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Biomedical subjects

H Yanagi

Publications and source records attributed to H Yanagi.

At least 55 records · Page 3Linked to original sources
PubMed2000

Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins.

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 microM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.

Journal Article↗
PubMed2000

A new type of glycoglycerolipids from Corynebacterium aquaticum.

A new type of glycoglycerolipids, S361A and S365A, were obtained from Corynebacterium aquaticum strains, S361 and S365, newly isolated from soils, and were identified as (2R)-1-[alpha-glucopyranosyl-(1alpha-3)-(6O-acyl-alpha-manno pyranosyl)]-3-O-acylglycerol and (2R)-1-[alpha-mannopyranosyl-(1alpha-3)-(6-O-acyl-alpha-mannopyran osyl)]-3-O-acylglycerol, respectively. S365A was identical to a novel glycoglycerolipid recently isolated from some bacteria, but S361A was a new analog having a glucosylmannosyl in place of the dimannosyl group. Our results indicate that this sn-2 lysotype of glyceroglycolipids may be widely distributed in bacteria.

Carbohydrate Conformation↗
PubMed2000

Autoantibodies against chaperonin CCT in human sera with rheumatic autoimmune diseases: comparison with antibodies against other Hsp60 family proteins.

Chaperonin CCT containing t-complex polypeptide 1 is a cytosolic molecular chaperone that assists in the folding of actin, tubulin, and other proteins and is a member of the 60-kDa heat shock protein (Hsp60) family. We examined antibody titers against human CCT and other Hsp60 family members in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematodes, Sjögren syndrome, and mixed connective tissue disease. Autoantibody titers against not only human mitochondrial Hsp60 but also CCT were significantly higher in the sera of patients with rheumatic autoimmune diseases than in healthy control sera. Although immunoglobulin G (IgG) titers against Escherichia coli GroEL were high in all the groups of sera tested, no significant differences in anti-GroEL responses were detected between patients and healthy controls. IgG titers against mycobacterial Hsp65 showed a similar pattern to titers of autoantibodies recognizing GroEL. Immunoabsorption experiments demonstrated that most of the autoantibodies recognizing CCT were cross-reactive with mitochondrial Hsp60, E coli GroEL, and mycobacterial Hsp65. Although most of the anti-Hsp60 IgG recognized CCT, anti-GroEL (or antimycobacterial Hsp65) IgG contained antibodies specific for GroEL (or mycobacterial Hsp65) in addition to antibodies cross-reactive with CCT and Hsp60. Results from immunoblot analyses, together with weak (15% to 20%) amino acid sequence identities between CCT and the other Hsp60 family members, suggested that CCT-reactive autoantibodies recognize conformational epitopes that are conserved among CCT and other Hsp60 family members.

Adult↗
PubMed1999

Cytosolic chaperonin is up-regulated during cell growth. Preferential expression and binding to tubulin at G(1)/S transition through early S phase.

The chaperonin containing t-complex polypeptide 1 (CCT) is a heterooligomeric molecular chaperone assisting in the folding of actin, tubulin, and other cytosolic proteins. The expression levels of CCT subunits varied among seven mouse cell lines tested but showed a close correlation with growth rate. Both the CCT protein and mRNA levels in the human promyelolytic cell HL60 decreased concomitant with growth arrest during differentiation. More rapid decrease in CCT level occurred when the mouse interleukin (IL)-3-dependent myeloid DA3 cells were starved for IL-3. Readdition of IL-3 caused rapid resumption of CCT synthesis during synchronous growth: the maximum CCT protein and mRNA levels were observed at G(1)/S transition through early S phase. The turnover rate of CCT was nearly constant regardless of growth. Gel filtration and immunoprecipitation analyses indicated that CCT in vivo is associated with tubulin at early S phase, but not at G(0)/G(1) phase. These results demonstrated that CCT expression is strongly up-regulated during cell growth especially from G(1)/S transition to early S phase and is primarily controlled at the mRNA level. CCT appears to play important roles for cell growth by assisting in the folding of tubulin and other proteins.

Animals↗
PubMed1999

Transcriptional activation of mouse cytosolic chaperonin CCT subunit genes by heat shock factors HSF1 and HSF2.

The chaperonin containing TCP-1 (CCT) is a eukaryotic molecular chaperone consisting of eight subunit species and assists in the folding of cytosolic proteins. We show here that all eight mouse CCT subunit genes contain sequences called heat shock elements for binding heat shock transcription factors (HSFs) by electrophoretic mobility shift assays and that these genes are transcriptionally activated by HSFs in reporter gene assays using HeLa cells transiently overexpressing HSFs. These results suggest that HSF1 and/or HSF2 play a role in Cct gene expression.

Animals↗
PubMed1999

Marked instability of the sigma(32) heat shock transcription factor at high temperature. Implications for heat shock regulation.

The heat shock response in Escherichia coli depends on a transient increase in the intracellular level of sigma(32) that results from both increased synthesis and transient stabilization of normally unstable sigma(32). Although the membrane-bound ATP-dependent protease FtsH (HflB) plays an important role in degradation of sigma(32), our previous results suggested that several cytosolic ATP-dependent proteases including HslVU (ClpQY) are also involved in sigma(32) degradation (Kanemori, M., Nishihara, K., Yanagi, H., and Yura, T. (1997) J. Bacteriol. 179, 7219-7225). We now report on the ATP-dependent proteolysis of sigma(32) by purified HslVU protease and its unusual dependence on high temperature: sigma(32) was rapidly degraded at 44 degrees C, but with much slower rates ( approximately 15-fold) at 35 degrees C. FtsH-dependent degradation of sigma(32) also gave similar results. In agreement with these results in vitro, the turnover of sigma(32) in normally growing cells at high temperature (42 degrees C) was much faster than at low temperature (30 degrees C). Taken together with other evidence, these results suggest that the sigma(32) level during normal growth is primarily determined by the stability (susceptibility to proteases) and synthesis rate of sigma(32) set by ambient temperature, whereas fine adjustment such as transient stabilization of sigma(32) observed upon heat shock is brought about through monitoring changes in the cellular state of protein folding.

ATP-Dependent Proteases↗
PubMed1999

Translational induction of heat shock transcription factor sigma32: evidence for a built-in RNA thermosensor.

Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock sigma-factor sigma32 encoded by the rpoH gene. Increased sigma32 levels result from both enhanced synthesis and stabilization. Previous work indicated that sigma32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5'-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of sigma32 synthesis further, we analyzed expression of rpoH-lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA-30S ribosome-tRNAfMet complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator.

Bacterial Proteins↗
PubMed1999

150-kDa oxygen-regulated protein (ORP150) suppresses hypoxia-induced apoptotic cell death.

To determine the contribution of 150-kDa oxygen-regulated protein (ORP150) to cellular processes underlying adaptation to hypoxia, a cell line stably transfected to overexpress ORP150 antisense RNA was created. In human embryonic kidney (HEK) cells stably overexpressing ORP150 antisense RNA, ORP150 antigen and transcripts were suppressed to low levels in normoxia and hypoxia, whereas wild-type cells showed induction of ORP150 with oxygen deprivation. Inhibition of ORP150 in antisense transfectants was selective, as hypoxia-mediated enhancement of glucose-regulated protein (GRP) 78 and GRP94 was maintained. However, antisense ORP150 transfectants displayed reduced viability when subjected to hypoxia, compared with wild-type and sense-transfected HEK cells. In contrast, diminished levels of ORP150 had no effect on cytotoxicity induced by other stimuli, including oxygen-free radicals and sodium arsenate. Although cellular ATP content was similar in hypoxia, compared with ORP150 antisense transfectants and wild-type HEK cells, suppression of ORP150 expression was associated with accelerated apoptosis. Hypoxia-mediated cell death in antisense HEK transfectants did not cause an increase in caspase activity or in cytoplasmic cytochrome c antigen. A well recognized inducer of apoptosis in HEK cells, staurosporine, caused increased caspase activity and cytoplasmic cytochrome c levels in both wild-type and antisense cells. These data indicate that ORP150 has an important cytoprotective role in hypoxia-induced cellular perturbation and that ORP150-associated inhibition of apoptosis may involve mechanisms distinct from those triggered by other apoptotic stimuli.

Apoptosis↗
PubMed1999

Current trends in restorative proctocolectomy: introduction of an ultrasonically activated scalpel.

PURPOSE: We evaluated the usefulness of an ultrasonically activated scalpel (Harmonic Scalpel) for mucosal proctocolectomy and ileal J-pouch-anal anastomosis. METHODS: Seventy-four patients with ulcerative colitis (70 patients) and familial adenomatous polyposis (4 patients) underwent mucosectomy using the Harmonic Scalpel since 1997. We compared the clinical and functional results with those of the monopolar electrocoagulator (forceps coagulation technique, 86 patients with colitis and 7 with polyposis). RESULTS: We performed graduated mucosal proctectomy by using the Harmonic Scalpel. The operative time (Harmonic Scalpel, 42 minutes vs. forceps coagulation technique, 85 minutes) and blood loss (Harmonic Scalpel, 33 ml vs. forceps coagulation technique, 86.8 ml) were significantly reduced by this method. The Harmonic Scalpel enabled restorative proctocolectomy by the synchronous approach within three hours. The functional results and complications were not significantly different between the two groups. CONCLUSION: The Harmonic Scalpel shortened the operative time, decreased blood loss, and was useful for restorative proctocolectomy in our study.

Adult↗
PubMed1999

Transanal mucosectomy using an ultrasonically activated scalpel for ulcerative colitis.

We describe herein our technique of using an ultrasonically activated scalpel (Harmonic Scalpel [HS]) to simplify the procedure of anorectal mucosectomy for ulcerative colitis (UC). This technique was successfully employed to perform restorative proctocolectomy and ileoanal anastomosis (IAA) in ten patients with UC. By using the HS, thermal injury of the internal anal sphincter was avoided during mucosectomy, and we were able to dissect the inflamed mucosa sharply without causing bleeding. HS reduced the operative time and blood loss compared with the traditional forceps-coagulation technique. These results indicate that the introduction of this new scalpel will facilitate an increase in the number of cases of mucosectomy for IAA.

Adolescent↗
PubMed1999

Compliance and medication knowledge among elderly Japanese home-care recipients.

OBJECTIVES: To investigate the risk factors for noncompliance in elderly home-care recipients; and to evaluate to what extent regular home visits and drug counseling by a pharmacist contribute to compliance. SUBJECTS: One hundred and sixty-three elderly home-care recipients aged 62 years and over took part in this study. All subjects were cognitively normal, and taking a regimen of one or more prescribed drugs. Medication use was observed by pharmacist-conducted interviews during home visits. Compliance was estimated by comparing prescribed regimens with medications actually being taken at home. RESULTS: The mean age with (SD) of the subjects was 78.7 (8.3) years. Eighteen per cent were regularly counseled by a pharmacist about medication. Poor compliance with prescribed medications was associated with subjects aged 80 years and over, who were administering their own medication, consuming less than three meals a day, did not have one dose packages, and who were not receiving pharmacist counseling. In multiple logistic regression analyses, frequency of meals (OR 5.99; 95% CI 1.25-28.79), pharmacist counseling (OR 5.32; 95% CI 2.00-14.20), and age (OR 0.96; 95% CI 0.92-1.00) were independent predictors of good compliance for home-care recipients with physical disabilities. Compliance correlated inversely with knowledge of drug names, and drug purposes in the uncounseled group. Compliance, however, positively correlated with knowledge of drug purposes in the counseled group. CONCLUSION: In this study, compliance among elderly Japanese home-care recipients was found to be associated with receiving pharmacist counseling, frequency of meals, and age.

Aged↗
PubMed1999

Structures and co-regulated expression of the genes encoding mouse cytosolic chaperonin CCT subunits.

The chaperonin-containing TCP-1 (CCT) is a hetero-oligomeric molecular chaperone that mediates protein folding in the cytosol of eukaryotes. Eight (or nine in testis) subunit species are assembled in the CCT hexadecamer complex. We have cloned seven CCT subunit genes, Cctb, Cctd, Ccte, Cctz-1, Cctz-2 (testis specific), Ccth and Cctq, from mouse genomic DNA libraries, in addition to the Ccta and Cctg genes reported previously, and the entire nucleotide sequences of these DNA clones were determined. These genes are approximately 15-20 kb in length except for Cctz-2 which is longer than 35 kb, and all the Cct genes consist of 11-16 exons. Primer extension analyses of testis RNA indicate one to several potential transcription start sites 50-150 bp upstream from the translation start codon of each Cct gene. There are several possible Sp1-binding sequences, but no obvious TATA box was observed around the potential start sites. From 5'-flanking regions to the first introns, the Cct genes are rich in CpG dinucleotides. In reporter gene assays using these regions, five of eight Cct genes showed strong transcriptional activity comparable with the combination of SV40 promoter and enhancer in HeLa cells. We also show, by Western and Northern blot analyses, that CCT expression levels vary widely among different tissues but the expression patterns are very similar among the eight subunit species. It is likely that expression levels of the eight different subunits are tightly co-regulated to maintain a constant ratio of these subunits which constitute the CCT hexadecamer complex with a fixed subunit arrangement.

Amino Acid Sequence↗
PubMed1999

Supportive evidence for contribution of the dopamine D2 receptor gene to heritability of stature: linkage and association studies.

Increases in height were reported in children chronically exposed prenatally and postnatally to D2 receptor-blocking drugs. A possible haplotypic association between stature and the DRD2 gene was also reported. In this study, we examined linkage between stature and DRD2 by genotyping a dinucleotide repeat polymorphism in 79 sib-pairs aged 8-17 years. An association between stature and a putative functional polymorphism in the promoter region of the DRD2 gene was examined in the sib-pairs and in 125 unrelated male adults. All the subjects were Japanese. Linkage (p = 0.004, SIBPAL) and an association (p = 0.009, paired t-test, in the sib-pairs; p = 0.006, ANOVA, in the adults) with stature were suggested. These findings indicate that DRD2 is one of the genes that contribute to heritability of stature.

Adolescent↗
PubMed1999

Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress.

The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in ER-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.

Activating Transcription Factor 6↗
PubMed1999

Frequent occurrence of hypoalphalipoproteinemia due to mutant apolipoprotein A-I gene in the population: a population-based survey.

To determine the frequency of familial hypoalphalipoproteinemia in the general population due to mutation of the apolipoprotein A-I (apo A-I) gene, we analyzed sequence variations in the apo A-I gene. The subjects were 67 children with a low high-density lipoprotein (HDL) cholesterol level (</=38 mg/dl, below the fifth percentile in the population of this study) who were screened from 1254 schoolchildren through a school survey. Four different mutations with deleterious potential, three frameshifts and one splice site mutation, were identified in four of the subjects. The plasma apo A-I levels of the four children with apo A-I gene mutations were reduced to approximately half of the normal levels and were below the first percentile of the general population distribution (80 mg/dl). Thus, the frequency of hypoalphalipoproteinemia due to a mutant apolipoprotein A-I gene was estimated at 6% (95% CI: 2.4-14. 4%) in the subjects with low HDL cholesterol levels and 0.3% (95% CI: 0.1-0.8%) in the Japanese population. This suggests that familial hypoalphalipoproteinemia due to apo A-I gene mutations is relatively common.

Adolescent↗
PubMed1999

The ATP-dependent HslVU/ClpQY protease participates in turnover of cell division inhibitor SulA in Escherichia coli.

Escherichia coli mutants lacking activities of all known cytosolic ATP-dependent proteases (Lon, ClpAP, ClpXP, and HslVU), due to double deletions [DeltahslVU and Delta(clpPX-lon)], cannot grow at low (30 degrees C) or very high (45 degrees C) temperatures, unlike those carrying either of the deletions. Such growth defects were particularly marked when the deletions were introduced into strain MG1655 or W3110. To examine the functions of HslVU and other proteases further, revertants that can grow at 30 degrees C were isolated from the multiple-protease mutant and characterized. The revertants were found to carry a suppressor affecting either ftsZ (encoding a key cell division protein) or sulA (encoding the SulA inhibitor, which binds and inhibits FtsZ). Whereas the ftsZ mutations were identical to a mutation known to produce a protein refractory to SulA inhibition, the sulA mutations affected the promoter-operator region, reducing synthesis of SulA. These results suggested that the growth defect of the parental double-deletion mutant at a low temperature was due to the accumulation of excess SulA without DNA-damaging treatment. Consistent with these results, SulA in the double-deletion mutant was much more stable than that in the Delta(clpPX-lon) mutant, suggesting that SulA can be degraded by HslVU. As expected, purified HslVU protease degraded SulA (fused to the maltose-binding protein) efficiently in an ATP-dependent manner. These results suggest that HslVU as well as Lon participates in the in vivo turnover of SulA and that HslVU becomes essential for growth when the Lon (and Clp) protease level is reduced below a critical threshold.

ATP-Dependent Proteases↗