Biomedical subjects
M Seki
Publications and source records attributed to M Seki.
[Severe hypoxia due to persistent left supra vena cava draining to the left atrium after weaning from cardiopulmonary bypass in a patient with endocardial cushion defect].
We report a case of severe hypoxia after weaning from cardiopulmonary bypass in a ten-month-old patient with endocardial cushion defect. The severe hypoxia was improved abruptly when the persistent left supra vena cava (PLSVC) was ligated. The hypoxia, therefore, was considered to be caused by venous blood which was directly drained into the left atrium through the PLSVC. In cases with large right-to-left shunt which is difficult to explain only by intracardiac shunt, attention must be paid to existence of PLSVC directly draining into the left atrium.
[A case of simple liver cyst with markedly elevated CEA level in the cystic fluid].
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[Bacterial infection and production of immunoglobulin in the tonsil].
Immunoglobulin (Ig) isotype switching is the process whereby B cells initially expressing IgM and/or IgD on their surface switch to other Ig heavy-chain loci. We demonstrated that TGF-beta, IL-10 and VIP in the presence of CD40 mAb, can induce isotype switching for IgA in human tonsillar B cells by the generation of switch circular DNA (S alpha/S mu), the induction of alpha germ-line transcripts and a significant amount of IgA production. IgA is the predominant immunoglobulin effector molecule of mucosal immunity that functions as the first line of specific immunologic defense against many microbial pathogens. However, IgA causes IgA nephropathy. We investigated here, whether tonsillar mononuclear cells from patients with IgA nephropathy produce HP-specific IgA and/or IgA-related cytokines by stimulation with components of HP outer membranes (OMHP). HP-specific IgA was predominately induced by tonsillar mononuclear cells of IgA nephropathy, compared to those from chronic tonsillitis. Production of IL-10 and TGF-beta was enhanced by stimulation with OMHP in tonsillar mononuclear cells from IgA nephropathy. These results suggested that local infection of HP and HP-specific IgA induction in the tonsil are associated with pathogens in IgA nephropathy.
Recovery of hepatic asialoglycoprotein receptors after major hepatic resection.
UNLABELLED: Although morphological restoration of the hepatic mass after partial hepatectomy has been well studied, fewer reports have appeared on the change of functional hepatic capacity during liver regeneration. Asialoglycoprotein receptor (ASGP-R) is a hepatic cell surface receptor specific for galactose-terminated glycoprotein. Kinetic modeling of 99mTc-labeled diethylenetriamine pentaacetic acid-galactosyl-human serum albumin (TcGSA) time-activity data yields estimates of ASGP-R concentration [R]o and amount R0, which are directly related to functional liver mass. We have investigated the changes in ASGP-R status as well as liver volume in regenerating human liver after major hepatic resection. METHODS: Twenty-two patients (18 noncirrhotic, 4 cirrhotic) had a TcGSA study before and 3 wk after major hepatic resection, with a mean hepatic parenchymal resection rate of 36.0%. RESULTS: [R]0 was significantly decreased from 0.683+/-0.024 micromol/L to 0.565+/-0.032 micromol/L (P < 0.001) after resection. The decrease in [R]0 was more prominent in cirrhotic patients. Recovery of ASGP-R was observed as a significantly increased R0 3 wk after the operation. Subsequent (long-term) restoration of ASGP-R appeared to be slower when compared with the volume restoration. CONCLUSION: ASGP-R concentration of the liver significantly decreased after major hepatic resection. Subsequent recovery of ASGP-R amount was shown by TcGSA study. By estimating hepatic functional reserve expressed by ASGP-R amount and concentration, one may detect a delayed or impaired liver regeneration with higher sensitivity.
Molecular cloning and characterization of three cDNAs encoding putative mitogen-activated protein kinase kinases (MAPKKs) in Arabidopsis thaliana.
We isolated three Arabidopsis thaliana cDNA clones (ATMKK3, ATMKK4 and ATMKK5) encoding protein kinases with extensive homology to the mitogen-activated protein kinase kinases (MAPKKs) of various organisms in the catalytic domain. ATMKK3 shows high homology (85% identity) to NPK2, a tobacco MAPKK homologue. ATMKK4 and 5 are closely related to each other (84% identity). Phylogenetic analysis showed that the plant MAPKKs constitute at least three subgroups. The recombinant ATMKK3 and ATMKK4 were expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Affinity purified GST-ATMKK3 and GST-ATMKK4 proteins contained phosphorylation activity, which shows that both the ATMKK3 and ATMKK4 genes encode functional protein kinases. Northern blot analysis revealed that the ATMKK3 gene expressed in all the organs. The levels of ATMKK4 and 5 mRNAs were relatively higher in steins and leaves than in flowers and roots. We determined the map positions of the ATMKK3, 4 and 5 genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.
Establishment of framework P1 clones for map-based cloning and genome sequencing: direct RFLP mapping of large clones.
Large insert capacity, clone stability and convenient propagation in Escherichia coli have made bacterial artificial chromosome and phage P1 vector-based libraries the first choice for large-scale sequencing projects, and these libraries have also proven useful for chromosome walking. The application of these libraries for either purpose is greatly facilitated by the establishment of a set of framework clones distributed across the genome. Using a P1-based library of Arabidopsis thaliana with genomic inserts of 70-90kb (Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., Whittier, R.F., 1995. Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking. Plant J. 7, 351-358), we have now established such a set of framework clones. To date, such clones have usually been identified by hybridization to smaller, previously mapped clones that detect restriction fragment length polymorphisms (RFLPs). In order to establish framework clones more efficiently, we refined protocols for P1 clone DNA isolation and RFLP detection in order to employ whole P1 clones directly as probes. This strategy enabled a very high rate of RFLP detection, and obviated the need to screen the P1 library with smaller RFLP probes. Altogether 95 clones were mapped providing a framework into which further clones can be integrated by physical overlap.
Cloning of two isoforms of mouse DNA helicase Q1/RecQL cDNA; alpha form is expressed ubiquitously and beta form specifically in the testis.
We cloned cDNAs encoding mouse homologues for the human DNA helicase Q1/RecQL (human helicase Q1) which has homology with the Escherichia coli RecQ protein and found that they encode two isoforms. The two isoforms are identical over the entire sequence except for the carboxyl terminal sequence spanning less than 30 amino acids. One of the two isoforms, alpha, contains a sequence, KKRK, in the carboxyl terminus, which is also contained in human helicase Q1 and was confirmed to function as the nuclear localization signal. The other form, beta, does not contain such a sequence. Expression of mouse helicase Q1 mRNA is extremely and relatively high in the testis and the thymus, respectively. RT-PCR analysis revealed that helicase Q1alpha was expressed in all tissues tested and the beta form was expressed only in the testis. A survey of expression of Q1alpha and Q1beta mRNA in the testis after birth revealed that Q1alpha mRNA is expressed in all testes of mice aged from 7 days to 8 weeks, and the expression of Q1beta mRNA begins 14 days after birth, corresponding to the appearance of cells in the pachytene stage.
Cloning of cDNA encoding a novel mouse DNA topoisomerase III (Topo IIIbeta) possessing negatively supercoiled DNA relaxing activity, whose message is highly expressed in the testis.
We cloned cDNA encoding a novel mouse homologue of DNA topoisomerase III (mTOP3beta). The nucleotide sequence contains an open reading frame of 863 amino acids, and the deduced molecular mass of the coded protein is 96.9 kDa. The overall sequence of mTOP3beta has a 48 and 36% identity with mouse TOP3alpha at the nucleotide and amino acid level, respectively. DNA topoisomerase IIIbeta was expressed using a baculovirus expression system and purified. The purified DNA topoisomerase IIIbeta had activity to relax negatively supercoiled DNA. Relaxation of supercoiled DNA was partial at 37 degreesC and complete relaxation was observed at higher temperatures. mTOP3beta mRNA was strongly expressed in the testis and relatively strongly in the brain. The levels of TOP3beta mRNA in the testis increased slightly 14 days and considerably 17 days after birth, when the cells in the pachytene phase begin to appear and increase.
An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression.
In higher plants, a cis-acting element, DRE/CRT, is involved in gene expression responsive to drought and low-temperature stress. To understand signal transduction pathways from the cold stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB1A and CBF1 in Arabidopsis thaliana. DREB1A and CBF1 were shown to be involved in low-temperature-responsive gene expression. We screened an Arabidopsis genomic DNA library with the cDNA fragment of DREB1A as a probe and isolated DREB1A and 2 related genes, DREB1B (= CBF1) and DREB1C. These were arrayed in the order B, A, C in an 8.7 kb region of Arabidopsis chromosome 4. Northern blot analysis using gene-specific probes showed that the 3 DREB1 genes are induced mainly by cold stress but not by osmotic stress in leaves, roots, and stems. Several conserved sequences were found in the promoter regions of all 3 genes. The beta-glucuronidase (GUS) reporter gene driven by the DREB1 promoters was induced at transcriptional level by low temperature in transgenic Arabidopsis plants.
cDNA cloning of mouse BLM gene, the homologue to human Bloom's syndrome gene, which is highly expressed in the testis at the mRNA level.
We cloned a cDNA encoding the mouse homologue to human Bloom's syndrome gene (BLM). The deduced amino acid sequence of mouse Blm showed 76% identity to the human sequence with very high homology in seven consecutive domains characteristic of DNA and RNA helicases. The expression of mBLM mRNA was examined in various tissues. Extremely high expression was observed in the testis as compared with other tissues. The mBLM mRNA level in the testis began to increase 12-14 days after birth, corresponding to the appearance of cells in the pachytene phase.
Human ecalectin, a variant of human galectin-9, is a novel eosinophil chemoattractant produced by T lymphocytes.
A 1.6-kilobase pair cDNA was isolated from a human T-cell-derived expression library that encodes a novel eosinophil chemoattractant (designated ecalectin) expressed during allergic and parasitic responses. Based on its deduced amino acid sequence, ecalectin is a 36-kDa protein consisting of 323 amino acids. Although ecalectin lacks a hydrophobic signal peptide, it is secreted from mammalian cells. Ecalectin is not related to any known cytokine or chemokine but rather is a variant of human galectin-9, a member of the large family of animal lectins that have affinity for beta-galactosides. Recombinant ecalectin, expressed in COS cells and insect cells, exhibited potent eosinophil chemoattractant activity and attracted eosinophils in vitro and in vivo in a dose-dependent manner but not neutrophils, lymphocytes, or monocytes. The finding that the ecalectin transcript is present in abundance in various lymphatic tissues and that its expression increases substantially in antigen-activated peripheral blood mononuclear cells suggests that ecalectin is an important T-cell-derived regulator of eosinophil recruitment in tissues during inflammatory reactions. We believe that this is the first report of the expression of an immunoregulatory galectin expressed by a T-cell line that is selective for eosinophils.
Critical point mutations for hepatitis C virus NS3 proteinase.
The hepatitis C virus NS3 proteinase plays an essential role in processing of HCV nonstructural precursor polyprotein. To detect its processing activity, we developed a simple trans-cleavage assay. Two recombinant plasmids expressing the NS3 proteinase region and a chimeric substrate polyprotein containing the NS5A/5B cleavage site between maltose binding protein and protein A were co-introduced into Escherichia coli cells. The proteinase processed the substrate at the single site during their polyprotein expression. Deletion analysis indicated that the functionally minimal domain of the NS3 proteinase was composed of 146 amino acids, 1059 to 1204. We isolated several cDNA clones encoding the functional domain of the NS3 proteinase from the sera of patients chronically infected with HCV and determined their proteinase activity by this trans-cleavage assay. Both active and inactive clones existed in the same patients. Comparative sequence analyses of these clones suggested that certain point mutations seemed to be related to the loss of proteolytic activity. This was confirmed by back mutation experiments. Among the critical mutations, Pro-1168 to Thr and Arg-1135 to Gly were intriguing. These amino acids, which are situated near the oxyanion hole, seem to be essential for maintaining the conformation of the active center of the NS3 proteinase.
Synaptology of the direct projections from the nucleus of the solitary tract to pharyngeal motoneurons in the nucleus ambiguus of the rat.
During the pharyngeal phase of the swallowing reflex, the nucleus of the solitary tract (NTS) receives peripheral inputs from the pharynx by means of the glossopharyngeal ganglion and is the location of premotor neurons for the pharyngeal (PH) motoneurons. The semicompact formation of the nucleus ambiguus (AmS) is composed of small and medium-sized neurons that do not project to the pharynx, and large PH motoneurons. We investigated whether the neurons in the NTS projected directly to the PH motoneurons or to the other kinds of neurons in the AmS by using the electron microscope. When wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the NTS after cholera toxin subunit B-conjugated HRP (CT-HRP) injections into the pharyngeal muscles of male Sprague-Dawley rats, many nerve terminals anterogradely labeled with WGA-HRP were found to contact PH motoneurons retrogradely labeled with CT-HRP. Most of the labeled axodendritic terminals (63%) contained pleomorphic vesicles with symmetric synaptic contacts (Gray's type II), and the remaining ones contained round vesicles with asymmetric synaptic contacts (Gray's type I). About 14% of the axosomatic terminals on PH motoneuron in a sectional plane were anterogradely labeled, and about 70% of the labeled axosomatic terminals were Gray's type II. Observations of serial ultrathin sections revealed that both the small and the medium-sized neurons received only a few labeled axosomatic terminals that were exclusively Gray's type I. These results indicate that the NTS neurons may send mainly inhibitory as well as a few excitatory inputs directly to the PH motoneurons in the AmS.
Isolation of a cDNA encoding mouse DNA topoisomerase III which is highly expressed at the mRNA level in the testis.
A cDNA (mTOP3) encoding mouse DNA topoisomerase III (topo III) was cloned using the human TOP3 cDNA as a probe. The deduced amino acid sequence of mouse topo III showed 86.8% identity with that of human topo III. Mouse TOP3 mRNA was highly expressed in the testis in comparison with other tissues. The TOP3 mRNA level in the testis increased slightly 14 days after birth and showed a marked increase in 17 days, times when the cells in pachytene phase begin to appear and increase in number.
Apoptosis of lymphocytes induced by glucocorticoids and relationship to therapeutic efficacy in patients with systemic lupus erythematosus.
OBJECTIVE: Glucocorticoids (GCs) are the drugs of first choice for treatment of systemic lupus erythematosus (SLE). However, the disease in some patients is resistant to these agents. This study evaluated the possibility of a relationship between response to GC treatment and the rate of apoptosis in vitro, and also analyzed Bcl-2 and Fas expression on peripheral blood mononuclear cells (PBMC) from patients with SLE in relation to GC-induced apoptosis. METHODS: Twenty-seven SLE patients and 13 normal controls were studied. Disease activity scores were determined before and after treatment and used to divide patients into 2 groups: GC-resistant and GC-responsive. Isolated PBMC were stimulated with anti-CD3 monoclonal antibodies, cultured with various concentrations of GC, and analyzed by alamar blue assay to determine the LC50, defined as the concentration of GC lethal to 50% of the cells. We measured the expression of CD4, CD8, Fas, and Bcl-2 by FACStar plus flow cytometry. RESULTS: Lymphocytes in the GC-resistant group of SLE patients showed a significantly lower percentage of GC-induced apoptotic cells than did lymphocytes from the responsive group or from normal controls. The LC50 in the resistant group was significantly higher than that in normal controls or the responsive group. The lymphocytes remaining in the resistant group after GC treatment were mainly CD8+, with a high expression of Bcl-2. There was no significant difference in Fas expression between the GC-responsive and GC-resistant groups. CONCLUSION: Determination of the LC50 may be useful in predicting the efficacy of GC treatment in SLE patients, and may be of use in considering other treatment options. CD8 and Bcl-2 double-positive lymphocytes that are insensitive to the apoptotic effect of GCs may play a role in the resistance to these agents in SLE patients.
Analysis of optimal range of socket orientations in total hip arthroplasty with use of computer-aided design simulation.
A three-dimensional computer-aided design model of a total hip replacement was used to study the effects of anteversion and abduction of the acetabular component and anteversion and varus-valgus angulation of the femoral component on the range of hip flexion and extension that could be obtained without component impingement. Impingement of the component was defined as impingement between the neck of the femoral component and the edge of the acetabular component. To achieve an angle of hip flexion greater than 90 degrees and an extension angle greater than 30 degrees without component impingement, the optimal angulations were found to be between 1 and 30 degrees of anteversion and 30 and 50 degrees of abduction of the acetabular component, as well as 10 degrees of anteversion of the femoral component. When the valgus angulation of the femoral component was reduced from 7 to 0 degrees, the allowable range of flexion without impingement increased under the same conditions of acetabular-component orientation and femoral-component anteversion. Significant inverse correlations were found between the anteversion angle of the acetabular component and both the lumbar lordosis angle and the sacrohorizontal angle.