Biomedical subjects

Y Matsubara

Publications and source records attributed to Y Matsubara.

At least 91 records · Page 5Linked to original sources

PubMed1999

Spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin during the development of chronic contact dermatitis.

In order to elucidate how chronic inflammation affects the organization of the extracellular matrix in the skin, a prolonged allergic contact dermatitis was induced in a mouse by repeated application to the ear of 2,4-dinitrofluorobenzene every 3 d for 66 d. Subsequently, the spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin were examined. In the acute phase of inflammation (day 3-day 12), the amount of fibronectin and tenascin-C increased markedly and were degraded, whereas the amount of fibulin-2 changed slightly. Abundant deposition of tenascin-C was observed in the connective tissue. Fibulin-1 and fibulin-2 distributed as fine fibrils. In contrast, the amounts of fibronectin and tenascin-C decreased and their degradation was suppressed in the chronic phase (day 15-day 66), but the amount of fibulin-2 increased. Tenascin-C was observed mainly at and underneath the epidermal basement membrane. In the subepidermal region, many fibulin-2-positive microfibrils were distributed. The amount and distribution of fibulin-1 did not change markedly in either phase. MMP-like enzymes of 62 kDa, probably activated MMP-2, were upregulated in the chronic phase, whereas components of 92, 85, or 67 kDa were highly induced in the acute phase. These results suggest that chronic inflammation in allergic contact dermatitis is associated with temporal changes in the expression, deposition, and degradation of inducible extracellular matrix components.

Animals↗

PubMed1999

A fluorogenic allele-specific amplification method for DNA-based screening for inherited metabolic disorders.

Rapidly growing mutation databases for various inherited metabolic diseases raise the possibility of neonatal screening by DNA-based diagnosis. It is therefore important to develop a simple DNA diagnostic method which is suitable for processing a large number of samples. We have devised an allele-specific amplification (ASA) method with a fluorogenic probe (TaqMan probe) to detect point mutations. A pairwise PCR amplification with two sets of allele-specific primers was performed in the presence of the TaqMan probe with real-time fluorescence monitoring on an ABI PRISM 7700 sequence detector. The difference in the amplification efficiency between two PCR reactions was determined by "threshold" cycles to differentiate mutant and normal alleles. The method, TaqMan-ASA, does not require post-PCR processing, thus obviating potential PCR contamination. Since the entire procedure can be carried out in a 96-well microtiter plate format, it is easy to automate. We successfully applied TaqMan-ASA to detect a common g727t mutation in Japanese patients with glycogen storage disease type Ia and a prevalent a985g mutation in Caucasian patients with medium-chain acyl-CoA dehydrogenase deficiency.

Alleles↗

PubMed1999

Reversal of hypopigmentation in phenylketonuria mice by adenovirus-mediated gene transfer.

Phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH) in the liver. Patients with PKU show increased L-phenylalanine in blood, which leads to mental retardation and hypopigmentation of skin and hair. As a step toward gene therapy for PKU, we constructed a replication-defective, E1/E3-deleted recombinant adenovirus harboring human PAH cDNA under the control of a potent CAG promoter. When a solution containing 1.2 x 10(9) plaque-forming units of the recombinant adenovirus was infused into tail veins of PKU model mice (Pah(enu2)), predominant expression of PAH activity was observed in the liver. The gene transfer normalized the serum phenylalanine level within 24 h. However, it also provoked a profound host immune response against the recombinant virus; as a consequence, the biochemical changes lasted for only 10 d and rechallenge with the virus failed to reduce the serum phenylalanine concentration. Administration of an immunosuppressant, FK506, to mice successfully blocked the host immune response, prolonged the duration of gene expression to more than 35 d, and allowed repeated gene delivery. We noted a change in coat pigmentation from grayish to black after gene delivery. The current study is the first to demonstrate the reversal of hypopigmentation, one of the major clinical phenotypes of PKU in mice as well as in humans, by adenovirus-mediated gene transfer, suggesting the feasibility of gene therapy for PKU.

Adenoviridae↗

PubMed1999

Differentiation of donor primordial germ cells into functional gametes in the gonads of mixed-sex germline chimaeric chickens produced by transfer of primordial germ cells isolated from embryonic blood.

This study was carried out to elucidate whether primordial germ cells, obtained from embryonic blood and transferred into partially sterilized male and female recipient embryos, could differentiate into functional gametes and give rise to viable offspring. Manipulated embryos were cultured until hatching and the chicks were raised until maturity, when they were mated. When the sex of the donor primordial germ cells and the recipient embryo was the same, 15 out of 22 male chimaeric chickens (68.2%) and 10 out of 16 female chimaeric chickens (62.5%) produced donor-derived offspring. When the sex of the donor primordial germ cells and the recipient embryo was different, 4 out of 18 male chimaeric chickens (22.2%) and 2 out of 18 female chimaeric chickens (11.1%) produced donor-derived offspring. The rates of donor-derived offspring from the chimaeric chickens were 0.6-40.0% in male donor and male recipient and 0.4-34.9% in female donor and female recipient. However, the rates of donor-derived offspring from the chimaeric chickens were 0.4-0.9% in male donor and female recipient and 0.1-0.3% in female donor and male recipient. The presence of W chromosome-specific repeating sequences was detected in the sperm samples of male chimaeric chickens produced by transfer of female primordial germ cells. These results indicate that primordial germ cells isolated from embryonic blood can differentiate into functional gametes giving rise to viable offspring in the gonads of opposite-sex recipient embryos and chickens, although the efficiency was very low.

Animals↗

PubMed1999

Determining factors of mortality in the elderly with hip fractures.

We conducted a retrospective study of the influence of various factors on the mortality of 114 patients with hip fractures. The mortality rate one year after surgery was 18%, which was 2.5 times larger than that of the general population. It was related to age, ECG abnormality, and post-operative complications.

Age Distribution↗

PubMed1999

[A case of secondary systemic amyloidosis associated with rheumatoid arthritis after 3-year disease duration].

A 63-year-old woman who started to have polyarthralgia in December 1993 has been diagnosed as rheumatoid arthritis (RA) and treated with muscular injection of gold sodium thiomalate. She began to have nausea, vomiting, anorexia and watery diarrhea in October 1995. A year later, she had to receive intravenous infusion on admission since more frequent watery diarrhea occurred more than ten times within a day. On admission in our hospital in December 1996, she had proteinuria in addition to gastrointestinal symptoms. The biopsy specimen from stomach, duodenum and kidney proved systemic amyloidosis associated with RA. In spite of steroid-pulse, dimethyl sulfoxide (DMSO) and colchicine therapy, profound proteinuria in nephrotic syndrome was continued in association with hypoproteinemia, anasarca and renal failure. She was treated on hemodialysis and intravenous hyperalimentation (IVH) until November 1997 when A-V shunt operation on left forearm was performed. However, the shunt was not available for HD and she suffered from septicemia and died on December 1997. This patient was a rare case of secondary systemic amyloidosis associated with RA in early clinical course.

Amyloidosis↗

PubMed1999

[A case of systemic lupus erythematosus associated with severe acute pancreatitis].

A 22-year-old woman began to have the symptoms of anorexia, high fever, cough and general fatigue from June of 1997. She was admitted in our hospital on Aug. 8th, 1997 for the further detail examination because of pancytopenia and positive antinuclear antibody (ANA). Her laboratory findings and clinical symptoms were compatible with systemic lupus erythematosus (SLE) such as leukopenia, proteinuria, hypocomplementemia, positive ANA, elevated titer of autoantibodies including anti-DNA, anti-Sm, anti-RNP antibodies, polyarthralgia and photosensitivity. The administration of oral prednisolone (40 mg/day) was started on Aug. 15th, 1997 under the diagnosis of SLE. However, she had severe abdominal pain in epigastrium with elevated serum amylase, ascites and dull shape of pancreas tail by CT scan compatible with acute pancreatitis. On Aug. 18th, her general condition was worsening with fever, epigastralgia, abdominal distension, anemia, weak palpation of radial artery, hypotension, tachycardia, shallow breathing and cold sensation on both extremities as shock. In spite of steroid pulse therapy with nafamostat mesilate intraarterial infusion, her condition was not improved. The dose of 50 mg/day of cyclophosphamide was added to the regimen on Aug. 22nd. Then, gradually her condition started to be restored. Anemia, leukopenia, hypocomplementemia continued. Second steroid pulse therapy was done on Sep. 5th. After then, she became better in her clinical symptoms and laboratory data. The dose of PSL was tapered to 15 mg/day and 7.5 mg/day update of Oct. 1998 without the pseudcysts found after pancreatitis. She is a rare case who recovered from severe acute pancreatitis due to SLE itself.

Acute Disease↗

PubMed1999

[Eosinophilic pneumonia without eosinophilia in BALF or peripheral blood and diagnosed by open lung biopsy].

A 47-year-old woman was referred to our hospital because of cough and an abnormal shadow in the left lung field. The infiltrate reduced without therapy and another infiltrate appeared in the right lung field. Bronchiolitis obliterans organizing pneumonia was clinically suspected due to the absence of signs of eosinophilia in peripheral blood and bronchoalveolar lavage fluid (BALF). Open lung biopsy specimens disclosed alveolitis with mononuclear cell infiltration and organization within the air spaces of bronchioli and alveolar ducts. The observation of pronounced eosinophil infiltration in the alveolar spaces of some specimens yielded a diagnosis of eosinophilic pneumonia. After steroid therapy, the abnormal shadows disappeared. BALF lymphocyte surface marker analysis detected no decrease in the CD4/CD8 ratio; activated CD4 and CD8 lymphocytes were notably higher than the corresponding levels in peripheral blood. IL-5, IL-3, and GM-CSF values in BALF were not significantly elevated. This was a case of borderline eosinophilic pneumonia that was difficult to diagnose on the basis of clinical parameters alone.

Anti-Inflammatory Agents↗

PubMed1999

Genotype frequency of plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism in healthy Japanese males and its relation to PAI-1 levels.

Plasminogen activator inhibitor-1 (PAI-1) plays an inhibitory role in the fibrinolytic enzyme system and is associated with thrombotic diseases. The gene for PAI-1 has an insertion/deletion polymorphism at the promoter region, the 4G/5G polymorphism, which is related to differences in transcription activity in vitro. Association of the 4G/5G polymorphism with plasma PAI-1 levels, however, has not been uniformly reported. We evaluated the relationship between the 4G/5G polymorphism and plasma PAI-1 antigen levels in 104 Japanese males not taking lipid-lowering drugs and without non-insulin-dependent (type 2) diabetes mellitus or coronary artery disease. The genotype frequency was 37.5%, 50.0%, and 12.5% for 4G/4G, 4G/5G, and 5G/5G genotypes, respectively, which differs from that reported for healthy Caucasian males (P < 0.01), with the 4G allele more frequently found in the Japanese population. No association was found between the 4G/5G polymorphism and plasma PAI-1 antigen levels. Multiple regression analysis revealed a significant (P < 0.01) contribution of triglyceride (TG) levels to variations in plasma PAI-1 antigen levels. The correlation between TG levels and plasma PAI-1 antigen levels was not 4G/5G genotype-specific. These findings suggest that PAI-1 4G/5G polymorphism is not associated with plasma PAI-1 antigen levels among healthy Japanese males and that TG levels correlate to plasma PAI-1 antigen levels in all PAI-1 4G/5G genotypes.

Adult↗

PubMed1998

Immunohistochemical detection of parasite antigens in Theileria parva-infected bovine lymphocytes.

Theileria parva is the causal agent of East Coast fever (ECF), a fatal disease of cattle characterized by pyrexia, transient lymphadenopathy and panleukopenia. We have evaluated monoclonal antibodies (mAb) against three distinct antigens (p67, PIM and p32) of the parasite as immunohistological reagents for monitoring the kinetics of infection in cattle. Bovine lymphocytes were stained with the mAb at various intervals after infection in vitro and in vivo. The p67 sporozoite surface antigen was detected in only a small percentage of both, in vitro and in vivo infected cells. In contrast, expression of the polymorphic immunodominant molecule (PIM) of the parasite proved a useful indicator of infection and staining was correlated with the results of Giemsa analysis. PIM was detected from day 3 in in vitro-infected cells, but was not detected until day 5 in vivo after challenge with a 70% lethal dose of stabilized sporozoite. The p32 antigen was expressed only late in infection in vivo and its expression was associated with the development of merozoites. Less than 20% of in vitro-infected cells expressed p32. The immunohistochemical staining with anti-PIM mAb was found to be a useful tool for analysis of T. parva infection kinetics in cattle.

Animals↗

PubMed1998

Hepatocyte growth factor activator: a possible regulator of morphogenesis during fetal development of the rat gastrointestinal tract.

The role played by the hepatocyte growth factor activator (HGFA) during morphogenesis of the gastrointestinal tract was investigated in fetal rats between days 16 and 21 of gestation. By our recently established method using chelation and dissecting microscope, samples could be separated into epithelium and mesenchyme, essentially without cross-contamination. The expression of the gene for HGFA together with those for hepatocyte growth factor (HGF) and its receptor, c-met, was investigated in each tissue element by RT-PCR. In the fetal rat gastrointestinal tract, mRNA signals for the HGFA gene were observed only in epithelia expressing c-met mRNA. In contrast, expression of HGF mRNA was limited to the mesenchymal elements, indicating the presence of a local HGF system in the gastrointestinal tract; an inactive form of HGF (proHGF) is secreted from the mesenchyme and then cleaved into the active form by HGFA secreted by the target epithelia. During the period of morphogenesis and histodifferentiation in the gastrointestinal tract, enhanced expression of the genes for HGF and its receptor/c-met was evident, with elevated HGFA mRNA level observed throughout the gastrointestinal tract except in the forestomach, where mRNA expression was barely detectable. These results strongly suggest the possibility that morphogenesis of the gastrointestinal tract is regulated not only by a local increase in production of HGF, but also by enhanced proteolytic activation of proHGF. Thus, it is probable that locally synthesized HGFA plays a significant role as a regulator of the morphogenic action of HGF during gastrointestinal tract development.

Animals↗

PubMed1998

Molecular analysis of glycogen storage disease type Ib: identification of a prevalent mutation among Japanese patients and assignment of a putative glucose-6-phosphate translocase gene to chromosome 11.

Glycogen storage disease type Ib (GSD-Ib) is an inborn error of metabolism with autosomal recessive inheritance, caused by defects in microsomal transport of glucose-6-phosphate. Recently, Gerin et al isolated a human cDNA encoding a putative transporter homologous to bacterial transporters of hexose-6-phosphate, and identified two mutations in its gene in two patients with GSD-Ib (9). Independently, a linkage analysis mapped the GSD-Ib gene on chromosome 11q23 (10). It remains to be elucidated whether the two genes are identical or GSD-Ib is genetically heterogeneous. We first mapped the transporter gene on chromosome 11 by using a DNA panel of human/hamster hybridoma cells. The result suggested that the GSD-Ib genes identified by the two distinct approaches may be identical and GSD-Ib was allelic. We then studied four unrelated Japanese families with GSD-Ib, and found three novel mutations: a four-base deletion/two-base insertion, a point mutation within a consensus splicing donor site, and a missense mutation (W118R). The W118R mutation was found in 4 out of 8 mutant alleles, suggesting that it is prevalent among Japanese patients.

Amino Acid Sequence↗

PubMed1998

Spontaneous transdifferentiation of quail pigmented epithelial cell is accompanied by a mutation in the Mitf gene.

An ectopic neural retina is formed at the outer layer of the retina in the silver homozygote (B/B) of the Japanese quail. In situ hybridization and immunohistochemical analysis revealed that cells in the outer layer of retina first expressed a pigment-cell-specific gene, mmp115, and then began to express a neural marker in B/B embryos, indicating that the ectopic neural retina is formed via transdifferentiation of differentiated pigmented epithelial cells (PECs). An in vitro study revealed that cultured retinal PECs (rPECs) from B/B embryos exhibit less pigment granule and a higher growth rate than cells from heterozygotes (B/+). B/+ PECs stopped proliferating when confluency was reached, while B/B PECs continued to proliferate. Some B/B cells overlaid other B/B cells and formed lentoid bodies. Immunological analysis revealed that B/B rPECs transdifferentiated to lens cells and neural cells in vitro with no addition of basic FGF (bFGF), while B/+ rPECs required bFGF to transdifferentiate. Expression of PEC-specific genes, mmp115, tyrosinase, and TRP-1, was downregulated, but that of Mitf and pax6 was upregulated in B/B PECs. Antibody against Mitf stained the nucleus of B/+ PECs but not that of B/B cells, suggesting that the normal Mitf is not present in the silver homozygote due to mutation. Sequence analysis revealed that Mitf from the silver homozygote has an amino acid substitution in the basic region and is truncated in the C-terminal region. Transient transfection analysis revealed that Mitf from the silver homozygote exhibits a lower level of activity than wild-type Mitf with respect to transactivation of the mmp115 promoter. Furthermore, overexpression of chicken Mitf induced normal pigmentation in B/B rPECs. These results strongly suggest that the silver phenotype is caused by the mutation of Mitf and that Mitf plays a critical role in rPEC differentiation and transdifferentiation.

Amino Acid Sequence↗

PubMed1998

Transient up-regulation of a prolyl endopeptidase activity in the microsomal fraction of rat liver during postnatal development.

To identify proteases which are involved in cell proliferation and differentiation of liver cells, we assayed various protease activities in rat liver during its postnatal development. We found that a protease activity specific to proline residues in the microsomal fraction of rat liver was transiently increased around postnatal day 8 and decreased thereafter, indicating that the enzyme activity is highly correlated to the proliferation and differentiation of liver cells. This protease was purified to an apparent homogeneity from the microsomal fraction of the postnatal-day-8 rat liver. The purified enzyme gave a single band with an apparent molecular mass of 65,000 on SDS/PAGE. It cleaved several peptide 4-methylcoumaryl-7-amide substrates and bioactive peptides at the C-terminus of proline residues. The enzyme did not hydrolyze any protein substrate examined suggesting that it is a peptidase rather than a proteinase. Although the best substrate among those tested was succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide (-NH-Mec), the purified enzyme did not hydrolyze succinyl-Gly-Pro-NH-Mec, indicating that the enzyme has different substrate specificity from any hitherto known prolyl endopeptidases. These results suggest that the purified enzyme is a unique prolyl endopeptidase which may be involved in proliferation and differentiation of liver cells.

Animals↗

PubMed1998

Two CPT2 mutations in three Japanese patients with carnitine palmitoyltransferase II deficiency: functional analysis and association with polymorphic haplotypes and two clinical phenotypes.

Carnitine palmitoyltransferase II (CPT II) deficiency manifests as two different clinical phenotypes: a muscular form and a hepatic form. We have investigated three nonconsanguineous Japanese patients with CPT II deficiency. Molecular analysis revealed two missense mutations, a glutamate (174)-to-lysine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPT II cDNA. Transfection experiments in COS-1 cells demonstrated that the two mutations markedly decreased the catalytic activity of mutant CPT II. Case 1 (hepatic form) was homozygous for the F383Y mutation, whereas case 3 (muscular form) was homozygous for the E174K mutation. Case 2 and her brother, who were compound heterozygotes for E174K and F383Y, exhibited the hepatic phenotype. We also identified a novel polymorphism in the CPT2 gene, a phenylalanine (352)-to-cysteine substitution (F352C), which did not alter CPT II activity in transfected cells. It was present in 21 out of 100 normal alleles in the Japanese population, but absent in Caucasian populations. Genotyping with the F352C polymorphism and the two previously reported polymorphisms, V368I and M647V, allowed normal Japanese alleles to be classified into five haplotypes. In all three families with CPT II deficiency, the E174K mutation resided only on the F1V1M1 allele, whereas the F383Y mutation was observed on the F2V2M1 allele, suggesting a single origin for each mutation.

Adult↗

PubMed1998

Immunological competence and nutritional status in patients with lung cancer.

The rate of infection in patients with malignant disease is significantly higher than in patients with benign disease. To investigate whether immunological competence is impaired in patients with lung cancer, we assessed neutrophil function (chemotaxis, phagocytosis, bacterial killing activity, and superoxide production), monocyte function (phagocytosis and killing activity), lymphocyte subsets using flow cytometry, and proliferation of lymphocytes stimulated by phytohemagglutinin, concanavalin A, and pokeweed mitogen. Studies were performed on 22 untreated patients with lung cancer and 21 age-matched healthy volunteers. Nutritional status was assessed by Niederman's nutritional index. In patients with lung cancer neutrophil chemotaxis, monocyte phagocytosis and killing, proliferation of lymphocytes stimulated by phytohemagglutinin and concanavalin A, but not pokeweed mitogen, and the number of natural killer cells were significantly lower than in healthy volunteers, whereas gammadelta T cells were increased (p < 0.05). The mean score on Niederman's nutritional index was worse in patients than in healthy volunteers (p < 0.001). Our results suggest that the impaired immunological competence and undernutrition may be among the mechanisms causing increased susceptibility to infection in patients with lung cancer.

Adenocarcinoma↗

PubMed1998

A missense mutation (His42Arg) in the T-protein gene from a large Israeli-Arab kindred with nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is caused by a mutation in the genes encoding the components of the glycine cleavage multi-enzyme system. More than 80% of the patients have defects in the gene encoding P-protein, whereas the rest of the patients have defects in the gene encoding T-protein. We have found a large Israeli-Arab kindred with NKH. At least 14 children were affected, and all the patients had seizures and respiratory failure within 2 days after birth. Enzymatic analysis revealed that T-protein activity was deficient in the liver specimen from one propositus. We screened this family for a mutation in the protein-coding region and exon/intron boundaries of T-protein gene by direct sequencing analysis. A missense mutation was found in exon 2; this resulted in an amino acid substitution from histidine to arginine at position 42 (H42R). Histidine 42 is conserved in human, bovine, chicken, pea, and Escherichia coli, suggesting that it has an important role in catalytic functions. Genotype analyses of 26 family members confirmed that the homozygous H42R mutation was completely associated with the onset of NKH. The availability of DNA testing facilitates the prenatal diagnosis of NKH and the identification of carriers, which is necessary for genetic counseling in the affected families.

Amino Acid Metabolism, Inborn Errors↗

PubMed1998

Mutation and polymorphic marker analyses of 65K- and 67K-glutamate decarboxylase genes in two families with pyridoxine-dependent epilepsy.

Pyridoxine-dependent epilepsy is a disease inherited as an autosomal recessive trait, characterized by rapid response to pharmacological dosages of pyridoxine. The defect has been suggested to reside in glutamate decarboxylase (GAD), since a mutant GAD with an abnormally high Km for a cofactor, pyridoxal phosphate, could not synthesize an adequate amount of gamma-amino butyric acid [Scriver and Whelan (1969) Ann NY Acad Sci 166: 83]. To test this hypothesis, we studied two affected families by screening for mutations in the GAD mRNA and by analyzing a polymorphic marker in the GAD gene. Since two forms of GAD, GAD65 and GAD67, have been identified in human brain, we analyzed both forms. To overcome the limited accessibility of brain tissues, we utilized the minute amounts of GAD mRNAs ectopically transcribed in lymphoblasts. The ectopic GAD transcripts were amplified by reverse-transcription-mediated, nested polymerase chain reaction for mutation analysis. Two and three base substitutions were found in GAD65 and GAD67 cDNAs, respectively. All of them were, however, polymorphisms that were also found in control subjects. We then examined a (CA) repeat polymorphism in the GAD65 gene and found that different maternal alleles were transmitted to two affected sibs in one family. Thus, an etiological mechanism other than a K(m) mutant GAD is responsible for pyridoxine-dependent epilepsy.

DNA Mutational Analysis↗