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Biomedical subjects

M Takenaka

Publications and source records attributed to M Takenaka.

At least 73 records · Page 4Linked to original sources
PubMed1996

The construction of novel mobilizable YAC plasmids and their behavior during trans-kingdom conjugation between bacteria and yeasts.

Trans-kingdom conjugation is an easy and efficient method for gene transfer from prokaryotes to eukaryotes since it does not require DNA extraction and purification. We constructed novel mobilizable plasmids pAY-YAC-B and pAY-YAC-E. The origin of conjugal transfer (oriT) was inserted at two different positions, pAY-YAC-B contains oriT region in between two telomeres whereas pAY-YAC-E has oriT at the cloning site of pYAC4. By conjugation, both plasmids were successfully transferred from E. coli to S. cerevisiae and S. kluyveri yeasts with the aid of helper plasmid pRH220 which harbors mob and tra genes. The plasmids were transferred more efficiently in S. cerevisiae compared to S. kluyveri. The analyses by restriction enzyme digestion and Southern hybridization indicated that both plasmids maintained their original structure and size in transconjugant yeasts, therefore, reflecting the faithful nicking and subsequent resealing of plasmids during conjugation. The comparison between conjugative transfer and transformation has also been performed and discussed.

Chromosomes, Artificial, Yeast↗
PubMed1996

Inhibition of TGF-beta 1 expression by antisense oligonucleotides suppressed extracellular matrix accumulation in experimental glomerulonephritis.

Overproduction of transforming growth factor-beta 1 (TGF-beta 1) has been implicated in the pathogenesis of fibrotic diseases. TGF-beta 1 plays a crucial role in the accumulation of extracellular matrix (ECM) in human and experimental glomerular diseases. However, it remains unclear whether inhibition of TGF-beta 1 overproduction would suppress TGF-beta 1-induced ECM accumulation. To inhibit the overproduction of TGF-beta 1 in experimental glomerulonephritis induced by anti-Thy 1.1 antibody, we introduced antisense oligodeoxynucleotides (ODN) for TGF-beta 1 into the nephritic kidney by the HVJ-liposome-mediated gene transfer method. Sense, scrambled or reverse ODN were also introduced as controls. Transfected ODN accumulated mainly in the nuclei of mesangial cells in the glomeruli of transfected kidneys. In the antisense ODN-transfected rats, a marked decrease in expression of TGF-beta 1 mRNA was confirmed by Northern analysis. Consequently, the expression of TGF-beta 1 protein in the glomerulus was markedly reduced in the antisense ODN-transfected kidney with a comparable effect in preventing glomerular ECM expansion in experimental glomerulonephritis. In contrast, sense, scrambled and reverse ODNs failed to suppress TGF-beta 1 expression and ECM accumulation. Thus, these results suggested that inhibition of TGF-beta 1 overproduction could suppress progression to glomerulosclerosis.

Animals↗
PubMed1996

Expression of betaine transporter mRNA: its unique localization and rapid regulation in rat kidney.

Betaine is a major compatible osmolyte in the renal medulla. It is taken up into cells via the betaine gamma-amino-n-butyric acid transporter (BGT-1). We investigated the localization of BGT-1 mRNA and its acute regulation by NaCl and furosemide administration. In situ hybridization revealed that BGT-1 mRNA is predominantly present in the outer medulla and papilla. Less intense signals were seen in the inner medulla and no signals were found in the cortex. Microscopic examination suggested that intense signals were present in the medullary thick ascending limbs of Henle's loop (MTAL) and the inner medullary collecting ducts (IMCD). A reverse transcription and polymerase chain reaction assay of individual microdissected segments along the nephron confirmed its localization. Intraperitoneal administration of NaCl rapidly increased the signal in the MTAL, and furosemide prevented the increase in BGT-1 mRNA by NaCl loading. In contrast, BGT-1 mRNA in the IMCD is less sensitive to these kinds of acute regulation. These results suggest that BGT-1 expression in the MTAL is rapidly regulated in response to the magnitude of NaCl absorption, as suggested for the expression of Na+/myo-inositol cotransporter.

Aldehyde Reductase↗
PubMed1996

[Tizanidine for preanesthetic medication].

The effect of tizanidine given as a premedication on perioperative hemodynamics (mean blood pressure, heart rate), sedation, hypnosis and midazolam requirements for induction were assessed in 68 patients scheduled for elective surgery under general anesthesia. Patients were assigned to three groups. Group 1 was premedicated 90 min prior to induction with tizanidine 4 mg po (n = 28); group 2 was premedicated with tizanidine 2 mg po (n = 12); group 3 received no premedication (n = 28). In group 1, increase of mean blood pressure on anesthesia induction was attenuated significantly and sedative and hypnotic effects were stronger significantly compared with other groups. We also found that the amounts of midazolam necessary for loss of consciousness were significantly less in patients who had received tizanidine 4 mg. In conclusion, tizanidine is a useful drug as preanesthetic medication for general anesthesia.

Adjuvants, Anesthesia↗
PubMed1996

[Regulation of osmolyte transporter gene expression by tonicity].

In response to exposure to hypertonic environment, cells accumulate small organic solutes termed osmolytes that protect them from the adverse effects of hypertonicity. Some osmolytes (myo-inositol, betaine and taurine) are accumulated by specific transporters. Those transporters have been cloned by expression cloning techniques. Transcription of osmolyte transporters is induced by hypertonicity. In situ hybridization reveals the localization of the Na+/myo-inositol cotransporter and the betaine transporter (BGT1) mRNAs in the kidney. The BGT1 gene was cloned and its 5' flanking region has been characterized A 13 bp tonicity responsive enhancer (TONE) has been identified. Analysis of transgenic mice harboring a 5' flanking region of the BGT1 gene, that included TONE, showed that it provides inducible reporter expression in kidney medulla.

Animals↗
PubMed1995

The canine betaine gamma-amino-n-butyric acid transporter gene: diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.

The Na(+)- and Cl(-)-coupled betaine transporter, designated BGT1, a member of the neurotransmitter transporter gene family, is responsible for accumulation of betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla. The canine gene for the betaine gamma-amino-n-butyric acid transporter has been cloned and analyzed. The gene extends over 28 kb and consists of 18 exons. The 5' end of the gene has three alternative first exons (1A, 1B, and 1C+D). Analysis of BGT1 mRNA revealed that there is considerable divergence in the 5' untranslated sequence resulting from three different 5' end motifs (A, B, and C) followed by an alternative motif (D) as well as two internal acceptor sites for splicing. Eight kinds of BGT1 mRNA were classified into three types (A, B, and C) according to the 5' end sequence. Northern blot analysis using probes specific for the A, B, or C motif revealed that hypertonicity induces all three types in MDCK cells. Reverse transcription and polymerase chain reaction showed that each type was expressed in a tissue-specific manner. Primer extension and/or RNase protection assays as well as transfection assays into MDCK cells demonstrated that exons 1A, 1B, and 1C+D have independent transcription initiation sites under control of independent promoters. Diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.

Animals↗
PubMed1995

High affinity IgE receptor (Fc epsilon RI) expression on eosinophils infiltrating the lesions and mite patch tested sites in atopic dermatitis.

Expression of the high affinity IgE receptor (Fc epsilon RI) on eosinophils has recently been reported. This led us to evaluate Fc epsilon RI expression on eosinophils in atopic dermatitis (AD). Double immunofluorescence stainings with an anti-Fc epsilon RI monoclonal antibody (mAb) and a polyclonal antieosinophil cationic protein (ECP) antibody were performed on lesional biopsy specimens from patients with AD and from patients with bullous pemphigoid (BP) as controls. In AD and BP lesions, 77% and 70% of eosinophils expressed Fc epsilon RI, respectively. However, the intensity of Fc epsilon RI staining in AD was much stronger than in BP, suggesting upregulation of Fc epsilon RI expression on eosinophils in AD. In addition, the eosinophils infiltrating AD lesions were stained strongly with anti-CD23 mAb and anti-IgE antibody. At the sites of mite patch testing in AD, Fc epsilon RI-, CD23- and IgE-positive eosinophils were observed to the same degree as in the lesions, and a considerable number of mite antigen-bearing eosinophils were detected. Fc epsilon RI and CD23 were both upregulated on the skin-infiltrating eosinophils in AD and bound IgE molecules.

Adult↗
PubMed1995

High affinity IgE receptor-mediated prostaglandin E2 production by monocytes in atopic dermatitis.

High affinity IgE receptor (Fc epsilon R I) expression on monocytes and its upregulation on monocytes from patients with atopic dermatitis (AD) have been recently reported. In this study, we investigated whether prostaglandin E2 (PGE2) release from AD monocytes was Fc epsilon R I-dependent or not. The monocytes were stimulated with anti-Fc epsilon R I monoclonal antibody (mAb) and anti-Fc epsilon R II mAb. Cross-linking of Fc epsilon R I, but not that of Fc epsilon R II induced PGE2 release from monocytes. In order to confirm that the PGE2 release is IgE-dependent, stimulation with IgE+anti-IgE, IgG+anti-IgG and immune complexes made by incubation with AD patients' serum and recombinant Der fII (rDer fII-IC) were carried out in the culture. Significant PGE2 release by all the stimulants was observed, as compared with spontaneous release. PGE2 release by immune complex made by incubation with IgE-absorbed AD serum and rDer fII was much lower than that by rDer fII-IC. In monocytes from nonatopic donors, significant PGE2 release was observed when stimulated with IgE+anti-IgE, IgG+anti-IgG, rDer fII-IC, but not with anti-FC epsilon R I mAb and anti-Fc epsilon R II mAb. However, the release were significantly lower than respective PGE2 releases from AD monocytes. Taken together, cross-linking of Fc epsilon R I and IgE induced a PGE2 release from monocytes. This is a first report demonstrating an Fc epsilon R I-dependent mediator release from monocytes of AD patients.

Antigen-Antibody Complex↗
PubMed1995

The construction of mobilizable YACs and their direct conjugative transfer from E. coli to yeasts.

YAC, yeast artificial chromosome, consists of Saccharomyces cerevisiae's ARS, CEN and nutrient requirement genes and TEL from Tetrahymena with ori and ampicillin resistant genes of pBR322. we have constructed two mobilizable pAY-YACs plasmids by inserting oriT at different positions. pAY-YAC-B (12.2 kbp) containes oriT in between two telomeres, whereas in pAY-YAC-E (14 kbp) oriT has been inserted at the cloning site of pYAC4. Both plasmids were mediated from E.coli to S.cerevisiae and S.kluyveri with the aid of helper plasmid pRH220 which resides mob and tra genes. The results of conjugal transmission, restriction enzyme digestion and Southern blotting analyses indicated that both plasmids were able to maintained their size and structure, thus reflected the faithfull accomplishment of nicking and resealing of plasmids during conjugation. The direct transformation of plasmid DNA into yeast showed that pAY-YACs plasmids, may be due to their small size, have tendency of circularization.

Chromosomes, Artificial, Yeast↗
PubMed1994

The tonicity-sensitive element that mediates increased transcription of the betaine transporter gene in response to hypertonic stress.

BGT1, the Na(+)-and Cl- coupled betaine transporter, is responsible for the accumulation of high concentrations of the non-perturbing osmolyte betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla. In MDCK cells, the increase in activity of the betaine transporter is preceded by an increase in transcription of BGT1 and in the abundance of BGT1 mRNA. To investigate the molecular mechanism of transcriptional regulation by tonicity, we have characterized the 5'-flanking region of the gene. Transient transfection assays in MDCK cells cultured in isotonic or hypertonic medium using luciferase reporter constructs containing various fragments of the 5'-flanking region revealed that the region spanning base pairs -69 to -50 5' to the transcription initiation site (-69/-50) has hypertonicity-responsive enhancer activity. A double-stranded -69/-50 concatemer cloned 5' to an SV40 basal promoter and luciferase reporter gene in hypertonic cells exhibited more than 11-fold the activity in isotonic cells. Expression assays and electrophoretic mobility shift assays of mutants of -69/-50 identified a smaller region that is required for hypertonicity to induce increased expression and a slowly migrating band on mobility shift assays.

Animals↗
PubMed1994

cDNA cloning of a cytosolic protein tyrosine phosphatase (RKPTP) from rat kidney.

A rat cDNA encoding a non-receptor type phosphotyrosine phosphatase (PTPase; EC 3.1.3.48) was identified. The 1608 bp cDNA contains a single open reading frame that predicts a 382 amino acid protein with M(r) 44,438. The predicted protein has no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein. The C-terminal region has a PTPase catalytic domain that has 40-50% nucleic acid homology to other known PTPases. The N-terminal region has little amino acid sequence homology to any other known sequences. The recombinant protein of the cloned cDNA expressed in Escherichia coli was shown to possess PTPase activity using myelin basic protein, tyrosine phosphorylated by p43v-abl tyrosine kinase, as a substrate.

Amino Acid Sequence↗
PubMed1994

An acrosomal protein, sp32, in mammalian sperm is a binding protein specific for two proacrosins and an acrosin intermediate.

An acrosomal protein, sp32, was completely purified from acid extracts of ejaculated porcine sperm. Purified sp32 gave a single 32-kDa protein band on SDS-polyacrylamide gel electrophoresis and was characterized as a binding protein specific for 55-, 53-, and 49-kDa forms of (pro)acrosin. This protein was not capable of binding a 43-kDa acrosin intermediate and 35-kDa mature acrosin. sp32 significantly accelerated autoactivation of proacrosin at a basic pH in vitro and affected the maturation pathway of proacrosin. In the presence of sp32, the 49-kDa acrosin intermediate from the 55- and 53-kDa proacrosins was accumulated, instead of the 43-kDa acrosin intermediate. These results suggest that sp32 interacts with both the amino- and carboxyl-terminal sequences of the 53-kDa proacrosin. The cDNA clones coding for porcine and guinea pig sp32 have been identified from testis cDNA libraries in lambda gt11. The deduced amino acid sequence indicates that sp32 is initially synthesized as a 61-kDa precursor protein with a putative signal peptide at the amino terminus. The carboxyl-terminal half of the precursor molecule corresponds to the mature sp32. Thus, sp32 is produced by post-translational modification of the precursor. The binding of sp32 to proacrosin may be involved in packaging the acrosin zymogen into the acrosomal matrix.

Acrosin↗