, which appear in mouse neural crest cells cultured with SCF from 9.5 d old mouse embryos. With an in situ apoptosis detection kit, a significant increase in apoptosis was detected after the removal of SCF, which further increased with the addition of ACK2 during SCF-dependent periods. The occurrence of apoptosis in the cultured cells was also demonstrated by a DNA analysis and electron microscopy. Immunohistochemical double staining confirmed that the apoptotic cells were c-KIT positive, and the electron microscopy showed that these apoptotic cells were melanocyte precursors. It was therefore demonstrated that apoptosis was induced in the SCF-dependent c-KIT-positive melanocytes in vitro when the SCF/c-KIT interaction was obstructed. These findings elucidate the mechanism of the regulation of melanocyte development, and the survival and proliferation of these precursor cells, by SCF/c-KIT interaction.

Animals↗
PubMed1998

Murine cutaneous mastocytosis and epidermal melanocytosis induced by keratinocyte expression of transgenic stem cell factor.

The growth and differentiation of mast cells and melanocytes require stem cell factor (SCF), the ligand for the kit receptor tyrosine kinase. SCF may exist as a membrane-bound or soluble molecule. Abnormalities of the SCF-kit signaling pathway, with increased local concentrations of soluble SCF, have been implicated in the pathogenesis of the human disease cutaneous mastocytosis, but have not yet been shown to play a causal role. To investigate both the potential of SCF to cause mastocytosis and its role in epidermal melanocyte homeostasis, we targeted the expression of SCF to epidermal keratinocytes in mice with two different transgenes controlled by the human keratin 14 promoter. The transgenes contained cDNAs that either produced SCF, which can exist in both membrane-bound and soluble forms, or SCF, which remains essentially membrane bound. Murine epidermal keratinocyte expression of membrane-bound/ soluble SCF reproduced the phenotype of human cutaneous mastocytosis, with dermal mast cell infiltrates and epidermal hyperpigmentation, and caused the maintenance of a population of melanocytes in the interadnexal epidermis, an area where melanocytes and melanin are found in human skin but where they are not typically found in murine skin. Expression of membrane-bound SCF alone resulted in epidermal melanocytosis and melanin production, but did not by itself cause mastocytosis. We conclude, first, that a phenotype matching that of human mastocytosis can be produced in mice by keratinocyte overproduction of soluble SCF, suggesting a potential cause of this disease. Second, we conclude that keratinocyte expression of membrane-bound SCF results in the postnatal maintenance of epidermal melanocytes in mice. Since the resulting animals have skin that more closely approximates human skin than do normal mice, their study may be more relevant to human melanocyte biology than the study of skin of normal mice.

Animals↗
PubMed1998

Accumulation of class I mutant p53 and apoptosis induced by carboplatin in a human glioma cell line.

Following DNA damage, wild-type p53 increases and mediates the multiple cellular responses for the repair of DNA damage or apoptosis. Inactivation of p53 by single-amino-acid substitutions contributes to the malignant phenotype and confers resistance to therapy. Among tumor-derived p53 mutants, class I mutants still retain a native-like three-dimensional structure, whereas class II mutants have unfolded DNA-binding domains. Sequencing analysis demonstrated that a human glioma cell line (U-373MG) had only a class I mutant form of p53 of His273, which targets an Arg273 that contacts DNA but retains the native structure. In this study, we investigated the metabolic alteration of the class I mutant p53 in apoptosis of U-373MG. The cell cycle progression of U-373MG cells was affected by the addition of carboplatin, while the amount of mutant p53 also increased in their nuclei. The treated cells underwent apoptosis 48h after exposure to 50 microg/ml carboplatin. Although the exact mechanism of the class I mutant p53 in the process of apoptosis has not yet been clarified, the fact that accumulation of the activated mutant p53 in the nucleus of U-373MG is concomitant with apoptosis, just as wild-type p53 does, implies that the class I mutant p53 might retain the ability to participate in apoptosis.

Antigens, Nuclear↗
PubMed1998

The inhibitory effect of glabridin from licorice extracts on melanogenesis and inflammation.

Glabridin is the main ingredient in hydrophobic fraction of licorice extract affecting on skins. In this study, we investigated inhibitory effects of glabridin on melanogenesis and inflammation using cultured B16 murine melanoma cells and guinea pig skins. The results indicated that glabridin inhibits tyrosinase activity of these cells at concentrations of 0.1 to 1.0 microg/ml and had no detectable effect on their DNA synthesis. Combined analysis of SDS-polyacrylamide gel electrophoresis and DOPA staining on the large granule fraction of these cells disclosed that glabridin decreased specifically the activities of T1 and T3 tyrosinase isozymes. It was also shown that UVB-induced pigmentation and erythema in the skins of guinea pigs were inhibited by topical applications of 0.5% glabridin. Anti-inflammatory effects of glabridin in vitro were also shown by its inhibition of superoxide anion productions and cyclooxygenase activities. These data indicated that glabridin is a unique compound possessing more than one function; not only the inhibition of melanogenesis but also the inhibition of inflammation in the skins. By replacing each of hydroxyl groups of glabridin with others, it was revealed that the inhibitory effect of 2'-O-ethyl glabridin was significantly stronger than that of 4'-O-ethyl-glabridin on melanin synthesis in cultured B16 cells at the concentration of 1.0 mg/ml. With replacement of both of two hydroxyl groups, the inhibitory effect was totally lost. Based on these data, we concluded that two hydroxyl groups of glabridin are important for the inhibition of melanin synthesis and that the hydroxyl group at the 4' position of this compound is more closely related to melanin synthesis.

Administration, Topical↗
PubMed1998

Development of melanocyte progenitors in murine Steel mutant neural crest explants cultured with stem cell factor, endothelin-3, or TPA.

Stem cell factor (SCF) has been suggested to be indispensable for the development of neural crest cells into melanocytes because Steel mutant mice (i.e., Sl/Sl(d)) have no pigmented hairs. On the other hand, it has been demonstrated that the addition of endothelin 3 (ET-3) or TPA to neural crest cell cultures can induce melanocyte differentiation without addition of extrinsic SCF. In this study, we excluded the influence of intrinsic SCF by using Sl/Sl mouse embryos to study more precisely the effects of natural cytokines, such as extrinsic soluble SCF or ET-3, or chemical reagents, such as TPA or cholera toxin. We found that SCF is supplied within the wild-type neural crest explants and that ET-3 cannot induce melanocyte differentiation or proliferation without SCF. These results indicate that SCF plays a critical role in survival or G1/S entry of melanocyte progenitors and that SCF initially stimulates their proliferation and then ET-3 accelerates their proliferation and differentiation. TPA has the ability to elicit neural crest cell differentiation into melanocytes without exogenously added SCF but it is not as effective as SCF because many more melanocytes developed in the wild-type neural crest explants cultured with TPA.

Animals↗
PubMed1998

Deimination of 70-kD nuclear protein during epidermal apoptotic events in vitro.

Peptidylarginine deiminase (PAD) is the enzyme responsible for converting protein-bound arginine residues to citrulline. It has recently been shown that a number of epidermal proteins, including filaggrin, trichohyalin, and keratins, are deiminated by the action of PAD, suggesting a possible role for protein deimination during the final stages of epidermal differentiation. We report here a novel PAD substrate found during the course of identifying deiminated proteins in cultured rat epidermal keratinocytes. We found that a 70-kD protein localized to the periphery of the nucleus was preferentially deiminated after ionomycin treatment in the presence of 2 mM calcium and was associated with apoptotic events in these cells. Furthermore, we discovered that the deimination of nuclear protein could be induced by transfection of a PAD cDNA into rat epidermal keratinocytes. These data suggest that PAD may act on the 70-kD nuclear protein to induce disassembly of the nuclear lamina and promote apoptosis during terminal epidermal differentiation.

Animals↗
PubMed1997

Depletion of cerebral D-serine in non-ketotic hyperglycinemia: possible involvement of glycine cleavage system in control of endogenous D-serine.

Tissue concentrations of D-serine and other chiral and non-chiral amino acids were measured post-mortem in the cerebral cortex of the neonatal or infantile individuals with (three cases) or without (seven cases) non-ketotic hyperglycinemia (NKH) using high performance liquid chromatography with fluorometric detection. In the cortical tissues of the NKH patients lacking activity of glycine cleavage system, there was a marked reduction and elevation of the contents of D-serine and glycine, respectively, compared to non-NKH controls. Systemic administration of an inhibitor of glycine cleavage system (GCS), cysteamine, mimicked the changes in the cortical concentrations of these amino acids in the 8-day-old rats. Augmentation of brain glycine levels by means of intraperitoneal injection of glycine itself resulted in an increase in cortical D-serine contents in the neonatal rats with normal activity of GCS. These findings provide the first evidence that GCS might be implicated in the biosynthesis or content regulation of endogenous D-serine in the mammalian brain.

Amino Acid Oxidoreductases↗
PubMed1997

Detection of SV40 T antigen genome in human gliomas.

The monkey polyomavirus simian virus 40 (SV40) has been reported to be associated with tumorigenesis of human neoplasms, mainly brain tumors. However, it remains controversial whether the virus really exists in human neoplasms and how the virus transforms human cells in vivo. We investigated the presence of SV40 T antigen genome in 33 human glioma tissue specimens from Japanese patients with different histopathologies by means of the polymerase chain reaction (PCR) followed by Southern blotting. The SV40 T antigen genome was amplified in 4 of the 13 ependymomas (31%) and 3 of the 20 other histotypes of gliomas (15%), whereas in the 22 nontumoral brain tissue specimens, only one case was found to be positive. DNA sequencing confirmed the PCR products to be those of SV40 T antigen. The findings thus suggest that the SV40 genome appears to exist in a certain population of brain tumors from Japanese patients, and that it may also play a role in the oncogenicity or maintenance of the transformed state.

Adolescent↗