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Biomedical subjects

M Ito

Publications and source records attributed to M Ito.

At least 235 records · Page 13Linked to original sources
PubMed2001

Screening for mitochondrial DNA heteroplasmy in children at risk for mitochondrial disease.

Temporal temperature gradient gel electrophoresis was used to screen 70% of the mtDNA, including all 22 tRNA genes, for heteroplasmy in 75 children with neuromuscular and/or multi-system dysfunction and elevated lactate levels, and in 95 controls. Standard PCR/ASO (allele specific oligonucleotide) and Southern analyses were also employed. Excluding common length variants, heteroplasmy was found in 22 patients and two controls (P < 0.001), with four patients demonstrating heteroplasmy in two locations each. Of the 23 heteroplasmic variants sequenced among the patients, 17 were novel point variants in the control region (CR) and only two involved tRNA genes. Heteroplasmy is highly associated with the disease group, and is predominately found in the CR, an area rarely studied in patient populations. These variants may be pathological mutations or disease markers.

Journal Article↗
PubMed2001

Bisphosphonate acts on osteoclasts independent of ruffled borders in osteosclerotic (oc/oc) mice.

We examined the effects of a third generation bisphosphonate [YM-175; disodium dihydrogen (cycloheptylamino)-methylene-1,1-bisphosphonate] on osteoclasts in osteosclerotic (oc/oc) mice to elucidate the cellular mechanism for incorporation of the bisphosphonate. Osteoclasts of oc/oc mice were in direct contact with bone matrix but devoid of ruffled borders. Tartrate-resistant acid phosphatase (TRAPase) showed spotty localization intercellularly, whereas vacuolar H(+)-ATPase (V-ATPase) immunoreactivity was observed homogeneously in the cytoplasm. Upon injection of bisphosphonate, most osteoclasts lost cell polarity and were detached from bone surfaces. The detached osteoclasts underwent apoptosis as characterized by condensation of chromatin, absence of Golgi apparatus, and formation of many vesicles in the cytoplasm. Both TRAPase and V-ATPase were evenly distributed in the cytoplasm. The pyknotic nuclei of osteoclasts revealed DNA fragments as evidenced by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The results indicate that osteoclasts lacking ruffled borders in oc/oc mice incorporated the bisphosphonate from a site different from ruffled borders and that bisphosphonate may directly affect osteoclasts without mediating its deposition to the bone matrix.

Animals↗
PubMed2001

Effect of trabecular bone contour on ultimate strength of lumbar vertebra after bilateral ovariectomy in rats.

To test the hypothesis that the effect of trabecular microarchitecture on bone strength varies with the duration of estrogen loss, we evaluated the relationship between three-dimensional (3D) parameters for trabecular microarchitecture and bone minerals with the compressive load of the lumbar vertebra in rats. Female Sprague-Dawley rats (n = 190) were divided into 19 groups. Ten rats were killed at day 0. Half of the remaining rats underwent bilateral ovariectomy (ovx), and the others were subjected to sham surgery. Ten rats from each group were killed at 3, 7, 11, 14, 28, 42, 56, 70, and 84 days postsurgery. Urinary deoxypyridinoline and serum osteocalcin increased significantly in the ovx group from days 28 and 11, respectively, compared with the sham group. Bone mineral content (BMC) and bone mineral density (BMD) of the fifth lumbar body diminished from days 42 and 84, respectively, compared with the sham group. In ovx rats, trabecular bone volume (BV/TV), measured using 3D images of microcomputed tomography, diminished from day 28 compared with both baseline control and sham. The trabecular bone pattern factor (TBPf) and structure model index (SMI) increased from day 28 in the ovx group compared with both baseline control and sham. Ultimate compression loads diminished at day 28 compared with baseline control and decreased progressively thereafter. Neither of these parameters changed in the sham group during the same period. Within 4 weeks post-ovx, TBPf, SMI, and BV/TV correlated with load (p < 0.01). BMC and BMD correlated with load from 6 weeks post-ovx (p < 0.01). Stepwise regression analysis showed that TBPf was the most significant determinant of load within 4 weeks post-ovx (coefficient of determination [R(2)] = 0.669; p < 0.01). SMI correlated with TBPf (R(2) = 0.968; p < 0.01). Moreover, R(2) for ultimate load indicated higher values of 0.975 with TBPf and SMI. However, BMC was the most significant determinant of load from 6 weeks post-ovx (R(2) = 0.511; p < 0.01), as it was in the sham group. These data suggest that changes in trabecular bone contour with increased bone turnover are critical for reducing lumbar bone strength during the early post-ovx period in rats.

Amino Acids↗
PubMed2001

Sesquiterpenes and monoterpenes from the bark of Inula macrophylla.

Eleven new sesquiterpenes (1--11) and two thymol derivatives (12, 13), along with 12 known sesquiterpenes and monoterpenes, were isolated from the bark of Inula macrophylla. Their structures were determined on the basis of spectral evidence (especially by HREIMS and 2D NMR) as well as chemical transformations. The structure of macrophyllic acid A (1) was confirmed by X-ray analysis, and the absolute configuration of 1 was determined on the basis of the appropriate chemical conversions and the application of a modified Mosher's method.

Crystallography, X-Ray↗
PubMed2001

Novel exon 11 skipping mutation in a patient with glycogen storage disease type IIId.

We report the molecular genetic abnormalities of a patient with GSD IIId presenting with progressive myopathy and cardiopathy leading to a fatal outcome. We identified two independent deletions including a 4 bp deletion (117-1120) and a 98 bp deletion (1135-1232) in cDNA. Sequencing of the genomic DNA of the corresponding region revealed a 4 bp deletion in exon 10; however, the other 98 bp deletion corresponding to exon 11, which was deleted in cDNA, was present in genomic DNA. We therefore concluded that skipping of exon 11 occurred in the cDNA of the patient. Intron/exon boundary analysis of the skipped exon 11 revealed no mutation in the consensus splice-site sequence. If normal splicing had occurred, a stop codon would have appeared within exon II due to frameshift mutation. The mechanism of exon skipping observed in our patient is as yet unknown, and it is still not clear whether intraexonal mutation of the preceding exon can influence splice-site selection. It is possible that a unique exon skipping occurred, preventing the appearance of a stop codon in our patient.

Adult↗
PubMed2001

Transition between two forms of heterochromatin at plant subtelomeres.

The manner of packing of the terminal DNA loci into nucleosomes and higher order structures may strongly influence their functional interactions. Besides the structural flexibility of telomeric DNA sequences, conserved features of their chromatin including short nucleosome phasing (157 bp) and nucleosome sliding have been described previously. To gain a complementary knowledge of subtelomeres, we have analysed the chromatin structure of two subtelomeric tandem repeats from the plant Silene latifolia: X43.1 and 15Ssp. X43.1 shows two distinct nucleosome periodicities--157 and 188 bp. Preferred positions of its two nucleosomes have been mapped at both low and high resolution and the experimental results correspond to computer-predicted positions. 15Ssp is a newly-discovered sequence showing a telomere-associated position by PCR and a subtelomeric location by pulsed-field gel electrophoresis and fluorescence in situ hybridisation. Its 159 bp sequence unit shows a tandem arrangement and the presence of micrococcal nuclease-hypersensitive sites when either naked DNA or chromatin is digested. Use of a chemical nuclease results in a regular nucleosome ladder of 157 bp periodicity. Moreover, 15Ssp mononucleosomes show instability and absence of specific positioning, features typical for telomeric chromatin.

Base Sequence↗
PubMed2001

Granulomatous and cytokine responses to pulmonary Cryptococcus neoformans in two strains of rats.

The present study was undertaken to elucidate the participation of Th1 and Th2 responses in granulomatous inflammation induced by Cryptococcus neoformans using Lewis and Brown Norway rats which have Th1-polarized and Th2-polarized innate immunity, respectively. Both strains demonstrated granulomatous inflammation in the lung, and the lesions were composed mainly of mononuclear phagocytes and surrounded by lymphocytes. Lewis rats demonstrated tuberculoid epithelioid cell granulomas with a central necrosis resembling caseation, and increased transcription of Th1 relevant cytokines. On the other hand, Brown Norway rats showed mature granulomas including eosinophils with increased transcription of IL-12 without increased transcription of not only IFN-gamma and IL-2 but also Th2 cytokines such as IL-4, IL-5, and IL-10, unexpectedly. The colony-forming unit of the lung was decreased exponentially in both strains, and that of Brown Norway rats was significantly lower than that of Lewis rats 10 days after the inoculation. This indicated that Brown Norway rats demonstrated more fungicidal activity than Lewis rats in the early stage of the infection. The role of eosinophils with humoral immunity may be considered to be resistant in Brown Norway rats in addition of the function of macrophages.

Animals↗
PubMed2001

The DSL domain in mutant JAG1 ligand is essential for the severity of the liver defect in Alagille syndrome.

Alagille syndrome (AGS) is a congenital multi-system anomaly mainly characterized by paucity of intrahepatic bile ducts caused by haploinsufficiency of the Jagged 1 gene (JAG1). To explore the relationship between genotype and phenotype, we analyzed the JAG1 gene in 25 Japanese AGS families at the genomic DNA level and identified 15 point mutations and one large deletion. Analysis of the genotype and phenotype strongly indicated that the Delta/Serrate/Lag-2 (DSL) domain in JAG1 protein played an essential role in determining the severity of the liver disorder. In four sporadic cases, missing an entire DSL domain in mutant JAG1 resulted in progressive liver failure and all 4 patients needed a liver transplant at a very young age. This correlation was further confirmed by statistical analysis (chi2=9.143, p<0.001). Our finding demonstrated that the DSL domain in JAG1 appears to be essential for normal liver development and function.

Alagille Syndrome↗
PubMed2001

Establishment and characterization of a mouse neural crest derived cell line (NCCmelan5).

Stem cell factor (SCF) and endothelin 3 (EDN3) are both necessary for melanocyte development. We have established an immortal cell population of neural crest cells from C57BL/6 mice, cultivating them with SCF, EDN3 and 15% fetal calf serum without feeder cells, and have designated that line as C57NCC SE. C57NCC SE consists of a population of melanocytes in various stages of differentiation. We used a single-cell cloning method, in which only one cell is transferred to each new culture plate, and succeeded in establishing an immortal cell line named NCCmelan5. All NCCmelan5 cells were positive for KIT (SCF receptor), HMB45 (human melanosomal antigen), tyrosinase-related protein-1 (TYRP1), tyrosinase-related protein-2 (TYRP2), tyrosinase and endothelin receptor B (EDNRB) and all could oxidize 3,4-dihydroxyphenylalanine (DOPA) to form melanin. Measurement of their DNA content revealed that 88.6% of the cells were in the G0-G1 phase, suggesting that they retained normal DNA ploidy. Thus, NCCmelan5 cells have the characteristics of mature melanocytes except that they are immortal; these cells may prove useful to study factors that directly affect melanogenesis and melanocyte development without the influence of feeder cells. It is clear that our attempt to establish immortal cell lines from murine neural crest cells would have never been successful without the addition of SCF and EDN3, since C57NCC SE and NCCmelan5 cells require those factors to proliferate.

Animals↗
PubMed2001

Hepatocyte growth factor secreted by periodontal ligament and gingival fibroblasts is a major chemoattractant for gingival epithelial cells.

To ascertain whether periodontal fibroblasts could be involved in the pathogenesis of periodontal pocket formation, the chemotactic activity of periodontal ligament fibroblast-conditioned medium (PLF-CM) and gingival fibroblast-conditioned medium (GF-CM) for gingival epithelial cells was examined using a modified Boyden chamber assay. Both PLF-CM and GF-CM possessed significant chemotactic activity, which was decreased markedly by treatment with anti-human hepatocyte growth factor (HGF) neutralizing antibody. Furthermore, the chemotactic activity of PLF-CM and GF-CM was well correlated with HGF content. These results show that PLF and GF secrete an HGF-like factor, and suggest that such a factor derived from periodontal fibroblasts might play a role in epithelial apical migration in periodontitis.

Cell Line↗
PubMed2001

Expression of the ets-1 proto-oncogene in human colorectal carcinoma.

The proto-oncogene, ets-1, is a transcription factor known to control the expression of a number of genes involved in extracellular matrix remodeling and has been postulated to play a role in cell migration and tumor invasion. To elucidate the involvement of ets-1 in human colorectal carcinomas, we examined 41 cases of colorectal adenoma and 122 cases of colorectal carcinoma by immunohistochemistry and compared the degree of Ets-1 expression with the depth of carcinoma invasion. In adenomas, 12 of 41 cases (29.3%) showed immuno-positivity for Ets-1. 12 of 27 cases (44.4%) of adenoma with high grade dysplasia showed immunopositivity for Ets-1. However, there was no positive case in low or moderate dysplasia of adenoma. In contrast, 103 of 122 cases (84.4%) of colorectal adenocarcinoma showed immunoreactivity for Ets-1 in the carcinoma cells themselves. We investigated the relationship between pathological features in colorectal carcinoma and Ets-1 immunoreactivity of the tumor cells. Among the 122 cases of invasive carcinomas, Ets-1 immunoreactivity was significantly correlated with the depth grading of tumor invasion (P < .0001), the presence of lymph node metastasis (P < .05), lymphatic invasion (P < .01) and venous invasion (P < .05). However, Ets-1 expression did not correlate with histological differentiation. In situ hybridization also confirmed the presence of ets-1 mRNA in colorectal carcinomas. Expression of ets-1 mRNA was also detected in two of three human colorectal carcinoma tissues and in four of six different kinds of carcinoma cell lines by the reverse transcription polymerase chain reaction method. These findings suggest that the expression of Ets-1 is one of the important factors related to carcinogenesis and/or tumor invasion of colorectal carcinoma.

Adenocarcinoma↗
PubMed2001

Purification and properties of the cell-associated beta-xylosidase from Aureobasidium.

beta-Xylosidase was extracted from Aureobasidium sp. ATCC 20524 and purified to homogeneity. The molecular mass was estimated at 411 kDa. The enzyme contained 15.3% (w/w) carbohydrate. The optimum pH and temperature were pH 3.5 and 80 degrees C, respectively. The enzyme was stable at pH 3.5-9 after 3 h and at 80 degrees C after 15 min. The Michaelis constant (K(m)) and maximum velocity (V(max)) toward p-nitrophenyl-beta-D-xyloside were 2.0 mmol l(-1) and 0.94 mmol min(-1) mg(-1) protein, respectively. The enzyme was inhibited strongly by mercury, lead, and copper ions.

Copper↗
PubMed2001

A novel differentiation-inducing therapy for acute promyelocytic leukemia with a combination of arsenic trioxide and GM-CSF.

Arsenic trioxide (As2O3) effectively induces clinical remission via apoptosis in relapsed acute promyelocytic leukemia (APL). However, because this new anti-leukemic drug is also considered to be a poison, its possible adverse effects are a highly important issue related to its clinical use. We here investigated, both in vitro and in vivo, the effects of a combination of As2O3 and GM-CSF as a novel therapeutic approach for the treatment of APL. Treatment of both retinoic acid (RA)-sensitive and -resistant APL cell lines (NB4 and UF-1 cells, respectively), as well as primary APL cells with a combination of As2O3 and GM-CSF for 4 days resulted in inducing differentiation, but not apoptosis, to mature granulocytes. In addition, a combination of both agents induced degradation of the PML/RARalpha protein. GM-CSF was found to be associated with increased tyrosine phosphorylation of Jak2 kinase in both NB4 and UF-1 cells, and a specific inhibitor of Jak2, AG490, completely blocked the ability of GM-CSF to prevent apoptosis and induce differentiation of As2O3-treated UF-1 cells. In in vivo analysis, As2O3 induced differentiation of APL cells in a RA-resistant APL model of human GM-CSF-producing transgenic SCID mice that had a high level of human GM-CSF in their sera. In contrast, As2O3 alone diminished tumors in UF-1 cells transplanted into NOD/SCID mice via induction of apoptosis. In conclusion, a combination of As2O3 and GM-CSF appears to be a novel differentiation-inducing therapy in patients with APL, including relapsed or RA-resistant cases.

Animals↗
PubMed2001

Histologic study of the regeneration process of human hair follicles grafted onto SCID mice after bulb amputation.

This study examines histologically the degeneration and subsequent regeneration processes of human hair follicles whose bulb is severely damaged. Human scalp hair follicles were isolated and grafted onto immunodeficient mice after their bulb was amputated. On day 14, thickening and corrugation of the vitreous membrane, apoptosis of follicular keratinocytes, and regression of the lower portion of the follicles were observed. By day 20, mesenchymal cells had accumulated around the lower end of the follicles. From day 14 through 50, the follicular regression and apoptosis continued, and between days 30 and 40 the follicles became maximally shortened, and the vitreous membrane disappeared. By day 50 the lower end of the follicles had become cup-shaped, and the cup surrounded an aggregate of mesenchymal cells that corresponded to the dermal papilla. By day 60, all the grafted follicles had developed into anagen VI follicles, and the apoptosis had ceased. These results indicate that human scalp hair follicles whose bulb is completely destroyed enter into dystrophic telogen after restoration of the dermal papilla, then into anagen, and that the duration of the dystrophic telogen is shorter than that of the normal hair cycle.

Animals↗
PubMed2001

Expression of calcium-binding S100 proteins A4 and A6 in regions of the epithelial sac associated with the onset of hair follicle regeneration.

In order to elucidate the onset mechanisms of hair regrowth, we characterized the expression of metastasis and cell-growth-associated calcium-binding S100 proteins in the regenerating follicular epithelium. Hair-cycle-dependent expression of S100A4 and S100A6 was found in the epithelial sac regions (bulge area and hair germ) of mouse pelage follicles. Protein localization of S100A4 was confined to the bulge area, the region where the presumed follicular stem cells reside, during the catagen-telogen-anagen transitional periods, whereas S100A6 protein was distributed throughout the epithelial sac regions during anagen phase. Prior to entering anagen phase, however, both S100 mRNAs were upregulated in the epithelial sac. Despite the induction of extensive cell death in the bulge region after plucking, a new hair cycle was initiated following transcription of S100A6 in the hair germ. Upon wounding stimuli, both the outer root sheath and the basal layer of epidermis expressed S100A6 mRNA prior to hyper-proliferation. Once the epithelial sac was induced to transcribe both S100 genes, resting follicles were concomitantly rejuvenated. These results suggest that S100A4 and S100A6 might play important roles in the activation of stem cells at the onset of follicle regeneration.

Animals↗
PubMed2001

Dystrophic scrotal calcinosis originating from benign eccrine epithelial cysts.

Scrotal calcinosis has been classified as a form of idiopathic calcinosis cutis. However, the pathogenesis of the calcified nodules has not been fully elucidated: it is still unclear whether the condition is truly idiopathic, or the result of breakdown of calcified epithelial cysts. We describe a 29-year-old Japanese patient with scrotal calcinosis originating from epithelial cysts. Light microscopy revealed a large epithelial cyst containing von Kossa-positive material and several small dilated ductal structures beside the cyst. The epithelia of the cyst and ductal structures were connected, showing similar eccrine duct differentiation on immunohistochemical staining and electron microscopy. In the cyst lumen, calcium was present as needle-shaped crystals. The pathogenetic mechanism of calcium deposition seemed to be due to excessive production and discharge of matrical debris and sulphated mucopolysaccharides, which derived from luminal cells, and their accumulation in the lumina.

Adult↗