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H Aiba

Publications and source records attributed to H Aiba.

At least 37 records · Page 2Linked to original sources
PubMed2000

Identification and characterization of a novel gene, hos3+, the function of which is necessary for growth under high osmotic stress in fission yeast.

hos3 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos3-M26 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. The hos3+ gene was cloned and identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. A hos3delta strain, which we then constructed, had the phenotype of high osmolarity sensitivity, as in the case of the original hos3-M26 mutant. More interestingly, when these hos- cells were grown in the non-permissive growth condition in the presence of 2 M glucose, we found that unusually many septated cells were accumulated after a prolonged incubation. A multicopy suppressor gene for hos- mutations was also isolated and identified as the dsk1+ gene encoding a protein kinase, which was previously suggested to be implicated in a process of the mitotic regulation of S. pombe. The function of the hos3+ gene is discussed from these results.

Cloning, Molecular↗
PubMed2000

Identification and characterization of a novel gene, hos2+, the function of which is necessary for growth under high osmotic stress in fission yeast.

hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon. A Hos2-defective (hos2delta) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant. For this hos2delta mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.

Amino Acid Sequence↗
PubMed2000

Measles virus-specific T helper 1/T helper 2-cytokine production in subacute sclerosing panencephalitis.

Live measles virus-specific T helper 1/T helper 2-cytokine productions by peripheral blood mononuclear cells in response to live measles, mumps or varicella virus were measured in 15 patients with subacute sclerosing panencephalitis and 15 controls by enzyme-linked immunosorbent assays. Most patients with subacute sclerosing panencephalitis had a defect in measles virus-specific production of interferon-gamma, one of the T helper 1 type cytokines, despite persistent presence of measles virus, with preserved interleukin-10 (T helper 2 type cytokine) synthesis. Patients with subacute sclerosing panencephalitis were divided into two groups: responders (group A) with significant interferon-gamma production (>20 pg/mL) in response to live measles virus and non-responders (group B) with a little or no interferon-gamma production. Comparison of the clinical courses between groups A and B revealed that all the patients of group A retained receptive function for a long time, while most patients of group B lost the function rapidly (P<0.01). An inverse correlation between interferon-gamma production by peripheral blood mononuclear cells and disease progression suggested that interferon-gamma plays an antiviral role in subacute sclerosing panencephalitis.

Adolescent↗
PubMed1999

The cta3+ gene that encodes a cation-transporting P-type ATPase is induced by salt stress under control of the Wis1-Sty1 MAPKK-MAPK cascade in fission yeast.

In Schizosaccharomyces pombe, the Wis1-Sty1 MAP (mitogen-activated protein) kinase signaling cascade is known to play a major role in cellular adaptation to adverse external stimuli, including osmotic stress, oxidative stress, nutrient deprivation, DNA-damaging agents, and heat stress. Nonetheless, it is not known whether or not this particular MAPK cascade is also involved in response to the most common stress, salinity. In this study, we provide evidence that the Wis1-Sty1 MAP cascade is implicated in salt stress response through regulating expression of a salinity-inducible gene. The downstream target gene thus identified is the cta3+ gene, which encodes a cation-transporting P-type ATPase. The salt stress-responsive nature of cta3+ expression was characterized extensively. It was found that not only the Sty1 MAP kinase but also the Atf1 transcription factor is crucial for the inducible expression of cta3+. As far as we know, this is the first instance that the stress-activated Wis1-Sty1 MAPK cascade plays a role in salt stress response in S. pombe.

Adenosine Triphosphatases↗
PubMed1999

An inactive open complex mediated by an UP element at Escherichia coli promoters.

A specific interaction between the alpha subunit of RNA polymerase and an A+T-rich upstream sequence (UP element) stimulates transcription at some promoters in Escherichia coli. We found that RNA polymerase formed a heparin-resistant nonproductive initiation complex at the malT promoter which has an A+T-rich upstream sequence that begins 9 bp upstream of the -35 region. Substitution of other sequences for the A+T-rich sequence eliminated both the formation of heparin-resistant complexes and alpha binding to the malT promoter. A 5-bp deletion between the A+T-rich sequence and the -35 region increased promoter activity. The UP element derived from the rrnB P1 promoter stimulated transcription of the malT core promoter when placed 4 bp upstream from the malT -35 region, but insertion of an additional 4 bp between the rrnB P1 UP element and the -35 element eliminated transcription activity without eliminating heparin-resistant complex formation. Similar UP element effects were observed in hybrids with the lac core promoter, even though the region around the transcription start site was melted in both productive and nonproductive complexes. We conclude that UP elements can mediate the formation of both productive and nonproductive open complexes, depending on their location with respect to the core promoter.

Base Sequence↗
PubMed1999

Isolation of multicopy suppressors of the calcium sensitivity of a mutant lacking the bZIP transcription factor Atf1 in fission yeast.

In Schizosaccharomyces pombe, recent studies have uncovered a set of putative transcription factors of the basic leucine zipper (bZIP) type (e.g., Atf1, Pcr1, Pap1), which function downstream of the Sty1 mitogen-activated protein kinase (MAPK) cascade which is involved in stress-activated signal transduction. Accordingly, a delta atf1 mutant is known to exhibit osmosensitivity for growth, since one of the targets of Atf1 is the gpd1+ gene, which is responsible for the osmoadaptive glycerol production mediated by the Sty1 MAPK cascade. During the course of our studies on the osmotic response in S. pombe, we found that growth of a delta atf1 mutant is highly sensitive to the level of Ca2+ ions in the medium (but less sensitive to Mg2+ and Na+ ions). This phenotype seemed to be relevant to the osmosensitivity, because an delta gpd1 mutant showed a similar phenotype. An attempt was therefore made to isolate multicopy suppressors of the calcium sensitivity exhibited by the delta atf1 cells. Among such suppressors were several bZIP factors, including two known proteins (Atf21 and Pcr1), and two new ones (named Atf31 and Zip1). These factors were characterized further, in comparison to Atf1, with special reference to the Sty1 MAPK signaling pathway.

Activating Transcription Factor 1↗
PubMed1999

Regulation of intrinsic terminator by translation in Escherichia coli: transcription termination at a distance downstream.

BACKGROUND: Rho-independent terminators in Escherichia coli are DNA sequences of 30-50 bp consisting of a GC-rich dyad symmetry sequence followed by a run of T residues in the nontemplate strand. The transcription termination at the Rho-independent terminator occurs within the T-tract in vitro. It has been believed that the transcription termination at the Rho-independent terminator occurs within the T-tract in vivo, as established in vitro, and therefore the 3' ends of mRNAs are mostly generated as a direct result of transcription termination. However, how the transcription termination occurs and how the 3' ends of mRNAs are formed in living cells remains to be studied. RESULTS: We developed a double terminator system in which a second Rho-independent terminator was placed downstream of the crp terminator. This system made it possible to detect transcripts that pass through the crp terminator by Northern blotting. We found that most of the crp transcripts extend beyond the crp terminator. The transcriptional read-through at the crp terminator was reduced when the translation of crp mRNA was interrupted. The level of the read-through transcript decreased with distance between the two terminators, suggesting that transcription termination occurs at multiple positions beyond the crp terminator. CONCLUSION: We conclude that most RNA polymerase reads through the crp terminator in the natural situation and terminates transcription over a wide region downstream of the crp terminator, resulting in heterogeneous primary transcripts that are subsequently processed back to the terminator hairpin. We propose that ribosome translation to the crp stop codon causes read-through of the terminator. The regulatory effect of translation on Rho-independent termination may be a general phenomenon at other operons.

Base Sequence↗
PubMed1999

Negative regulation of the pts operon by Mlc: mechanism underlying glucose induction in Escherichia coli.

BACKGROUND: The pts operon of Escherichia coli consists of three genes ptsH, ptsI and crr, each encoding for central components of the phosphoenolpyruvate: carbohydrate phosphotransferase system, HPr, enzyme I and IIAGlc, respectively. Transcription of the pts operon is stimulated when glucose is present in the culture medium. One of the two major promoters, P0, is responsible for this glucose induction. However, no regulatory protein responsible for the glucose induction of the pts operon has been identified yet and molecular mechanism by which glucose stimulates the pts transcription is not known. RESULTS: We found by Northern blotting that the pts mRNA levels in cells lacking Mlc, a new global repressor of carbohydrate metabolism, were increased without external glucose and that the addition of glucose had no effect on the pts mRNA levels in the mutant cells. Western blotting revealed that the enzyme I level in the mlc- cells was also elevated without glucose and no further increase in the enzyme I level was observed in the presence of glucose. S1 analysis revealed that transcription of the glucose-sensitive promoter, P0, occurs constitutively in the mlc- cells independently from the external glucose. In vitro transcription studies indicated that Mlc strongly inhibited P0 transcription. DNase I footprinting experiment revealed that Mlc bound to P0 promoter region to prevent RNA polymerase binding at P0. CONCLUSION: We conclude that Mlc is a repressor for the pts transcription acting as a major regulatory protein involved in the glucose induction of pts operon. We propose that glucose induces the pts transcription by modulating the Mlc activity. The mechanism by which glucose modulates the Mlc action remains to be studied.

Bacterial Proteins↗
PubMed1999

A fission yeast gene (prr1(+)) that encodes a response regulator implicated in oxidative stress response.

An inspection of the Schizosaccharomyces pombe genome database revealed that this eukaryotic microorganism possesses a gene that may encode a bacterial type of histidine-to-aspartate (His-Asp) phosphorelay component, namely, a response regulator. The predicted gene, named prr1(+) (S. pombe response regulator), encodes a protein that contains a typical phospho-accepting receiver domain, preceded by a mammalian heat shock factor (HSF)-like DNA-binding domain. Inactivation of this prr1(+) gene resulted in mutant cells defective in some aspects of stress responses, including sensitivity to oxidative stress, cold-temperature, and heavy metal toxicity. It was also demonstrated that Prr1 is required for the transcription of some genes (e.g., trr1(+), ctt1(+)), which are induced by oxidative stress. These results suggest that a His-Asp phosphorelay system may be involved in a stress-activated signaling pathway in S. pombe.

Aspartic Acid↗
PubMed1998

A common role of CRP in transcription activation: CRP acts transiently to stimulate events leading to open complex formation at a diverse set of promoters.

We have shown previously that the cyclic AMP receptor protein (CRP) is not required after the formation of the open complex at the lac promoter (Tagami and Aiba, 1995, Nucleic Acids Res., 19, 6705-6712). In this paper, we investigate the role of CRP in transcription activation at the malT and gal promoters. At the malT promoter, RNA polymerase (RNAP) forms a nonproductive RNAP-promoter binary complex in the absence of CRP and a productive CRP-RNAP-promoter ternary complex in the presence of CRP. CRP can be removed from the malT ternary complex by a moderate concentration of heparin. The resulting binary complex is functionally identical to the ternary complex. At the gal promoter, RNAP predominantly forms a binary complex at the P2 promoter in the absence of CRP and a ternary complex at the P1 promoter in the presence of CRP. A very high concentration of heparin is able to dissociate CRP from the galP1 ternary complex without changing the properties of the complex. These data indicate that CRP is not required for the maintenance of the ternary complex and plays no role in the subsequent steps, irrespective of the promoter. We conclude that the common role of CRP in the activation of transcription is to stimulate events leading to the formation of a productive open complex at a diverse set of CRP-dependent promoters. We suggest that the interaction between CRP and RNAP is needed only transiently for the activation of transcription.

Bacterial Proteins↗
PubMed1998

CRP down-regulates adenylate cyclase activity by reducing the level of phosphorylated IIA(Glc), the glucose-specific phosphotransferase protein, in Escherichia coli.

The cellular cAMP level is markedly down-regulated by cAMP receptor protein (CRP) in Escherichia coli. CRP regulates adenylate cyclase both at the level of transcription of its structural gene cya and at the level of enzyme activity. We established a method to determine the phosphorylation state of IIA(Glc), the glucose-specific phosphotransferase protein, in intact cells. We found that IIA(Glc) exists predominantly in the unphosphorylated form in wild-type cells growing in LB medium, while it is largely phosphorylated in crp or cya cells. Disruption of the ptsG gene that codes for the membrane component of the major glucose transporter (IICB(Glc)), and/or the fruF gene coding for FPr (fructose-specific hybrid phosphotransferase protein), did not affect the phosphorylation state of IIA(Glc). When IICB(Glc) was overproduced in the presence of glucose, the levels of both cAMP and phosphorylated IIA(Glc) in crp cells were concomitantly decreased to wild-type levels. In addition, when His-90 in IIA(Glc) was replaced by glutamine, both phosphorylation of IIA(Glc) and the overproduction of cAMP in crp cells were eliminated. We also found that extracts of crp+ cells markedly stimulate dephosphorylation of IIA(Glc)-P in vitro. We conclude that CRP-cAMP down-regulates adenylate cyclase primarily by reducing the level of phosphorylated IIA(Glc). The data suggest that unspecified proteins whose expression is under the control of CRP-cAMP are responsible for this regulation.

Adenylyl Cyclases↗
PubMed1998

Inducer exclusion by glucose 6-phosphate in Escherichia coli.

The main mechanism causing catabolite repression by glucose and other carbon sources transported by the phosphotransferase system (PTS) in Escherichia coli involves dephosphorylation of enzyme IIA(Glc) as a result of transport and phosphorylation of PTS carbohydrates. Dephosphorylation of enzyme IIA(Glc) leads to 'inducer exclusion': inhibition of transport of a number of non-PTS carbon sources (e.g. lactose, glycerol), and reduced adenylate cyclase activity. In this paper, we show that the non-PTS carbon source glucose 6-phosphate can also cause inducer exclusion. Glucose 6-phosphate was shown to cause inhibition of transport of lactose and the non-metabolizable lactose analogue methyl-beta-D-thiogalactoside (TMG). Inhibition was absent in mutants that lacked enzyme IIA(Glc) or were insensitive to inducer exclusion because enzyme IIA(Glc) could not bind to the lactose carrier. Furthermore, we showed that glucose 6-phosphate caused dephosphorylation of enzyme IIA(Glc). In a mutant insensitive to enzyme IIA(Glc)-mediated inducer exclusion, catabolite repression by glucose 6-phosphate in lactose-induced cells was much weaker than that in the wild-type strain, showing that inducer exclusion is the most important mechanism contributing to catabolite repression in lactose-induced cells. We discuss an expanded model of enzyme IIA(Glc)-mediated catabolite repression which embodies repression by non-PTS carbon sources.

Biological Transport↗
PubMed1998

A global repressor (Mlc) is involved in glucose induction of the ptsG gene encoding major glucose transporter in Escherichia coli.

Glucose stimulates the expression of ptsG encoding the major glucose transporter in Escherichia coli. We isolated Tn 10 insertion mutations that confer constitutive expression of ptsG. The mutated gene was identified as mlc, encoding a protein that is known to be a repressor for transcription of several genes involved in carbohydrate utilization. Expression of ptsG was eliminated in a mlc crp double-negative mutant. The Mlc protein was overproduced and purified. In vitro transcription studies demonstrated that transcription of ptsG is stimulated by CRP-cAMP and repressed by Mlc. The action of Mlc is dominant over that of CRP-cAMP. DNase I footprinting experiments revealed that CRP-cAMP binds at two sites centred at -40.5 and -95.5 and that Mlc binds at two regions centred around -8 and -175. The binding of CRP-cAMP stimulated the binding of RNA polymerase to the promoter while Mlc inhibited the binding of RNA polymerase but not the binding of CRP-cAMP. Gel-mobility shift assay indicated that glucose does not affect the Mlc binding to the ptsG promoter. Our results suggest that Mlc is responsible for the repression of ptsG transcription and that glucose modulates the Mlc activity by unknown mechanism.

Bacterial Proteins↗
PubMed1998

Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc.

The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIAGlc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIAGlc causes inhibition of uptake of a number of non-PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIAGlc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIAGlc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6-phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIAGlc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]-[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIAGlc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.

Bacterial Proteins↗
PubMed1998

Isolation and characterization of high-osmolarity-sensitive mutants of fission yeast.

For the fission yeast Schizosaccharomyces pombe, adaptation to high-osmolarity medium is mediated by a mitogen-activated protein (MAP) kinase cascade, involving the Wis1 MAP kinase kinase and the Sty1 MAP kinase. The MAP kinase pathway transduces an osmotic signal and accordingly regulates the expression of the downstream target gene (gpd1(+)) that encodes NADH-dependent glycerol-3-phosphate dehydrogenase, in order to adaptively accumulate glycerol inside the cells as an osmoprotectant. We previously characterized a set of high-osmolarity-sensitive S. pombe mutants, including wis1, sty1, and gpd1. In this study, we attempted to further isolate novel osmolarity-sensitive mutants. For some of the mutants isolated, profiles of glycerol production in response to the osmolarity of the growth medium were indistinguishable from that of the wild-type cells, suggesting that they are novel types. They were classified into three distinct types genetically and, thus, were designated hos1, hos2, and hos3 (high osmolarity sensitive) mutants. One of them, the hos1 mutant, was characterized in detail. The hos1 mutant was demonstrated to have a mutational lesion in the known ryh1(+) gene, which encodes a small GTP-binding protein. Disruption of the ryh1(+) gene results not only in osmosensitivity but also in temperature sensitivity for growth. It was also found that the delta ryh1 mutant is severely sterile. These results are discussed with special reference to the osmoadaptation of S. pombe.

Culture Media↗
PubMed1998

Electrophysiological study of myoclonus in pediatric practice.

Myoclonus can be caused from a variety of central nervous system disorders. With regard to its pathophysiology, much work has been done in adult patients, but reports from pediatric clinics are scarce. We describe the clinical and electrophysiological features of four different types of myoclonus in children using back averaging technique. In a patient with cortical reflex myoclonus, myoclonic jerks were induced in the distal right arm by taps on the ipsilateral fingers. Myoclonus was preceded by the cortical spike which was localized posterior to the central sulcus contralateral to the myoclonus, and the cortical spike was time-locked to the myoclonus. In a patient with subcortico-cortical myoclonus, myoclonus occurred as a focal motor seizure. Myoclonic jerks were preceded by generalized cortical waves which were of maximal amplitude over the cortex contralateral to the myoclonus, and cortical waves time-locked to the myoclonus. A patient with spinal myoclonus had rhythmical and segmental myoclonus. Myoclonus was induced in both arms after electrical stimulation at the wrist with a latency which was slightly longer than that of the F wave. In a patient with brainstem myoclonus, muscle activity was bilaterally synchronous and was predominant in the proximal flexors. The pattern of muscle recruitment suggested that the myoclonus signal travelled downward along the spinal cord and upward through the brainstem. The back averaging technique is essential in the differential diagnosis of myoclonus.

Adolescent↗
PubMed1997

cAMP receptor protein-cAMP plays a crucial role in glucose-lactose diauxie by activating the major glucose transporter gene in Escherichia coli.

The inhibition of beta-galactosidase expression in a medium containing both glucose and lactose is a typical example of the glucose effect in Escherichia coli. We studied the glucose effect in the lacL8UV5 promoter mutant, which is independent of cAMP and cAMP receptor protein (CRP). A strong inhibition of beta-galactosidase expression by glucose and a diauxic growth were observed when the lacL8UV5 cells were grown on a glucose-lactose medium. The addition of isopropyl beta-D-thiogalactoside to the culture medium eliminated the glucose effect. Disruption of the crr gene or overproduction of LacY also eliminated the glucose effect. These results are fully consistent with our previous finding that the glucose effect in wild-type cells growing in a glucose-lactose medium is not due to the reduction of CRP-cAMP levels but is due to the inducer exclusion. We found that the glucose effect in the lacL8UV5 cells was no longer observed when either the crp or the cya gene was disrupted. Evidence suggested that CRP-cAMP may not enhance directly the lac repressor action in vivo. Northern blot analysis revealed that the mRNA for ptsG, a major glucose transporter gene, was markedly reduced in a delta crp or delta cya background. The constitutive expression of the ptsG gene by the introduction of a multicopy plasmid restored the glucose effect in delta cya or delta crp cells. We conclude that CRP-cAMP plays a crucial role in inducer exclusion, which is responsible for the glucose-lactose diauxie, by activating the expression of the ptsG gene.

Culture Media↗