Biomedical subjects

T Horiuchi

Publications and source records attributed to T Horiuchi.

At least 37 records · Page 2Linked to original sources

PubMed2001

Development of orally active nonpeptidic inhibitors of human neutrophil elastase.

5-Amino-2-phenylpyrimidin-6-ones, some of their desamino derivatives, and miscellaneous derivatives were synthesized and biologically evaluated on both in vitro activity and oral activity in an acute hemorrhagic assay. These compounds contained an alpha-keto-1,3,4-oxadiazole moiety to bind covalently to the Ser-195 hydroxy group of human neutrophil elastase (HNE). Among those tested, compounds 11a-c,e,i-l(F), 11d,e,k(H), 21d,e,k(F), and 21d,e(H) showed a good oral profile. RS-Mixture 3(H) was selected for clinical evaluation based on its oral potency, duration of action, enzyme selectivity, safety profile, and ease of synthesis. Structure-activity relationships (SARs) are discussed.

Administration, Oral↗

PubMed2001

Detection of electrochemical enzymatic reactions by surface plasmon resonance measurement.

We describe the surface plasmon resonance (SPR) detection of an enzymatic turnover reaction and the measurement of glucose concentration using a multienzyme layer modified gold electrode. We constructed an osmium redox polymer mediated enzyme sensor on a gold thin-film electrode and monitored electrochemical reaction by SPR measurement. Unlike the usual binding assay with SPR, here we used SPR to detect the redox state of an electron mediator that was the result of the electron-transfer reaction of sequential enzymatic reactions. Therefore, the degree of refractive index change was independent of the dielectric property of the substrate and enzymatic molecular recognition was converted to refractive index change with amplification. For the quantitative evaluation of glucose with this method, we used chronopotentiometry and a linear relation was obtained between the glucose concentration and the rate of refractive index change.

Electrochemistry↗

PubMed2001

Atpase activity and multimer formation of Pilq protein are required for thin pilus biogenesis in plasmid R64.

Plasmid R64 pilQ gene is essential for the formation of thin pilus, a type IV pilus. The pilQ product contains NTP binding motifs and belongs to the PulE-VirB11 family of NTPases. The pilQ gene was overexpressed with an N-terminal His tag, and PilQ protein was purified. Purified His tag PilQ protein displayed ATPase activity with a V(max) of 0.71 nmol/min/mg of protein and a K(m) of 0.26 mm at pH 6.5. By gel filtration chromatography, PilQ protein was eluted at the position corresponding to 460 kDa, suggesting that PilQ protein forms a homooctamer. To analyze the relationship between structure and function of PilQ protein, amino acid substitutions were introduced within several conserved motifs. Among 11 missense mutants, 7 mutants exhibited various levels of reduced DNA transfer frequencies in liquid matings. Four mutant genes (T234I, K238Q, D263N, and H328A) were overexpressed with a His tag. The purified mutant PilQ proteins contained various levels of reduced ATPase activity. Three mutant PilQ proteins formed stable multimers similar to wild-type PilQ, whereas the PilQ D263N multimer was unstable. PilQ D263N monomer exhibited low ATPase activity, while PilQ D263N multimer did not. These results indicate that ATPase activity of the PilQ multimer is essential for R64 thin pilus biogenesis.

Adenosine Triphosphatases↗

PubMed2001

Outside-to-inside signal through the membrane TNF-alpha induces E-selectin (CD62E) expression on activated human CD4+ T cells.

The membrane TNF-alpha is known to serve as a precursor of the soluble form of TNF-alpha. Although it has been reported the biological functions of the membrane TNF-alpha as a ligand, the outside-to-inside (reverse) signal transmitted through membrane TNF-alpha is poorly understood. Here we report a novel function mediated by outside-to-inside signal via membrane TNF-alpha into the cells expressing membrane TNF-alpha. Activation by anti-TNF-alpha Ab against membrane TNF-alpha on human T cell leukemia virus (HTLV) I-infected T cell line, MT-2, or PHA-activated normal human CD4(+) T cells resulted in the induction of an adhesion molecule, E-selectin (CD62E), on the cells with the peak of 12-24 h, which completely disappeared by 48 h. When wild-type or mutant membrane TNF-alpha (R78T/S79T) resistant to proteolytic cleavage was introduced into Jurkat or HeLa cells, E-selectin was induced by the treatment with anti-TNF-alpha Ab with the similar kinetics. Membrane TNF-alpha-expressing Jurkat cells also up-regulated E-selectin when brought into cell-to-cell contact with TNF receptor-expressing HeLa cells. Northern blot analysis and RT-PCR analysis showed that the membrane TNF-alpha-mediated E-selectin expression was up-regulated at the level of transcription. These results not only confirmed our previous findings of reverse signaling through membrane TNF-alpha, but also presented evidence that E-selectin was inducible in cell types different from endothelial cells. It is strongly suggested that membrane TNF-alpha is a novel proinflammatory cell surface molecule that transmits bipolar signals in local inflammation.

CD4-Positive T-Lymphocytes↗

PubMed2001

Association of tumor necrosis factor receptor type II polymorphism 196R with Systemic lupus erythematosus in the Japanese: molecular and functional analysis.

OBJECTIVE: To investigate whether a polymorphism(s) or mutation(s) in the tumor necrosis factor receptor II (TNFRII) gene is involved in the pathogenesis of systemic lupus erythematosus (SLE). METHODS: All 10 exons of the TNFRII gene were analyzed by exon-specific polymerase chain reaction-single-strand conformation polymorphism, followed by nucleotide sequencing of exons that displayed aberrant bands. To analyze the function of the TNFRII polymorphisms, the full-length TNFRII complementary DNA of each allele was transfected in HeLa cells and then studied for specific binding of 125I-TNFalpha, as well as interleukin-6 (IL-6) production and cytotoxic activity after treatment with recombinant human TNFalpha. RESULTS: We identified 4 polymorphisms, at codons 56, 181, 196, and 232. The latter 2 had amino acid substitutions M196R and E232K, respectively. Only the 196R allele was significantly associated with SLE in our 105 Japanese SLE patients, with an allele frequency of 20.5%, compared with 12.6% in 99 healthy controls (P = 0.0335). More importantly, using TNFRII-transfected HeLa cells, we demonstrated significantly increased IL-6 production by 196R TNFRII compared with 196M TNFRII. The cytotoxic activity induced by 196R TNFRII was also increased compared with that of 196M TNFRII. This increase was achieved without affecting the binding affinity of TNFalpha to TNF-RII, as demonstrated by the finding that specific TNFalpha binding to the HeLa transfectants of 196R and 196M TNFRII was similar, with Kd values of 3.12 x 10(-10)M and 4.34 x 10(-10)M, respectively. CONCLUSION: These results suggest that 196R TNFRII, which transduces the signals of TNFalpha more effectively than does 196M TNFRII, is involved in the pathogenesis of SLE.

Adolescent↗

PubMed2001

Metallosphaera sedula TA-2, a calditoglycerocaldarchaeol deletion strain of a thermoacidophilic archaeon.

A spherical thermoacidophilic archaeon, strain TA-2, was obtained from acidic hot springs located in Ohwaku Valley, Hakone, Japan. This isolate is an obligate aerobic chemoorganoheterotroph that grows optimally at about 75 degrees C, pH 2.8. The G + C content of DNA from TA-2 is 47 mol%. The 16S rRNA gene from TA-2 showed more than 99% similarity with those of Metallosphaera sedula and Metallosphaera prunae and less than 92% similarity with other members of the order Sulfolobales. DNA-DNA hybridization experiments showed more than 93% genomic DNA homology among TA-2, M. sedula DSM5348T, and M. prunae DSM10039T. However, TA-2 lacks calditoglycerocaldarchaeol derivatives, which are usually found in the membrane lipids of members of the order Sulfolobales. Therefore, calditoglycerocaldarchaeol may not be essential for survival in thermophilic and acidophilic environments. The isolate was deposited as Metallosphaera sedula TA-2 (JCM 9064, IFO 15160).

Adaptation, Biological↗

PubMed2001

Factors associated with anxiety, depression and suicide ideation in female outpatients with SLE in Japan.

The subjects consisted of 84 female SLE outpatients who were all over 20 years of age. These patients were able to maintain relatively stable physical conditions and lead normal daily lives, and they were regularly treated at the outpatient clinic. All subjects were Japanese. Psychological features (trait anxiety, state anxiety, depression and suicide ideation) were evaluated using psychological tests, and the relationships between the respective psychological features and background factors were statistically evaluated using stepwise multiple logistic regression analyses. In this study, we found that 'the self-evaluation of not having understood SLE at the time of starting SLE treatment' was the background factor significantly affecting depression or trait anxiety. 'No spouse' had a statistically significant effect on depression, and 'self-awareness as problems of side-effects due to steroids' had a statistically significant effect on state anxiety. We also found 'human relations among family members' and 'high daily steroid dosage' to be significantly correlated with suicide ideation. However, there were no correlations between the psychological features and 'disease activity at the time of investigation' or 'history of neuropsychiatric diseases'. In female SLE outpatients, performing psychological approaches focusing on 'understanding SLE at the beginning of treatment', 'the human relationships among family members', or 'issues related to steroid therapy' may be useful for the early treatment or prevention of various major mental problems.

Adult↗

PubMed2001

International Child Care Practices Study: infant sleeping environment.

BACKGROUND: The International Child Care Practices Study (ICCPS) has collected descriptive data from 21 centres in 17 countries. In this report, data are presented on the infant sleeping environment with the main focus being sudden infant death syndrome (SIDS) risk factors (bedsharing and infant using a pillow) and protective factors (infant sharing a room with adult) that are not yet well established in the literature. METHODS: Using a standardised protocol, parents of infants were surveyed at birth by interview and at 3 months of age mainly by postal questionnaire. Centres were grouped according to geographic location. Also indicated was the level of SIDS awareness in the community, i.e. whether any campaigns or messages to "reduce the risks of SIDS" were available at the time of the survey. RESULTS: Birth interview data were available for 5488 individual families and 4656 (85%) returned questionnaires at 3 months. Rates of bedsharing varied considerably (2-88%) and it appeared to be more common in the samples with a lower awareness of SIDS, but not necessarily a high SIDS rate. Countries with higher rates of bedsharing appeared to have a greater proportion of infants bedsharing for a longer duration (>5 h). Rates of room sharing varied (58-100%) with some of the lowest rates noted in centres with a higher awareness of SIDS. Rates of pillow use ranged from 4% to 95%. CONCLUSIONS: It is likely that methods of bedsharing differ cross-culturally, and although further details were sought on different bedsharing practices, it was not possible to build up a composite picture of "typical" bedsharing practices in these different communities. These data highlight interesting patterns in child care in these diverse populations. Although these results should not be used to imply that any particular child care practice either increases or decreases the risk of SIDS, these findings should help to inject caution into the process of developing SIDS prevention campaigns for non-Western cultures.

Beds↗

PubMed2001

Mutation of DNASE1 in people with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a highly prevalent human autoimmune diseases that causes progressive glomerulonephritis, arthritis and an erythematoid rash. Mice deficient in deoxyribonuclease I (Dnase1) develop an SLE-like syndrome. Here we describe two patients with a heterozygous nonsense mutation in exon 2 of DNASE1, decreased DNASE1 activity and an extremely high immunoglobulin G titer against nucleosomal antigens. These data are consistent with the hypothesis that a direct connection exists between low activity of DNASE1 and progression of human SLE.

Adolescent↗

PubMed2001

Extensive clonal expansion of T lymphocytes causes contracted diversity of complementarity-determining region 3 and skewed T cell receptor repertoires after allogeneic hematopoietic cell transplantation.

We previously described skewed repertoires of the T cell receptor-beta chain variable region (TCRBV) and the TCR-alpha chain variable region (TCRAV) soon after allogeneic hematopoietic cell transplantation. To determine the characteristics of skewed TCRBV after transplantation, we examined the clonality of T lymphocytes carrying skewed TCRBV subfamilies and determined the CDR3 sequences of expanded T cell clones. In all 11 recipients examined, TCR repertoires were skewed, with an increase of certain TCRBV subfamilies that differed among individuals. In nine of 11 patients, clonal/oligoclonal T cell expansion was observed, although the expanded T cells were not necessarily oligoclonal. The extent of expansion after transplantation appeared to predict clonality. The arginine (R)-X-X-glycine (G) sequence was identified in clonally expanded T cells from four of five recipients examined, and glutamic acid (E), aspartic acid (D) and alanine (A) were frequently inserted between R and G. These results suggest that T lymphocyte expansion may result from the response to antigens widely existing in humans, and that the extensive clonal expansion of a limited number of T cells leads to contracted CDR3 diversity and post-transplant skewed TCR repertoires.

Clone Cells↗

PubMed2001

Identification of the T cell clones expanding within both CD8(+)CD28(+) and CD8(+)CD28(-) T cell subsets in recipients of allogeneic hematopoietic cell grafts and its implication in post-transplant skewing of T cell receptor repertoire.

We have previously reported that skewed repertoires of T cell receptor-beta chain variable region (TCRBV) and TCR-alpha chain variable region (TCRAV) are observed at an early period after allogeneic hematopoietic cell transplantation. Furthermore, we found that T lymphocytes using TCRBV24S1 were increased in 28% of the recipients of allogeneic grafts and an increase of TCRBV24S1 usage was shown to result from clonal expansions. Interestingly, the arginine residue was frequently present at the 3' terminal of BV24S1 segment and was followed by an acidic amino acid residue within the CDR3 region. These results suggest that these clonally expanded T cells are not randomly selected, but are expanded by stimulation with specific antigens. This study was undertaken to elucidate the mechanisms of the post-transplant skewing of TCR repertoires. Since the CD8(+)CD28(-)CD57(+) T cell subset has been reported to expand in the peripheral blood of patients receiving allogeneic hematopoietic cell grafts, we examined the TCRAV and TCRBV repertoires of the CD8(+)CD28(-) T cell and CD8(+)CD28(+) T cell subsets, and also determined the clonality of both T cell populations. In all three recipients examined, the CD8(+)CD28(-) T cell subset appeared to define the post-transplant TCR repertoire of circulating blood T cells. Moreover, the CDR3 length of TCRBV imposed constraints in both CD8(+)CD28(-) T cell and CD8(+)CD28(+) T cell subsets. The DNA sequences of the CDR3 region were determined, and the same clones were identified within both CD8(+)CD28(-) and CD8(+)CD28(+) T cell subsets in the same individuals. These results suggest that the clonally expanded CD8(+)CD28(-) T cells after allogeneic hematopoietic cell transplantation derive from the CD8(+)CD28(+) T cell subset, possibly by an antigen-driven mechanism, resulting in the skewed TCR repertoire.

Amino Acid Sequence↗

PubMed2001

Oligoclonal expansion of CD4(+)CD28(-) T lymphocytes in recipients of allogeneic hematopoietic cell grafts and identification of the same T cell clones within both CD4(+)CD28(+) and CD4(+)CD28(-) T cell subsets.

Recipients of allogeneic bone marrow grafts have clonally expanded CD8(+)CD28(-) T lymphocytes during the early period after transplantation, which leads to skewing of T cell receptor (TCR) repertoires. Here, we have addressed the question of whether clonal expansion of CD28(-) T cells is also observed in CD4(+) T lymphocytes after human allogeneic hematopoietic cell transplantation. We found that the fraction of T cells lacking CD28 expression in the CD4(+) subset was increased after transplantation, and expanded CD4(+)CD28(-) T lymphocytes carrying certain TCRBV subfamilies showed limited TCR diversity. In order to further study the ontogeny of CD4(+)CD28(-) T cells, we analyzed the complementarity-determining region 3 (CDR3) of the TCR-beta chain of CD4(+)CD28(+) and CD4(+)CD28(-) cells. We identified the same T cell clones within both CD4(+)CD28(-) and CD4(+)CD28(+) T cell subsets. These results suggest that both subsets are phenotypic variants of the same T cell lineage.

Amino Acid Sequence↗

PubMed2001

Physical principles in therapeutic apheresis.

Physical modality could have some impact on new apheresis technologies because it is nonbiologic and controllable for its operating conditions. In this article, the application of electromagnetic force and light scattering force to bioseparation and biostimulation was explored.

Blood Component Removal↗

PubMed2001

Platelet-derived growth factor is involved in the augmentation of airway responsiveness through remodeling of airways in diesel exhaust particulate-treated mice.

BACKGROUND: Thickening of the region adjacent to the basement membrane is a key component of the remodeling of the asthmatic airway and is caused by collagen deposition in the region. OBJECTIVE: We sought to clarify the role of platelet-derived growth factor (PDGF), a competence factor of fibroblast, in the enhanced airway responsiveness and remodeling in a murine model. METHODS: Diesel exhaust particulates (DEPs) were administered intranasally every other day for 2 weeks with or without anti-PDGF-beta neutralizing antibody or goat IgG. Pulmonary function was then analyzed by using whole-body plethysmography before and after acetylcholine inhalation. RESULTS: Anti-PDGF-beta neutralizing antibody significantly inhibited both the elevation of airway resistance elicited by 1.25 and 2.5 mg/mL acetylcholine and the increase in the airway wall thickening induced by DEPs. In addition, bronchoalveolar lavage fluid cell analysis revealed that anti-PDGF-beta neutralizing antibody did not affect cellular infiltration at the airways. CONCLUSION: PDGF plays an important role in the process of remodeling brought about by DEP exposure in mice.

3T3 Cells↗

PubMed2001

Age-related changes in cortical and trabecular bone mineral status. A quantitative CT study in lumbar vertebrae.

PURPOSE: To investigate the age and sex dependence of the bone mineral status of human lumbar vertebrae with special regard to differences between cortical and trabecular bone. MATERIAL AND METHODS: The study group comprised 125 normal Japanese healthy volunteers (54 males and 71 females), and was subdivided into adult male and female groups (subjects younger than 40 years), intermediate male and female groups (ages ranging between 41 and 64 years) and old male and female groups (subjects older than 65 years). The cortical bone mineral status was estimated using a single-energy quantitative CT (SE-QCT) technique, whereas trabecular bone mineral density (BMD) was estimated using a dual-energy (DE-QCT) technique. RESULTS: A considerable gender difference in the age-related cortical bone status was found. There was a significant reduction of the mean values of the cortical volume and BMD in the old female group compared with those obtained in the old male group. CONCLUSION: The results suggest that in men, cortical and trabecular bone volume decrease very little with age. In women, cortical volume and BMD and trabecular BMD decrease with age while trabecular bone volume does not. The study showed that all variables had higher values in men than in women and that the difference increased with age.

Adult↗

PubMed2001

The amino acid residues affecting the activity and azole susceptibility of rat CYP51 (sterol 14-demethylase P450).

The amino acid residues affecting the function of rat sterol 14-demethylase P450 (CYP51) were examined by means of point mutation. Forty-five mutants with respect to 27 amino acid sites were constructed and expressed in Escherichia coli. Substitution of highly conserved Y131, E369, R372, or R382 decreased the expression of CYP51 protein, indicating some structural importance of these residues. Substitution of H314, T315, or S316 caused considerable effects on the catalytic activity, and T315 was identified as the "conserved threonine" of CYP51. H314 was important for maintenance of the activity of CYP51 and was a characteristic residue of this P450, because the position corresponding to this residue is occupied by an acidic amino acid in most other P450 species. A144 was identified as a residue affecting the interaction of CYP51 with ketoconazole. Substitution of A144 with I, which occupies the corresponding position in fungal CYP51, enhanced the ketoconazole susceptibility of rat CYP51 with little change in the catalytic activity, indicating an important role of this residue in determination of the ketoconazole susceptibility of CYP51. Alteration of the catalytic activity was caused by the substitution at some other sites, whereas substitution of a few highly conserved amino acids caused little alteration of the activity of CYP51.

Amino Acid Substitution↗

PubMed2001

Human sperm aster formation and pronuclear decondensation in bovine eggs following intracytoplasmic sperm injection using a Piezo-driven pipette: a novel assay for human sperm centrosomal function.

In human fertilization, the sperm introduces the centrosome; the microtubule-organizing center and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8-12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.

Animals↗

PubMed2001

Helicobacter pylori DNA in drinking water in Japan.

Helicobacter pylori has been detected in drinking water in Peru and Sweden, suggesting the possibility of water-borne transmission. To date there have been few reports of H. pylori being detected in water; one was of the ureA gene of H. pylori in wells and springs in rural Japan. We examined water sampled in or near urban areas of Japan for H. pylori DNA by three assays using the polymerase chain reaction (PCR). Near Tokyo, samples were obtained: 10 of tap water, 6 of well water, 10 of river water, and 10 of sea water. Samples were filtered with membranes with 0.05- or 0.22-microm pores, which bacterial cells are caught by. Bacterial nucleic acids were extracted and purified and the PCR was done to amplify adhesin specific for H. pylori and the ureA gene, if present. Real-time PCR that measured the yield in terms of fluorescence was done with primers for 16S rRNA. None of the samples of tap, river, or sea water contained adhesin, ureA or 16S rRNA. None of the 6 samples of well water contained adhesin or ureA, but 2 of the 6 samples contained 16S rRNA. Some of the users of the well had had H. pylori infection in the past. H. pylori DNA was detected in well water and the users had been infected, so water-borne transmission via well water may occur even in towns in Japan.

Adhesins, Bacterial↗