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T Yano

Publications and source records attributed to T Yano.

At least 73 records · Page 4Linked to original sources
PubMed2001

Gonadal regulation of PrRP mRNA expression in the nucleus tractus solitarius and ventral and lateral reticular nuclei of the rat.

We investigated the prolactin-releasing peptide (PrRP) gene expression quantitatively in the rat brain and the involvement of estrogen and progesterone using in situ hybridization. The strongest signals were observed in the nucleus tractus solitarius (NTS), which showed approximately 70% of total PrRP mRNA in the brain. Moderate expression was observed in the ventral and lateral reticular nuclei (VLRN) of the medulla oblongata. PrRP mRNA signals in the hypothalamic ventromedial- and dorsomedial nuclei showed only 5% of total signals. The PrRP mRNA expression among female rats showing normal gonadal cycle and male rats showed that the highest levels were in female rats in proestrus. Administration of estrogen or progesterone after ovariectomy induced an increase in PrRP mRNA expression in the NTS. PrRP mRNA content in the NTS increased with the progress of the pregnancy and reached a peak on the 14th day, the mid-period of pregnancy, when plasma progesterone increases. We also observed the colocalization of PrRP and estrogen receptor alpha in the neurons distributed in the NTS by double labeling immunocytochemistry. These findings indicate that PrRP gene expression is regulated by gonadal steroid hormones in the medulla oblongata, and parts of PrRP synthesizing neurons are considered to be directly influenced by estrogen in the NTS.

Animals↗
PubMed2001

Directed evolution of ampicillin-resistant activity from a functionally unrelated DNA fragment: A laboratory model of molecular evolution.

To establish an experimental system to directly observe molecular evolution, a DNA fragment that confers ampicillin resistance on Escherichia coli was cloned from an archaeal genomic DNA. The activity of this clone was enhanced by 50 rounds of directed evolution by using DNA shuffling. Analysis of the evolved DNA fragments shows that two genetic regions have coevolved: One region, which has no obvious ORF, is essential for the activity, whereas the other, which appears to encode a protein, is not essential but enhances the activity of the former region. Analysis of the evolutionary intermediates shows that negative mutations are effectively removed while beneficial mutations accumulate and illustrates how a protein has evolved over the course of the evolution experiments. Although the mechanism of the activity remains unclear, the evolved DNA fragments also confer resistance to other drugs that inhibit bacterial cell-wall synthesis. The present system would serve as an experimental model to study evolutionary dynamics in the laboratory and provide the concept of screening natural libraries to obtain starting materials for directed evolution.

Ampicillin↗
PubMed2001

Regulation of cellular invasion and matrix metalloproteinase activity in HepG2 cell by connexin 26 transfection.

We previously reported that connexin (Cx) 26 expression is involved in negative growth control of HepG2 cells established from a human hepatoma. We also found that induction of E-cadherin and subsequent formation of a cell adhesion complex were induced in HepG2 cells by Cx 26 expression. To examine the exact role of Cx 26-induced E-cadherin junctions in regulating appearance of malignant phenotypes of HepG2 cells, we expressed a Cx 26 antisense oligodeoxynucleotide (AS-ODN) in an established HepG2 cell clone that has stable expression of Cx 26 genes. We investigated changes in the expression of E-cadherin, the localization of beta-catenin, and some malignant phenotypes of HepG2 clone after the suppression of Cx 26 expression by AS-ODN treatment. The AS-ODN treatment prevented the expression of Cx 26 and E-cadherin, and the localization of beta-catenin was changed from cytoplasmic membrane to the cytoplasm. In parallel, a morphological change from a monolayer of polygonal cells to multilayered colonies was induced by the treatment, indicating a change of a malignant phenotype of HepG2 cells. The activity of matrix metalloproteinase 9 (MMP-9) was elevated by the AS-ODN treatment. A concomitant increase in invasiveness of the Cx 26-expressing cells by the treatment was also observed in an in vitro assay with Matrigel matrix. These results suggest that the induction of E-cadherin and formation of the cell adhesion complex by Cx 26 expression contribute to the reversal of some malignant phenotypes of HepG2 cells. Furthermore, the Cx 26-dependent expression of E-cadherin leads to reduction of the invasiveness of the cells through suppression of MMP-9 activity.

Animals↗
PubMed2001

Molecular cloning of the complement (C1r/C1s/MASP2-like serine proteases from the common carp (Cyprinus carpio).

The classical pathway of complement composed of C1, C4, and C2 is an antibody-dependent activation cascade that is present in jawed vertebrates. C1 is a Ca2+-dependent complex of C1q, C1r, and C1s, and analogous to an initiation complex of the lectin pathway of complement, which consists of the mannose-binding lectin (MBL) homologous to C1q and the MBL-associated serine proteases (MASPs) homologous to C1r and C1s. Thus divergence of Clq and MBL and that of C1r, C1s and the MASPs are considered to be crucial events in the establishment and evolution of the classical complement pathway. However, molecular information on the C1 subcomponents is very limited in lower vertebrates. Here we describe two distinct C1r/C1s/MASP2-like cDNA clones (C1r/s-A, C1r/s-B) isolated from the common carp (Cyprinus carpio). They share 83% identity at the amino acid level and have a domain structure similar to that of C1r/C1s/MASPs from other species. The serine protease domain of the carp homologues lacks the histidine loop and is encoded by a single exon containing an AGY codon for the active serine residue, as in mammalian C1r, C1s, and MASP2. Southern blot and PCR analyses indicated that the carp has at least three copies of the C1r/s-A gene and a single C1r/s-B gene. Although phylogenetic tree analysis does not definitively assign carp C1r/s-A and C1r/s-B, they might represent ancestral molecules which later diverged into C1r, C1s, and MASP2 of higher vertebrates.

Alleles↗
PubMed2001

Cyst fluid levels of carcinoembryonic antigen, carbohydrate antigen 19-9, squamous cell carcinoma antigen, and amylase in thyroglossal duct and branchial cleft cysts.

Cyst fluid was aspirated from five thyroglossal duct cysts (TDCs) and four branchial cleft cysts (BCCs). The cyst fluid levels of carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, squamous cell carcinoma antigen (SCC), and amylase were measured and compared between the two types of cyst. The enzyme immunoassay technique was used for CEA and CA19-9, while SCC and amylase levels were measured by the immunoradiometric assay and nitrophenol method, respectively. Immunostaining for CEA, CA19-9, and amylase was also performed. The serum levels of these markers in both groups of patients were within the normal ranges. In contrast, their cyst fluid levels were extremely high. There was no significant difference in the cyst fluid levels of CEA and SCC between the two types of cyst; however, the cyst fluid from the TDCs showed significantly higher levels of CA19-9 and significantly lower levels of amylase compared with that from the BCCs. Immunostaining revealed expression of CA 19-9 in nearly half the columnar epithelial cells in the TDCs, but not in the squamous epithelial cells in the BCCs. CEA and amylase were not found in the epithelial cells of either type of cyst. These findings seem to reflect the difference in etiology between TDCs and BCCs.

Adult↗
PubMed2001

Video-assisted extended thymectomy in patients with thymoma by lifting the sternum.

We present use of minimally invasive video thoracoscopic surgery to perform complete extended thymectomy in patients with thymoma. These procedures were performed using a sternum-elevating method that provides a wide field of vision between the sternum and heart. Indications for this method are Masaoka Stage I, II and some Stage III (invasion to the lung and pericardium). This new method may be useful from the standpoint of minimal access, rapid recovery, less pain, and good cosmetic results.

Adult↗
PubMed2001

Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization.

To develop tools for analysis of the acute phase response, we used suppression subtractive hybridization of cDNAs from the livers of trout in an unchallenged state and in the course of a response to injection with a Vibrio bacterin emulsified in Freund's Incomplete Adjuvant. The resulting cDNA library contains 300-600bp long fragments of 25 or more immune-relevant genes. Fifteen were previously unreported for salmonids, and 12 were not known from any fish species. Known acute phase proteins include serum amyloid A, transferrin and precerebellin-like protein; trout C-polysaccharide-binding protein 1 is probably also an acute phase protein. Components of both the complement system (n=5) and the clotting system (n=3), as well as lectins, various binding proteins, a putative antibacterial peptide, a chemotaxin, an anti-oxidant enzyme, as well as some likely cell-surface receptors and metabolic and lysosomal enzymes are represented in the library. One clone closely resembles a group of Toll-like receptors, including the human IL-1 receptor. Three cDNAs appear to represent complete open reading frames.

Acute-Phase Proteins↗
PubMed2001

Organization of the NKEF gene and its expression in the common carp (Cyprinus carpio).

Natural killer enhancing factor (NKEF) is a member of the newly defined peroxiredoxin (Prx) family. Its functions are to enhance the cytotoxic capacity of natural killer cells and to prevent DNA and protein from being damaged by oxidative stress in the presence of thiol compounds. However, little is known about the structure and function of NKEF in lower vertebrates. We have recently cloned a cDNA encoding NKEF from the common carp (Cyprinus carpio) by use of suppression subtractive hybridization (SSH). In the present study, we used PCR to obtain a genomic DNA which covers the entire coding region of carp NKEF. In the 3363bp-long genomic sequence, six exons and five introns were identified. The carp NKEF gene has splice donor/acceptor site sequences at the boundaries of exons and introns, and contains two Val-Cys-Pro (VCP) motifs. The exon/intron organization of the carp NKEF gene shows complete conservation with other members of the Prx family. Genomic Southern blotting analyses suggest that carp has multiple copies of the NKEF gene. RT-PCR analyses reveal that carp NKEF has very different expression levels not only in tissues but also from individuals.

Amino Acid Sequence↗
PubMed2001

Dantrolene ameliorates delayed cell death and concomitant DNA fragmentation in the rat hippocampal CA1 neurons subjected to mild ischemia.

The present study investigated whether dantrolene, which inhibits the Ca(2+) release from the intracellular Ca(2+) store sites, reduced nuclear DNA fragmentation and produced neuronal protection in a model of global forebrain ischemia. Male Wistar rats were subjected to four-vessel occlusion (4VO) for 5 min and then infused continuously with dantrolene or vehicle into the cerebral ventricle for 3 days. The intact rats did not undergo any intervention. The number of viable and DNA nick-end-labeled neurons in the hippocampal CA1 were evaluated 4 days after the ischemia. The number of viable neurons in the dantrolene-treated rats was significantly higher than that in the vehicle-treated rats and lower than that in the intact animals (P<0.01 and <0.05, respectively). The number of DNA nick-end-labeled nuclei was significantly lower in dantrolene-treated rats compared with the vehicle-treated animals (P<0.0001). No nick-end labeling was observed in the intact animals. A linear correlation was found between the number of viable cells and nick-end labeled nuclei in the CA1 (r=0.91, P<0.0001). These results suggest that the postischemic intraventricular dantrolene is effective in precluding neuronal death and concomitant nuclear DNA fragmentation following transient global ischemia.

Animals↗
PubMed2001

Diffusion-weighted echo-planar MR imaging of CNS involvement in systemic lupus erythematosus.

RATIONALE AND OBJECTIVES: The purpose of this study was to determine the range of findings at diffusion-weighted magnetic resonance (MR) imaging in patients with systemic lupus erythematosus (SLE) and central nervous system involvement. MATERIALS AND METHODS: Diffusion-weighted MR images were reviewed in 20 patients with SLE and correlated with clinical symptoms and findings at computed tomography, conventional MR imaging, MR angiography, or conventional angiography. RESULTS: Diffusion-weighted MR imaging showed acute or subacute lesions in nine of 20 patients (45%). In the other 11, it showed no abnormal findings or chronic lesions. In four of the nine patients with lesions, diffusion-weighted imaging primarily showed hyperintense lesions with decreased apparent diffusion coefficient (ADC), which indicates acute or subacute infarcts. In four other patients, it primarily showed iso- or slightly hyperintense lesions with increased ADC, suggesting vasogenic edema. In two of these four patients, the findings were consistent with hypertensive encephalopathy. In the other two, small hyperintense foci on diffusion-weighted images with decreased ADC were seen within the vasogenic edema. These foci presumably represent microinfarcts associated with SLE vasculopathy. In the ninth patient, diffusion-weighted imaging showed a small linear hyperintense lesion with normal ADC in the left parietooccipital region. CONCLUSION: Diffusion-weighted imaging shows primarily two patterns of acute or subacute parenchymal lesions in patients with SLE: acute or subacute infarction and vasogenic edema with or without microinfarcts.

Adult↗
PubMed2001

The origin of cluster N2 of the energy-transducing NADH-quinone oxidoreductase: comparisons of phylogenetically related enzymes.

NADH-quinone (Q) oxidoreductase is a large and complex redox proton pump, which utilizes the free energy derived from oxidation of NADH with lipophilic electron/proton carrier Q to translocate protons across the membrane to generate an electrochemical proton gradient. Although its molecular mechanism is largely unknown, recent biochemical, biophysical, and molecular biological studies have revealed that particular subunits and cofactors play an essential role in the energy-coupling reaction. Based on these latest experimental data, we exhaustively analyzed the sequence information available from evolutionarily related enzymes such as [NiFe] hydrogenases. We found significant and conserved sequence differences in the PSST/Nqo6/NuoB, 49kDa/Nqo4/NuoD, and ND1/Nqo8/NuoH subunit homologs between complex I/NDH-1 and [NiFe] hydrogenases. The alterations, especially in the postulated ligand motif for cluster N2 in the PSST/Nqo6/NuoB subunits, appear to be evolutionarily important in determining the physiological function of complex I/NDH-1. These observations led us to propose a hypothetical evolutionary scheme: during the course of evolution, drastic changes have occurred in the putative cluster N2 binding site in the PSST/Nqo6/NuoB subunit and the progenitors of complex I/NDH-1 have concurrently become to utilize a lipophilic electron/proton carrier such as Q as its physiological substrate. This scheme provides new insights into the structure and function relationship of complex I/NDH-1 and may help us understand its energy-coupling mechanism.

Amino Acid Sequence↗
PubMed2001

Reduction of malignant phenotype of HEPG2 cell is associated with the expression of connexin 26 but not connexin 32.

Connexin (Cx) genes have a negative growth effect on tumour cells with certain specificity. However, it is not clear whether each Cx gene can act similarly in growth control. Hepatocytes normally express Cx26 and Cx32 as their major gap junction genes, but HepG2 cells, a hepatoma cell line, are deficient in gap junctional intercellular communication (GJIC) based on the down-regulation of Cx26 and aberrant localization of Cx32. In this study, we showed that some of the expressed Cx26 protein in HepG2 cells localized in the plasma membrane and contributed to recovery of GJIC, while the Cx32 protein remained localized in the cytoplasm. The Cx26-transfected clones showed a significantly slower growth in vivo as well as in vitro and reduced anchorage-independent growth ability compared with a mock-transfected clone. Cx26-transfected cells had more regular cell layers due to the re-establishment of the E-cadherin cell adhesion complex. E-cadherin expression following Cx26 transfection was induced. Cx26 expression simultaneously brought E-cadherin and beta-catenin proteins into the plasma membrane without any change in the expression level of beta-catenin protein. These results suggest that the expression of Cx26 contributes to negative growth control of HepG2 cells and the morphological change through the induction of E-cadherin and subsequent formation of cell adhesion complex.

Animals↗
PubMed2001

Evidence for the expression of interleukin (IL)-18, IL-18 receptor and IL-18 binding protein in the human endometrium.

To see whether the interleukin (IL)-18 system is operative in the endometrium, we examined the expression of IL-18, IL-18 receptor (IL-18R) and IL-18 binding protein (IL-18BP), the substance known to neutralize IL-18 activity, in this tissue. Reverse transcription-polymerase chain reaction analyses showed that IL-18, IL-18R and IL-18BP mRNA were constitutively expressed without significant fluctuation throughout the menstrual cycle. When epithelial cells and stromal cells were cultured separately, the expression levels of IL-18 mRNA in epithelial cells were about 18-fold higher compared to those in stromal cells. Furthermore, the IL-18 precursor protein was detected by Western blot analysis in cultured epithelial cells but not in stromal cells. Recombinant human IL-18 stimulated the secretion of interferon (IFN)-gamma by resident bone marrow-derived cells in the endometrium. On the other hand, IFN-gamma up-regulated the IL-18BP expression both in cultured epithelial cells and stromal cells. Thus, we have presented evidence for the presence of the IL-18 system in the human endometrium. In light of its immunomodulatory roles in a variety of tissues, this system may afford protection against pathogenic micro-organisms and provide a regulatory mechanism for controlled trophoblast invasion by modulating a local cytokine network.

Bone Marrow Cells↗
PubMed2001

Demonstration of the importance and usefulness of manipulating non-active-site residues in protein design.

Do non-active-site residues participate in protein function in a more direct way than just by holding the static framework of the protein molecule? If so, how important are they? As a model to answer these questions, ATB17, which is a mutant of aspartate aminotransferase created by directed evolution, is an ideal system because it shows a 10(6)-fold increase in the catalytic efficiency for valine but most of its 17 mutated residues are non-active-site residues. To analyze the roles of the mutations in the altered function, we divided the mutations into four groups, namely, three clusters and the remainder, based on their locations in the three-dimensional structure. Mutants with various combinations of the clusters were constructed and analyzed, and the data were interpreted in the context of the structure-function relationship of this enzyme. Each cluster shows characteristic effects: for example, one cluster appears to enhance the catalytic efficiency by fixing the conformation of the enzyme to that of the substrate-bound form. The effects of the clusters are largely additive and independent of each other. The present results illustrate how a protein function is dramatically modified by the accumulation of many seemingly inert mutations of non-active-site residues.

Amino Acid Substitution↗
PubMed2001

Dissociation of Bax from a Bcl-2/Bax heterodimer triggered by phosphorylation of serine 70 of Bcl-2.

Serine 70 in the loop region of Bcl-2 is specifically phosphorylated by paclitaxel-treatment in tumor cells and BHK cells expressing Bcl-2. The phosphorylation of serine 70 of Bcl-2 (pS70-Bcl-2) peaks 24 to 48 h after paclitaxel treatment and accelerates apoptosis. Phosphorylation is effectively inhibited in the presence of actinomycin D or cycloheximide, which restore cell viability to the same level as control cells not expressing Bcl-2. These results indicate that paclitaxel-induced kinase(s) and/or its activator(s) are synthesized de novo and play an important role in paclitaxel-induced apoptosis by phosphorylating Bcl-2. In binding assays using the phosphorylation-specific antibody against pS70-Bcl-2, the induction of serine 70 phosphorylation 70 results in a loss of the binding ability of Bcl-2 to Bax, a pro-apoptotic partner, and induces subsequent cell death. When the pS70-Bcl-2 antibody was added to human breast cancer tissue, serine 70 phosphorylation was also detected, even prior to treatment with anticancer agents. Further study of breast cancers revealed 83% of tumors with high pS70-Bcl-2 expression responded to paclitaxel or docetaxel treatment, whereas 57% of those with low expression not respond. These findings suggest that pS70-Bcl-2 might be a predictive factor for prognosis and sensitivity to paclitaxel treatment for breast cancer.

Animals↗
PubMed2001

Vaccination effect of interleukin-6-producing pancreatic cancer cells in nude mice: a model of tumor prevention and treatment in immune-compromised patients.

In an effort to explore properties important in hematogenous metastasis of pancreatic adenocarcinoma, we previously demonstrated that tumor-derived interleukin (IL)-6 is a crucial factor that conveys resistance to liver metastasis. Here we extend the study to examine a possible vaccination effect of tumor-derived IL-6 in T-cell-deficient nude mice, as a model for predicting the effect in immune-compromised patients. We used a pair of IL-6-nonproducing and highly producing pancreatic adenocarcinoma cell lines, PCI-43 and PCI-43h, respectively. The reaction intensity of anti-PCI IgG antibodies in host nude mice was maximal 28 days after inoculation of PCI-43h cells, and remained high thereafter. A fraction of the pancreatic carcinoma cell lines, namely, PCI-6, -10, and -43, expressed surface antigenic determinant(s) reactive with the IgG; but the others, PCI-19, -24, -55, -64, -66, -68, -72, and -79, did not. Inoculation of PCI-43h but not PCI-43 suppressed growth of simultaneously inoculated PCI-43, but not PCI-24 xenografts. In addition, administration of PCI-43h, but not PCI-43 suppressed the growth of PCI-43 that was xenografted 4 weeks later, thus revealing a vaccination effect of IL-6-producing PCI-43h, but not IL-6-nonproducing PCI-43. These data, obtained from T-cell-deficient nude mice, suggest an in vivo role for IL-6 in inducing IgG-mediated, pancreatic carcinoma-specific vaccination against a thymus-independent antigen.

Animals↗