Biomedical subjects

M Sekiguchi

Publications and source records attributed to M Sekiguchi.

At least 55 records · Page 3Linked to original sources

PubMed2000

Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein.

Escherichia coli MutT protein hydrolyzes 8-oxo-7,8-dihydro-2'-dGTP (8-oxo-dGTP) to the monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine (8-oxo-G) into nascent DNA. Bacterial and mammalian homologs of MutT protein share the phosphohydrolase module (MutT: Gly37-->Gly59). By saturation mutagenesis of conserved residues in the MutT module, four of the 10 conserved residues (Gly37, Gly38, Glu53 and Glu57) were revealed to be essential to suppress spontaneous A:T-->C:G transversion mutation in a mutT(-) mutator strain. For the other six residues (Lys39, Glu44, Thr45, Arg52, Glu56 and Gly59), many positive mutants which can suppress the spontaneous mutation were obtained; however, all of the positive mutants for Glu44 and Arg52 either partially or inefficiently suppressed the mutation, indicating that these two residues are also important for MutT function. Several positive mutants for Lys39, Thr45, Glu56 and Gly59 efficiently decreased the elevated spontaneous mutation rate, as seen with the wild-type, hence, these four residues are non-essential for MutT function. As Lys38 and Glu55 in human MTH1, corresponding to the non-essential residues Lys39 and Glu56 in MutT, could not be replaced by any other residue without loss of function, different structural features between the two modules of MTH1 and MutT proteins are evident.

Amino Acid Sequence↗

PubMed2000

Accumulation of adenine DNA glycosylase-sensitive sites in human mitochondrial DNA.

The mitochondrial respiratory chain inevitably produces reactive oxygen species as byproducts of aerobic ATP synthesis. Mitochondrial DNA (mtDNA), which is located close to the respiratory chain, is reported to contain much more 8-oxoguanine (8-oxoG), an oxidatively modified guanine base, than nuclear DNA. Despite such a high amount of 8-oxoG in mtDNA (1-2 8-oxoG/10(4) G), mtDNA is barely cleaved by an 8-oxoG DNA glycosylase or MutM, which specifically excises 8-oxoG from a C:8-oxoG pair. We find here that about half of human mtDNA molecules are cleaved by another 8-oxoG-recognizing enzyme, an adenine DNA glycosylase or MutY, which excises adenine from an A:8-oxoG pair. The cleavage sites are mapped to adenines. The calculated number of MutY-sensitive sites in mtDNA is approximately 1.4/10(4) G. This value roughly corresponds with the electrochemically measured amount of 8-oxoG in mtDNA (2.2/10(4) G), raising the possibility that 8-oxoG mainly accumulates as an A:8-oxoG pair.

Adenine↗

PubMed2000

Contribution of cardiac muscle cell disorganization to the clinical features of hypertrophic cardiomyopathy.

Heart failure rarely develops in the setting of hypertrophic cardiomyopathy (HCM). Because of this, cardiac muscle cell disorganization (CD), which is a histologic characteristic of HCM, is not believed to be responsible for the development of systolic dysfunction. The aim of the present study was to clarify whether CD can cause systolic dysfunction and ventricular dilation in patients with HCM. Sixteen hearts from patients with HCM obtained at autopsy were divided into two groups: group A (n = 11), without biventricular dilation, and group B (n = 5), with dilation. Specimens consisting of transverse and longitudinal tissue sections of the ventricles were prepared, and the extent of CD and interstitial fibrosis was quantified, using light microscopy. None of the patients in group A had had chronic congestive heart failure, while all of the patients in group B had died of congestive heart failure. In group B, CD was not limited to the interventricular septum. Rather, diffuse CD was observed in both ventricular free walls. The extent of CD was significantly greater in group B than in group A, while the degree of interstitial fibrosis was similar in the two groups (13.6% in group A vs 14.6% in group B). These results suggest that CD may be responsible for systolic dysfunction and ventricular dilation.

Adolescent↗

PubMed2000

Detection of the association between a deletion polymorphism in the gene encoding angiotensin I-converting enzyme and advanced diabetic retinopathy.

We investigated the relationship between advanced diabetic retinopathy (ADR) and an angiotensin-converting enzyme (ACE) gene polymorphism in subjects with type 2 diabetes and ADR, pre-proliferative (PrePDR) or proliferative diabetic retinopathy (PDR) without overt nephropathy. Polymerase chain reactions were used to detect insertion/deletion (I/D) polymorphisms of the ACE gene. There was no difference in the frequency of II, ID, or DD genotypes, or of I and D alleles among subjects with type 2 diabetes without diabetic retinopathy (NDR) or with simple diabetic retinopathy (SDR) and non-diabetic controls. There was also no difference in the frequency of ACE genotypes among subjects with type 2 diabetes with NDR, or SDR and ADR. However, the frequency of the ACE DD genotype in ADR was significantly higher than that in controls (chi(2)=6.64, P=0.036). On the other hand, the frequency of the D allele in ADR was significantly higher than that in controls (chi(2)=6.33, P=0.012), NDR (chi(2)=4.18, P=0.041) and SDR (chi(2)=4. 89, P=0.027), respectively. These results indicate a significant relationship between the presence of the D allele polymorphism in the ACE gene and ADR in Japanese subjects with type 2 diabetes and no overt nephropathy.

Albuminuria↗

PubMed2000

Spontaneous B-cell IgE production in a patient with remarkable eosinophilia and hyper IgE.

BACKGROUND: The pathophysiology of eosinophilia and hyper-IgE is not fully elucidated yet. OBJECTIVE: To clarify the pathophysiology of a patient with remarkable eosinophilia and hyper IgE, we examined cytokine levels in serum, surface antigens of peripheral blood eosinophils and IgE production in vitro. RESULTS: Concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), and granulocyte/macrophage-colony stimulating factor (GM-CSF) in the serum were 21 pg/mL, <15 pg/mL, <15 pg/mL, 8 pg/mL, and <5 pg/mL pg/mL, respectively. Newly expressed surface antigens CD4, CD25, CD69, and HLA-DR, but not CD54, were observed on peripheral blood eosinophils. Extremely high levels of IgE secretion was found in the patient's mononuclear cells without stimuli; this was not enhanced by IL-4 or IL-4 plus anti-CD40 monoclonal antibody stimulation. Furthermore, highly purified B cells spontaneously produced large amounts of IgE and the production was not enhanced in addition of his T cells. CONCLUSION: The eosinophils were activated, and the B cells spontaneously produced IgE independently of T cells or cytokines, suggesting that intrinsic abnormality of B cells leading to dysregulated production of IgE in this disease.

Aged↗

PubMed2000

Echinophyllins C-F, new nitrogen-containing clerodane diterpenoids from Echinodorus macrophyllus.

Four new nitrogen-containing clerodane diterpenoids, echinophyllins C-F (1-4), were isolated from the leaves of the Brazilian medicinal plant Echinodorus macrophyllus ("Chapéu-de-couro"), and their structures and relative stereochemistry were elucidated from their spectroscopic data. Compounds 1-4 possess an alpha,beta-unsaturated gamma-lactam ring consisting of a clerodane diterpene unit and an amine moiety.

Diterpenes↗

PubMed2000

Chapecoderins A-C, new labdane-derived diterpenoids from Echinodorus macrophyllus.

A new seco-labdane-type diterpenoid, chapecoderin A (1), and two new rearranged labdane-type diterpenoids, chapecoderins B (2) and C (3), were isolated from the leaves of the Brazilian medicinal plant Echinodorus macrophyllus ("Chapéu-de-couro"), and their structures and relative stereochemistry were elucidated by spectroscopic data. Chapecoderins A-C (1-3) possess in common an alpha,beta-unsaturated gamma-lactone ring in the side chain.

Molecular Structure↗

PubMed2000

Identification of the major Abeta1-42-degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition.

Alzheimer amyloid beta-peptide (Abeta) is a physiological peptide constantly anabolized and catabolized under normal conditions. We investigated the mechanism of catabolism by tracing multiple-radiolabeled synthetic peptide injected into rat hippocampus. The Abeta1-42 peptide underwent full degradation through limited proteolysis conducted by neutral endopeptidase (NEP) similar or identical to neprilysin as biochemically analyzed. Consistently, NEP inhibitor infusion resulted in both biochemical and pathological deposition of endogenous Abeta42 in brain. This NEP-catalyzed proteolysis therefore limits the rate of Abeta42 catabolism, up-regulation of which could reduce the risk of developing Alzheimer's disease by preventing Abeta accumulation.

Amino Acid Sequence↗

PubMed2000

Use of domain-swapped molecules for conformational epitope mapping of desmoglein 3 in pemphigus vulgaris.

Pemphigus vulgaris is an autoimmune blistering disease caused by autoantibodies against desmoglein 3, a member of the desmosomal cadherin family. These autoantibodies recognize conformation-dependent epitopes on desmoglein 3. In this study we attempted to map the conformational epitopes of desmoglein 3 in pemphigus vulgaris using recombinant desmoglein 3 produced by the baculovirus expression system. We developed a series of domain-swapped molecules between desmoglein 3 and desmoglein 1, which have similar structures but distinct epitopes. These were developed by substituting deleted segmental regions of desmoglein 3 by the corresponding desmoglein 1. Thus domain-swapped molecules containing desmoglein 3 residues 1-403, 1-161, 163-566, and 405-566 were constructed and used as competitors for competition enzyme-linked immunosorbent assay against the entire extracellular domain of desmoglein 3 with 25 pemphigus vulgaris sera. Considering more than 50% absorption as significant, residues 1-403 and 1-161 showed significant absorption in 24 out of 25 (96%) and 18 out of 25 (72%) pemphigus vulgaris sera, respectively, whereas only one serum and no sera showed significant absorption by residues 163-566 and 405-566, respectively. Furthermore, no apparent change in their major epitopes was seen during the time course in four pemphigus vulgaris cases tested. These findings indicate that the domain-swapping approach is useful for conformational epitope mapping in pemphigus and that amino-terminal residues 1-161, which are considered to include a region essential for cell-cell adhesion in cadherins, contain the critical residues of the conformational epitope of desmoglein 3 recognized by the autoantibodies in pemphigus vulgaris sera.

Autoantigens↗

PubMed2000

Concept for organ engineering: a reconstruction method of rat liver for in vitro culture.

In the past decade, there have been remarkable advances in tissue engineering technology toward the goal of creating organoids in vitro from cells and cellular scaffolding. Indeed, tissue-engineered organoids such as skin and cartilage, each with comparatively simple architectures, are presently at the clinical stage. However, conventional tissue engineering techniques have not allowed for the reconstruction of an organoid that mimics an organ of complex architecture of abundant vascular networks. We established a method for organ engineering that can remodel a rat liver into a reconstructed organoid without separating the majority of liver cells by a continuous three-step perfusion. The liver was perfused through its vascular system with a buffered balanced salt solution to cleanse blood from the organ, with a collagenase/dispase medium to deconstruct cellular scaffolds, and with a culture medium containing collagen type I to reorganize the multicellular architecture. The reconstructed organoid was then prepared by excising the perfused liver from the rat and culturing it at 37 degrees C for 2 h. Histologically healthy parenchymal hepatocytes expressing albumin were observed in the excised organoid even after culture for 3 weeks. Furthermore, a fibroblast-implanted organoid was prepared by using a culture medium containing suspended fibroblasts in the third step of the perfusion procedure, demonstrating the efficacy of heterogeneous cells for the reconstruction of an organoid. This method may be applicable to the formation of organoids from other organs, such as kidney and spleen, each of which have abundant capillaries, and therefore the method provides a novel concept for the development of lab-grown organs, i. e., organ engineering.

Animals↗

PubMed2000

Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase.

O(6)-methylguanine-DNA methyltransferase plays vital roles in preventing induction of mutations and cancer as well as cell death related to alkylating agents. Mice defective in the MGMT: gene, encoding the methyltransferase, were used to evaluate cell death-inducing and tumorigenic activities of therapeutic agents which have alkylation potential. MGMT(-/-) mice were considerably more sensitive to dacarbazine, a monofunctional triazene, than were wild-type mice, in terms of survival. When dacarbazine was administered i.p. to 6-week-old mice and survival at 30 days was enumerated, LD(50) values of MGMT(-/-) and MGMT(+/+) mice were 20 and 450 mg/kg body wt, respectively. Increased sensitivity of MGMT(-/-) mice to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACNU), a bifunctional nitrosourea, was also noted. On the other hand, there was no difference in survival of MGMT(+/+) and MGMT(-/-) mice exposed to cyclophosphamide, a bifunctional nitrogen mustard. It appears that dacarbazine and ACNU produce O(6)-alkylguanine as a major toxic lesion, while cyclophosphamide yields other types of modifications in DNA which are not subjected to the action of the methyltransferase. MGMT(-/-) mice seem to be less refractory to the tumor-inducing effect of dacarbazine than are MGMT(+/+) mice. Thus, the level of O(6)-methylguanine-DNA methyltransferase activity is an important factor when determining susceptibility to drugs with the potential for alkylation.

Adenoma↗

PubMed2000

A defect in a single allele of the Mlh1 gene causes dissociation of the killing and tumorigenic actions of an alkylating carcinogen in methyltransferase-deficient mice.

Mice with mutations in both alleles of the Mgmt and the Mlh1 gene, the former encoding a DNA repair methyltransferase and the latter a protein functioning at an early step of mismatch repair, are as resistant to the killing action of alkylating agents as are wild-type mice. These mice yielded a large number of tumors when exposed to alkylating carcinogens, but this characteristic was subdued since they also showed a relatively high level of spontaneous tumorigenicity, as a consequence of the defect in mismatch repair. This complexity is now resolved by introducing the Mlh1(+/-) mutation, instead of Mlh1(-/-), in these methyltransferase-deficient mice. Mgmt(-/-) Mlh1(+/-) mice, with about half the amount of MLH1 protein as Mgmt(-/-) Mlh1(+/+) mice, were resistant to the killing action of N-methyl-N-nitrosourea (MNU), up to the level of 30 mg/kg body wt. Eight weeks after exposure to this dose of MNU, 40% of MNU-treated Mgmt(-/-) Mlh1(+/-) mice had thymic lymphomas and there were no tumors in those mice not given the treatment. It seems that the cellular content of MLH1 protein is a critical factor for determining if damaged cells enter into either one of the two pathways leading to mutation induction or to apototic cell death. Loss of Mlh1 expression was frequently observed in tumors of Mgmt(-/-) Mlh1(+/-) mice and this might be related to progression of the tumors.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Six-year disease-free survival of a patient with metastatic eyelid squamous cell carcinoma and colon adenocarcinoma after repeated postoperative adoptive immunotherapy.

A 74-year-old male was affected concurrently with squamous cell carcinoma of the left eyelid and adenocarcinoma of the colon, both with lymph node metastasis. He underwent exenteration of the left orbit with left modified radical neck dissection and subsequently resection of the transverse colon with regional lymph node dissection. The patient has been treated by an adoptive immunotherapy as a sole postoperative modality without receiving any chemotherapeutic agents causing immunosuppression. For the adoptive immunotherapy, autologous peripheral blood lymphocytes were activated with an immobilized anti-CD3 antibody and IL-2 for 14 days (the CD3-AT cells). The infusion with 1.38 x 10(10) CD3-AT cells has been repeated 150 times in total at the time of writing. Neither recurrence nor additional metastasis has been detected for 6 years after surgery.

Adenocarcinoma↗

PubMed2000

Synthesis and triplex forming ability of conformationally locked oligonucleotides containing unnatural nucleobases: efficient recognition of a C.G interruption.

In order to develop a novel nucleoside analogue which recognizes C.G interruption in homopurine.homopyrimidine DNA, we designed and synthesized a conformationally locked nucleoside analogue, 1-(2-O,4-C-methylene-beta-D-ribofuranosyl)pyridin-2-one (4), and introduced it into a triplex-forming oligonucleotide (TFO). On melting temperature (Tm) measurements, the unprecedented C.G base recognition ability of 4 was observed.

Base Pairing↗

PubMed2000

Two distinct mechanisms underlie the stimulation of neurotransmitter release by phorbol esters in clonal rat pheochromocytoma PC12 cells.

Phorbol ester treatment induces the phosphorylation of SNAP-25 at Ser(187) and the potentiation of Ca(2+)-induced dopamine (DA) and acetylcholine (Ach) release from PC12 cells. In order to evaluate the functional consequences of phosphorylation, quantitative analysis was carried out using an anti-phosphopeptide antibody that specifically recognizes SNAP-25 phosphorylated at Ser(187). DA and ACh release, assayed in low-K(+) as well as high-K(+) solution, increased by treating the cells with phorbol-12-myristate-13-acetate (PMA); however, the stimulation of high-K(+)-dependent release occurred at lower concentrations and with shorter exposures to PMA than that of the basal release in low-K(+)-solution. The PMA-induced phosphorylation of SNAP-25 did not correlate with the potentiation of high-K(+)-dependent neurotransmitter release. The potentiation of high-K(+)-dependent DA release by phorbol 12,13-diacetate (PDA), a water soluble phorbol ester, almost completely disappeared within 1 min after washing PDA in the presence of okadaic acid, conditions under which the phosphorylation of SNAP-25 persisted for at least 15 min. PMA-induced phosphorylation of SNAP-25 was inhibited by staurosporine, however, the potentiation of high-K(+)-dependent DA release was suppressed only partially. These results indicate that protein kinase activation does not account for a large fraction of the phorbol ester-induced potentiation of depolarization-dependent neurotransmitter release from PC12 cells.

Acetylcholine↗

PubMed2000

Growth factor expression in the osteophytes of the human femoral head in osteoarthritis.

Osteoarthritis is characterized by marked osteophyte formation consisting of new cartilage and bone. Because several growth factors are known to be involved in chondrogenesis and osteogenesis, the expression of transforming growth factor-beta 1 and basic fibroblast growth factor in the osteophytes of the human femoral heads in osteoarthritis were examined. Transforming growth factor-beta 1 messenger ribonucleic acid was detected in the osteophytes by reverse transcription-polymerase chain reaction. All of the nine examined osteophytes expressed transforming growth factor-beta 1 messenger ribonucleic acid, whereas one of four osteoarthritic femoral heads and none of four osteonecrotic femoral heads expressed transforming growth factor-beta 1 messenger ribonucleic acid. The extent of transforming growth factor-beta 1 messenger ribonucleic acid expression varied among the osteophytes. Transforming growth factor-beta 1 or basic fibroblast growth factor was analyzed in osteophytes immunohistochemically. Transforming growth factor-beta 1 was localized in the superficial cells in the osteophyte cartilage, but it was scarcely detected in the superficial cells in the degenerative articular cartilage. Basic fibroblast growth factor was detected in the cells in the whole layer of osteophyte cartilage and in the articular cartilage. There was a difference in the localization, which suggests the different roles of transforming growth factor-beta 1 and basic fibroblast growth factor in bone and cartilage metabolism in osteophyte formation.

Fibroblast Growth Factor 2↗

PubMed2000

FTY720 prevents development of experimental autoimmune myocarditis through reduction of circulating lymphocytes.

FTY720 is a new immunosuppressant agent and selectively decreases the number of circulating lymphocytes. In this study, we compared the effects of FTY720 with those of tacrolimus on experimental autoimmune myocarditis (EAM) in rats. A significant decrease in circulating lymphocyte counts was noted after a single administration of FTY720 in normal rats. At day 0, 7-week-old male Lewis rats were immunized with purified porcine cardiac myosin emulsified in complete Freund's adjuvant. FTY720 or tacrolimus was administered intraperitoneally daily. The number of myocarditis-affected areas in the FTY720 treatment groups with doses of 0.1 mg/kg/ day was significantly lower than those in control groups at days 14 and 28. In addition, at day 28, the myocarditis-affected areas in the FTY720 treatment group were significantly smaller than those in the tacrolimus treatment group receiving the same dose. Effects of early administration (days 0-10) and delayed administration (days 11-20) of FTY720 also were examined. At day 28, the myocarditis-affected areas in the early therapy group were significantly lower than those in the control group. In conclusion, we demonstrated that the development of EAM could be prevented by FTY720. These data also indicated that lymphocyte-mediated immunity is critically involved in the development of EAM.

Analysis of Variance↗

PubMed2000

Characterization of human polymorphic DNA repair methyltransferase.

The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis. More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts. We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3. Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation. In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme. Escherichia coli strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence. The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples. On the other hand, E. coli and human cells expressing V2 cDNA showed a significantly reduced level of survival. In these cells, V2 protein was hardly detected, even though mRNA was produced normally. An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences. There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo. Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would support these views. From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare.

Blotting, Northern↗