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Hiroshi Sakurai

Publications and source records attributed to Hiroshi Sakurai.

15 recordsLinked to original sources
PubMed2006

The DNA-binding domain of yeast Hsf1 regulates both DNA-binding and transcriptional activities.

The heat shock transcription factor (HSF) is a key regulator of the heat shock response. In Saccharomyces cerevisiae, the transcription activating ability of Hsf1 is repressed by its DNA-binding domain, but the detailed mechanism by which the inhibitory function is relieved in response to stress remains unknown. In this study, we isolated and characterized three hsf1 mutants with temperature-sensitive mutations in the DNA-binding domain. Two mutations inhibited DNA-binding activity, leading to decreased expression of target genes. The third mutation caused transcriptional defects without affecting DNA binding, and its suppressor mutation was located in a region important for sensing heat shock. These results indicate that the DNA-binding domain regulates both the DNA-binding and transcriptional activities of Hsf1, and suggest that these functions are located within discrete regions of the DNA-binding domain.

Amino Acid Substitution↗
PubMed2006

Effects of a new extracorporeal system using CTR on mortality and inflammatory responses to bacterial toxin-induced multiple organ dysfunction syndrome in rabbits.

BACKGROUND/AIMS: In the pathogenesis of multiple organ dysfunction syndrome (MODS) caused by bacterial infection, a complex series of systemically secreted bacterial toxins and cytokines are intensely associated. Our previous study demonstrated that a new adsorbent, CTR, was capable of removing cytokines and toxic shock syndrome toxin-1 (TSST-1) in vitro. Moreover, extracorporeal treatment with CTR reduced the high mortality rate and inhibited inflammatory responses in endotoxin-induced shock in rats. However, it is unclear whether CTR treatment will be an effective therapy for MODS. Here, we demonstrated the efficacy of a new extracorporeal system using CTR on MODS induced by bacterial toxins in rabbits. METHODS: Direct hemoperfusion (DHP) apheresis with or without CTR for 120 min was performed in rabbits that had been intravenously infused with endotoxin and TSST-1. The mean arterial pressure was recorded and the plasma toxin and cytokine concentrations were measured during the treatment period. Mortality was assessed up to 7 days after exposure to the toxins. In addition, tissues specimens were examined using microscopy. RESULTS: The mortality rates at 7 days after the injection of the toxins were 90 and 10% for the control and CTR groups, respectively. The plasma concentrations of TSST-1, tumor necrosis factor and interleukin-1 beta in the CTR group were significantly lower than those in the control group. Histopathological examination revealed that tissue damage, such as necrosis and depletion of lymphocytes in the spleen and mesenteric lymph node, was reduced in the CTR group, compared with that in the control group, at 24 h after toxin infusion. CONCLUSION: The new adsorbent CTR improved the mortality rate in a MODS rabbit model by adsorbing TSST-1 and cytokines. Further development of CTR may expand the scope of extracorporeal therapies for patients with MODS.

Animals↗
PubMed2006

A novel adsorbent of circulating bacterial toxins and cytokines: the effect of direct hemoperfusion with CTR column for the treatment of experimental endotoxemia.

OBJECTIVES: The current study examined the ability of a new adsorbent, CTR, to remove enterotoxins, toxic shock syndrome toxin-1 (TSST-1), and cytokines from blood and/or serum in vitro and the effects of the extracorporeal treatment with CTR column on mortality rate and inflammatory responses to endotoxic shock in vivo. DESIGN: Laboratory investigation. SETTING: University and company experimental laboratory. MATERIALS: CTR is composed of porous cellulose beads to which a hydrophobic organic compound with a hexadecyl alkyl chain has been covalently bound to the surface as a ligand. Human/bovine serum and human blood samples in vitro and Male Wistar rats were used. INTERVENTIONS: CTR's ability to adsorb bacterial toxins and cytokines related to sepsis in serum and/or blood was examined with an in vitro batch adsorption protocol. In vivo, male Wistar rats were anesthetized and assigned to one of three groups (n=14 per group): Escherichia coli endotoxin (15 mg/kg intravenously) alone (endotoxemic), apheresis with control column without CTR for 120 mins (control column), or extracorporeal treatment with CTR column for 120 mins (CTR treatment). MEASUREMENTS AND MAIN RESULTS: With use of the CTR adsorbent, the adsorption rates were 50% to 90% for enterotoxins, TSST-1, and cytokines such as TNF-alpha and interleukin (IL)-6 in the batch tests. In vivo, the mortality rates at 8 hrs after endotoxin injection were 92%, 92%, and 14% for the endotoxemic, control column, and CTR treatment groups, respectively. Hypotension and elevated plasma cytokine concentrations and the infiltration of neutrophils of the lungs were less conspicuous in the CTR treatment group than in the other two groups. CONCLUSIONS: CTR, a novel adsorbent, effectively adsorbed small- to middle-sized proteins, such as cytokines, enterotoxins, and TSST-1 in vitro. Direct hemoperfusion apheresis with CTR column reduced mortality and had inhibitory effects on the inflammatory responses during endotoxemia in vivo. These findings suggest that extracorporeal blood purification with CTR column may be available to use for patients with sepsis and/or endotoxemia.

Adsorption↗
PubMed2005

Mutated yeast heat shock transcription factor activates transcription independently of hyperphosphorylation.

The homotrimeric heat shock transcription factor (HSF) binds to the heat shock element of target genes and regulates transcription in response to various stresses. The Hsf1 protein of Saccharomyces cerevisiae is extensively phosphorylated upon heat shock; a modification that is under positive regulation by its C-terminal regulatory domain (CTM). Hyperphosphorylation has been implicated in gene-specific transcriptional activation. Here, we surveyed genes whose heat shock response is reduced by a CTM mutation. The CTM is indispensable for transcription via heat shock elements bound by a single Hsf1 trimer but is dispensable for transcription via heat shock elements bound by Hsf1 trimers in a cooperative manner. Intragenic mutations located within or near the wing region of the winged helix-turn-helix DNA-binding domain suppress the temperature-sensitive growth phenotype associated with the CTM mutation and enable Hsf1 to activate transcription independently of hyperphosphorylation. Deletion of the wing partially restores the transcriptional defects of the unphosphorylated Hsf1. These results demonstrate a functional link between hyperphosphorylation and the wing region and suggest that this modification is involved in a conformational change of a single Hsf1 trimer to an active form.

Base Sequence↗
PubMed2005

Stress-induced transcription of the endoplasmic reticulum oxidoreductin gene ERO1 in the yeast Saccharomyces cerevisiae.

Gene transcription changes dramatically in response to various stresses. This event is an obligatory step for adaptation of cells to certain environments. Endoplasmic reticulum (ER) oxidoreductin encoded by the ERO1 gene of the yeast Saccharomyces cerevisiae is essential for the formation of protein disulfide bonds in the ER and for cell viability. We show that transcription of ERO1 is regulated by two transcriptional activators in response to different stresses. In the unfolded protein response induced by the reductant dithiothreitol, transcription factor Hac1 activates ERO1 transcription through a sequence that diverges from the consensus Hac1-binding sequence. Heat shock transcription factor Hsf1 activates ERO1 in response to heat, ethanol, and oxidative stresses. Using cells containing mutations in the Hac1- and Hsf1-binding sequences of the chromosomal ERO1 promoter, we demonstrate that Hac1-regulated transcription of ERO1 confers resistance to dithiothreitol. Although mutations in the Hsf1-binding sequences do not affect the sensitivity of cells to heat, ethanol, or oxidative stresses, both the Hac1- and Hsf1-regulated pathways are critical for normal growth under complex stress conditions.

Basic-Leucine Zipper Transcription Factors↗
PubMed2005

Identification of a novel class of target genes and a novel type of binding sequence of heat shock transcription factor in Saccharomyces cerevisiae.

In response to hyperthermia, heat shock transcription factor (HSF) activates transcription of a set of genes encoding heat shock proteins (HSPs). The promoter regions of HSP genes contain the HSF binding sequence called the heat shock element (HSE), which consists of contiguous inverted repeats of the sequence 5'-nGAAn-3' (where n is any nucleotide). We have constructed an hsf1 mutant of Saccharomyces cerevisiae and analyzed genome-wide changes in heat shock response in the mutant cells. The results have revealed that Hsf1 is necessary for heat-induced transcription of not only HSP but also genes encoding proteins involved in diverse cellular processes such as protein degradation, detoxification, energy generation, carbohydrate metabolism, and maintenance of cell wall integrity. Approximately half of the Hsf1-regulated genes lacked the typical HSE in their promoter regions. Instead, several of these genes have a novel Hsf1 binding sequence that contains three direct repeats of nTTCn (or nGAAn) interrupted by 5 bp. The number and spacing of the repeating units are critical determinants for heat-induced transcription as well as for recognition by Hsf1. In the yeast genome, the presence of the sequence is enriched in Hsf1-regulated genes, suggesting that it is generally used as an HSE in the Hsf1 regulon.

Base Sequence↗
PubMed2005

Saccharomyces cerevisiae heat shock transcription factor regulates cell wall remodeling in response to heat shock.

The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates expression of genes encoding heat shock proteins and a variety of other proteins as well. To better understand the cellular roles of Hsf1, we screened multicopy suppressor genes of a temperature-sensitive hsf1 mutation. The RIM15 gene, encoding a protein kinase that is negatively regulated by the cyclic AMP-dependent protein kinase, was identified as a suppressor, but Rim15-regulated stress-responsive transcription factors, such as Msn2, Msn4, and Gis1, were unable to rescue the temperature-sensitive growth phenotype of the hsf1 mutant. Another class of suppressors encoded cell wall stress sensors, Wsc1, Wsc2, and Mid2, and the GDP/GTP exchange factor Rom2 that interacts with these cell wall sensors. Activation of a protein kinase, Pkc1, which is induced by these cell wall sensor proteins upon heat shock, but not activation of the Pkc1-regulated mitogen-activated protein kinase cascade, was necessary for the hsf1 suppression. Like Wsc-Pkc1 pathway mutants, hsf1 cells exhibited an osmotic remedial cell lysis phenotype at elevated temperatures. Several of the other suppressors were found to encode proteins functioning in cell wall organization. These results suggest that Hsf1 in concert with Pkc1 regulates cell wall remodeling in response to heat shock.

Calcium-Binding Proteins↗
PubMed2004

Basal jawed vertebrate phylogeny inferred from multiple nuclear DNA-coded genes.

BACKGROUND: Phylogenetic analyses of jawed vertebrates based on mitochondrial sequences often result in confusing inferences which are obviously inconsistent with generally accepted trees. In particular, in a hypothesis by Rasmussen and Arnason based on mitochondrial trees, cartilaginous fishes have a terminal position in a paraphyletic cluster of bony fishes. No previous analysis based on nuclear DNA-coded genes could significantly reject the mitochondrial trees of jawed vertebrates. RESULTS: We have cloned and sequenced seven nuclear DNA-coded genes from 13 vertebrate species. These sequences, together with sequences available from databases including 13 jawed vertebrates from eight major groups (cartilaginous fishes, bichir, chondrosteans, gar, bowfin, teleost fishes, lungfishes and tetrapods) and an outgroup (a cyclostome and a lancelet), have been subjected to phylogenetic analyses based on the maximum likelihood method. CONCLUSION: Cartilaginous fishes have been inferred to be basal to other jawed vertebrates, which is consistent with the generally accepted view. The minimum log-likelihood difference between the maximum likelihood tree and trees not supporting the basal position of cartilaginous fishes is 18.3 +/- 13.1. The hypothesis by Rasmussen and Arnason has been significantly rejected with the minimum log-likelihood difference of 123 +/- 23.3. Our tree has also shown that living holosteans, comprising bowfin and gar, form a monophyletic group which is the sister group to teleost fishes. This is consistent with a formerly prevalent view of vertebrate classification, although inconsistent with both of the current morphology-based and mitochondrial sequence-based trees. Furthermore, the bichir has been shown to be the basal ray-finned fish. Tetrapods and lungfish have formed a monophyletic cluster in the tree inferred from the concatenated alignment, being consistent with the currently prevalent view. It also remains possible that tetrapods are more closely related to ray-finned fishes than to lungfishes.

Animals↗
PubMed2004

A spectroscopic study on defluorination of poly(tetrafluoroethylene) by alkyllithium/electron-donating solvents.

Defluorination of PTFE by alkyllithium/electron-donating solvents such as N,N,N',N'-tetramethylethylenediamine (TMEDA), hexamethylphosphoramide (HMPA) was studied by means of spectroscopy such as ESR, 7Li- and 13C-NMR, XPS, UV-Vis and IR. Based on the experimental results, it was concluded that an electron from radical species, which was generated in the alkyllithium/electron-donating solvent, was transferred onto PTFE molecule so as to eliminate fluorine atoms from the PTFE and to form carbon-centered radicals on the PTFE; concomitantly, the alkyl group of the alkyllithium was transferred onto the PTFE. Combined with the experimental results of the phenyllithium/HMPA system, mechanism of the fluorine atom elimination reactions from PTFE by the radical species is discussed.

Alkanes↗
PubMed2004

Phosphorylation of the yeast heat shock transcription factor is implicated in gene-specific activation dependent on the architecture of the heat shock element.

Heat shock transcription factor (HSF) binds to the heat shock element (HSE) and regulates transcription, where the divergence of HSE architecture provides gene- and stress-specific responses. The phosphorylation state of HSF, regulated by stress, is involved in the activation and inactivation of the transcription activation function. A domain designated as CTM (C-terminal modulator) of the Saccharomyces cerevisiae HSF is required for the activation of genes containing atypical HSE but not typical HSE. Here, we demonstrate that CTM function is conserved among yeast HSFs and is necessary not only for HSE-specific activation but also for the hyperphosphorylation of HSF upon heat shock. Moreover, both transcription and phosphorylation defects due to CTM mutations were restored concomitantly by a set of intragenic suppressor mutations. Therefore, the hyperphosphorylation of HSF is correlated with the activation of genes with atypical HSE but is not involved in that of genes with typical HSE. The function of CTM was circumvented in an HSF derivative lacking CE2, a yeast-specific repression domain. Taken together, we suggest that CTM alleviates repression by CE2, which allows HSF to be heat-inducibly phosphorylated and presume that phosphorylation is a prerequisite for the activator function of HSF when it binds to an atypical HSE.

Amino Acid Sequence↗
PubMed2003

Carboxy-terminal region of the yeast heat shock factor contains two domains that make transcription independent of the TFIIH protein kinase.

BACKGROUND: Phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is implicated in transition from initiation to elongation in the transcription cycle. In yeast cells, Kin28, a subunit of the general transcription factor TFIIH, is responsible for the CTD phosphorylation. Although Kin28 is indispensable for transcription of many genes, its requirement is bypassed in certain genes such as SSA4 or CUP1, whose transcription is activated by the heat shock factor Hsf1. RESULTS: We show that C-terminal region of Hsf1, which consists of an activation domain AR2 and a regulatory domain CTM, mediates the Kin28-independent transcription. The AR2 domain, when fused to the DNA-binding domain of Gal4 and recruited to the GAL7 gene via the Gal4-binding sequence, is sufficient for activating GAL7 in the absence of Kin28. We have further found that AR2 has an ability to recruit TATA box-binding protein-associated factors (TAFs) to the promoter. Consistently, transcription from promoters occupied naturally or artificially with TAFs is sustained in the absence of Kin28 function. CONCLUSIONS: These results show that CTM modulates activation function of AR2 in the Hsf1 molecule. We also suggest that recruitment of TAFs to a promoter is involved in the Kin28-independent transcription.

Carrier Proteins↗
PubMed2003

Artificial recruitment of certain Mediator components affects requirement of basal transcription factor IIE.

BACKGROUND: Basal transcription factors are essential for RNA polymerase II (RNAPII)-catalysed transcription of many but not all the mRNA-encoding genes in vivo as well as in vitro. For example, copper-inducible transcription of the copper metallothionein gene CUP1 occurs independently of basal factor TFIIE in budding yeast. To gain insight into the mechanism by which the requirement for TFIIE is bypassed, we artificially recruited certain constituents of Mediator, a large protein complex transmitting signals from various activators to the RNAPII machinery, to the CUP1 promoter by protein fusions with Ace1, the copper-inducible activator. RESULTS: Fusions with Med2 or Pgd1 activated CUP1 independently of TFIIE. Surprisingly, fusions with neither Srb5 nor Med9 circumvented TFIIE requirement for the CUP1 activation. Components of TFIID were similarly recruited to the CUP1 promoter without activation. By using a chromatin immunoprecipitation technique, we found that TFIIE is necessary for stable binding of TFIIH and RNAPII to the ADH1 promoter, whose activation requires TFIIE. However, binding of TFIIH and RNAPII to CUP1 upon its activation did not require TFIIE. CONCLUSIONS: Our results strongly suggest that the TFIIE requirement of a gene is determined by a target(s) in Mediator through which the signal of the cognate activator is transmitted.

Carrier Proteins↗
PubMedDate not supplied

Changes of near-UV CD spectrum of human hemoglobin upon oxygen binding: a study of mutants at alpha 42, alpha 140, beta 145 tyrosine or beta 37 tryptophan.

The deoxy-form of human adult hemoglobin (Hb A) exhibits a distinct negative CD band at 287 nm that disappears in the oxy-form. It has been suggested that the negative CD band is due to the environmental alteration of Tyr-alpha 42 or Trp-beta 37 at the alpha(1)beta(2) contact upon deoxygenation. To evaluate the contributions of the aromatic residues at the alpha(1)beta(2) contact and the penultimate tyrosine residues of the alpha and beta subunits (alpha 140 and beta 145) to the negative CD band, three recombinant (r) Hbs (rHb Ser-alpha 42, rHb His-beta 37, and rHb Thr-beta 145) were produced in Escherichia coli, and we compared the near-uv CD spectra of these three rHbs and Hb Rouen (Tyr-alpha 140-->His) with the spectra of Hb A under the condition in which all mutant Hbs were able to undergo the T-->R transition (Hill's n > 2.0). The contributions of Tyr-alpha 42, Trp-beta 37, Tyr-alpha 140, and Tyr-beta 145 to the negative CD band were estimated from changes in the ellipticity of the negative CD band at 287 nm to be 4, 18, 32, and 27%, respectively. These results indicate that environmental alteration of the penultimate tyrosine residues caused by the formation of salt bridges upon the R-->T transition is primarily responsible for the negative CD band.

Circular Dichroism↗