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Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 37 records · Page 2Linked to original sources
PubMed1998

Overexpression of human glutathione peroxidase protects transgenic mice against focal cerebral ischemia/reperfusion damage.

As stroke is a major cause of disability and death in the western world, there is great interest in the basic mechanisms by which ischemia/reperfusion (I/R) causes damage. To this end, extensive research has been carried out which identifies reactive oxygen species (ROS) as key participants in brain damage resultant from I/R. Brain tissue is protected from ROS damage by antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GP). Overexpression of SOD in transgenic mice has already been demonstrated to confer protection against I/R damage in murine stroke models. We are using transgenic mice overexpressing the intracellular form of glutathione peroxidase (GP1) to determine the protective capacity of overexpression of this enzyme on stroke damage. 1 h of focal cerebral ischemia followed by 24 h of reperfusion was induced using the intraliminal suture method. Volume of infarction was reduced by 48% in GP1 mice compared to nontransgenic littermates. Brain edema was reduced by 33%. Behavioral deficits agreed with histologic data. Overexpression of glutathione peroxidase confers significant protection against I/R damage in our stroke model possibly through direct scavenging of ROS or through the influencing of signalling mechanisms which lead to tissue damage.

Animals↗
PubMed1998

Hierarchical and co-operative binding of OmpR to a fusion construct containing the ompC and ompF upstream regulatory sequences of Escherichia coli.

BACKGROUND: OmpR is a transcription factor that regulates the expression of the porin genes ompF and ompC in Escherichia coli. The phosphorylation state of OmpR, directed by the osmosensor EnvZ, determines its ability to bind to the upstream regulatory regions of these genes, a total of 14 phospho-OmpR binding sites. While it has been possible to study the stoichiometry and hierarchy of the OmpR-DNA interaction in the upstream regions of ompF and ompC, their disunited location on the bacterial chromosome has made it difficult to compare the individual binding affinities of respective sites. RESULTS: Using 1,10-phenanthroline-Cu+ footprinting on a fused construct containing both the ompF and ompC upstream regulatory sequences, and gel shift experiments on oligomers corresponding to individual sites, we have established a comparative hierarchy for OmpR binding, as F1, C1 > F2, F3 > C2 > C3. In addition, the binding patterns reveal an apparent co-operative relationship between OmpR molecules bound at several upstream motifs. Densitometric analyses of the footprinted regions provide support for these observations. Mutational analysis of this construct reveals that the alteration of a conserved cytidine in the F1 motif (-86) causes a loss of OmpR affinity and disrupts hierarchical OmpR-binding in the entire ompF region. CONCLUSIONS: The present results provide a unique view of the OmpR interaction with the two respective promoters, ompF and ompC, and an insight into the question of how the expression of ompF and ompC are reciprocally regulated by medium osmolarity.

Bacterial Proteins↗
PubMed1998

The CspA family in Escherichia coli: multiple gene duplication for stress adaptation.

CspA was originally found as the major cold-shock protein in Escherichia coli, consisting of 70-amino-acid residues. It forms a beta-barrel structure with five anti-parallel beta-strands and functions as an RNA chaperone. Its dramatic but transient induction upon cold shock is regulated at the level of transcription, mRNA stability and translation. Surprisingly, E. coli contains a large CspA family, consisting of nine genes from cspA to cspI. Phylogenetic analysis of these gene products and the cold-shock domain of human YB-1 protein reveals that there are two major branches in the evolution of CspA homologues: one branch for CspF and CspH, and another for all the other known CspA homologues from both prokaryotes and eukaryotes. The locations of these genes on the E. coli chromosome suggest that the large CspA family probably resulted from a number of gene duplications and, after subsequent adaptation, resulted in specific groups of genes that respond to different environmental stresses; for example, cspA, cspB and cspG for cold-shock stress and cspD for nutritional deprivation. The E. coli CspA family will be discussed in terms of their structures and functions, and their gene structures and regulation.

Adaptation, Physiological↗
PubMed1998

Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice.

Normal cellular metabolism produces oxidants which are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidants and antioxidants has been postulated to lead to the degeneration of specific populations of neurons in neurodegenerative diseases, e.g. Parkinson's disease. The present study investigates whether overexpression of glutathione peroxidase, the enzyme which metabolizes hydrogen peroxide to water, can prevent or slow down neuronal injury in an animal model of Parkinson's disease. Transgenic mice overexpressing the human glutathione peroxidase gene under the control of the mouse hydroxymethylglutaryl-coenzyme A promoter and genetically matched control mice were injected intracerebroventricularly with the dopaminergic neurotoxin 6-hydroxydopamine. Seven days after injection, the number of tyrosine hydroxylase-positive nigral dopaminergic neurons was decreased by 52.4% and 20.5% in 6-hydroxydopamine-injected control and glutathione peroxidase transgenic mice, respectively. Similarly, 3 days after injection of the neurotoxin, striatal dopamine was decreased by 71.2% and 56.5%, respectively. Overexpression of glutathione peroxidase therefore partially protects dopaminergic neurons against 6-hydroxydopamine-induced toxicity.

Acyl Coenzyme A↗
PubMed1998

Role of the cold-box region in the 5' untranslated region of the cspA mRNA in its transient expression at low temperature in Escherichia coli.

Upon temperature downshift, a group of proteins called cold shock proteins, such as CspA, CspB, and CsdA, are transiently induced in Escherichia coli. However, when the 5' untranslated region (5' UTR) of cspA mRNA is overproduced at low temperature, the expression of cold shock genes is prolonged or derepressed. It has been proposed that this effect is due to highly conserved 11-base sequences designated the "cold box" existing in the 5' UTRs of cspA, cspB, and csdA. Here, we demonstrate that the overproduction of the 5' UTR of not only cspA but also cspB and csdA mRNAs causes derepression of all three genes at the same time. Conversely, when the cold-box region was deleted from the cspA 5' UTR its derepression function was abolished. The amount of mRNA from the chromosomal cspA gene was much higher in cells overproducing the wild-type 5' UTR by means of a plasmid than it was in cells overproducing the cold-box-deleted 5' UTR. The stability of the chromosomal cspA mRNA in cells overproducing the wild-type 5' UTR was almost identical to that in cells overproducing the cold-box-deleted 5' UTR. Therefore, the derepression of cspA caused by overproduction of 5' UTR at the end of the acclimation phase occurs at the level of transcription but not by mRNA stabilization, indicating that the cold-box region plays a negative role in cspA transcription in cold shock-adapted cells. The role of the cold-box region was further confirmed with a cspA mutant strain containing a cold-box-deleted cspA gene integrated into the chromosome, which showed a high level of constitutive production of CspA but not CspB during exponential growth at low temperature.

Bacterial Proteins↗
PubMed1998

In vitro synthesis of multicopy single-stranded DNA, using separate primer and template RNAs, by Escherichia coli reverse transcriptase.

A minor population of wild strains of Escherichia coli contains a retron, a retroelement responsible for the synthesis of multicopy single-stranded DNA (msDNA). The retron is a genetic element consisting of the gene for reverse transcriptase (RT) and the msr-msd region under a single promoter. A single RNA transcript from the msr-msd region serves not only as a template but also as a primer for msDNA synthesis. Here, using a cell-free system with purified RT from retron Ec73, we examined whether the reaction can occur in a bimolecular reaction with use of separately expressed msr and msd transcripts. DNA sequencing of the cell-free product revealed that the sequence of the 5'-end region was identical to that of msDNA-Ec73, indicating that the cDNA synthesis was primed from the 2'-OH group of the specific internal G residue of the primer RNA, identical to the branching G residue in the RNA molecule of msDNA-Ec73. The present results raise an intriguing possibility for a role of bacterial retrons in vivo, the possibility that cellular mRNAs can be converted into cDNAs in retron-harboring cells if the mRNAs contain a sequence complementary to the sequence directly upstream of the branching G residue of the msr RNA transcript.

DNA, Bacterial↗
PubMed1998

The gene for 16S rRNA methyltransferase (ksgA) functions as a multicopy suppressor for a cold-sensitive mutant of era, an essential RAS-like GTP-binding protein in Escherichia coli.

Era, a Ras-like GTP-binding protein in Escherichia coli, has been shown to be essential for growth. However, its cellular functions still remain elusive. In this study, a genetic screening of an E. coli genomic library was performed to identify those genes which can restore the growth ability of a cold-sensitive mutant, Era(Cs) (E200K), at a restrictive temperature when expressed in a multicopy plasmid. Among eight suppressors isolated, six were located at 1 min of the E. coli genomic map, and the gene responsible for the suppression of Era(Cs) (E200K) was identified as the ksgA gene for 16S rRNA transmethylase, whose mutation causes a phenotype of resistance to kasugamycin, a translation initiation inhibitor. This is the first demonstration of suppression of impaired function of Era by overproduction of a functional enzyme. A possible mechanism of the suppression of the Era cold-sensitive phenotype by KsgA overproduction is discussed.

Chromosome Mapping↗
PubMed1997

Identification and developmental characterization of a novel Y-box protein from Drosophila melanogaster.

The Y-box proteins are a family of highly conserved nucleic acid binding proteins which are conserved from bacteria to human. In this report we have identified a new member of this family from Drosophila melanogaster. Degenerate-PCR was used to identify a conserved region within the highly conserved cold-shock domain (CSD) of Y-box proteins. Subsequently, the cDNA for this gene was sequenced, and the identified open reading frame was named ypsilon schachtel (yps). The expression pattern of yps indicates that this gene is expressed throughout development with the highest level of expression found in adult flies. In situ hybridization shows that the yps mRNA is maternally loaded into the egg cytoplasm. In addition, there appears to be expression of yps mRNA in mesodermal tissue during embryogenesis. YPS, while containing a conserved CSD, is novel in that it completely lacks the alternating acidic and basic regions found in the C-terminus of the other vertebrate eukaryotic Y-box proteins. The CSD of yps was purified and gel-shift analysis showed that this domain can interact with RNA. We predict that YPS would be an RNA-binding protein due to these results and the motifs which have been identified within the amino acid sequence.

Amino Acid Sequence↗
PubMed1997

Overexpression of antioxidant enzymes in transgenic mice decreases cellular ploidy during liver regeneration.

Reactive oxygen species (ROS) and antioxidant enzymes have been implicated in control mechanisms of cellular growth and proliferation. We investigated the influence of levels of endogenous antioxidant enzymes on liver regeneration in transgenic mice overexpressing human Cu,Zn-superoxide dismutase (SOD) and intracellular glutathione peroxidase (GP1) as a model system. After a two-thirds partial hepatectomy (PH), no significant difference was observed in rate of liver mass restoration among nontransgenic, SOD, and GP1 mice. In contrast, the level of polyploidization was significantly reduced in transgenic animals after PH, with a concomitant increase in 2N nuclei. The portion of 8N nuclei after 72 h reached 33.1, 15.8, and 22.1%, whereas the portion of 2N nuclei reached 7.5, 13.8, and 12.3% in nontransgenic, SOD, and GP1 mice, respectively. A similar effect was observed in another model of liver proliferation, during normal development around weaning time. Measurements of ROS production during PH indicate that overexpression of SOD leads to the decreased production of O2- and elevation of H2O2. Unexpectably, overexpression of GP in transgenic mice also results in increased production of H2O2 in hepatocytes. Finally, our data demonstrate that levels of endogenous antioxidant enzymes might influence the rate of hepatocyte polyploidization during liver proliferation.

Animals↗
PubMed1997

Protein memory through altered folding mediated by intramolecular chaperones.

The 77-residue propeptide of subtilisin acts as an intramolecular chaperone that organizes the correct folding of its own protease domain. Similar folding mechanisms are used by several prokaryotic and eukaryotic proteins, including prohormone-convertases. Here we show that the intramolecular chaperone of subtilisin facilitates folding by acting as a template for its protease domain, although it does not form part of that domain. Subtilisin E folded by an intramolecular chaperone with an Ile(-48)-to-Val mutation acquires an 'altered' enzymatically active conformation that differs from wild-type subtilisin E. Although both the altered and wild-type subtilisins have identical amino-acid sequences, as determined by amino-terminal sequencing and mass spectrometry, they bind their cognate intramolecular chaperones with 4.5-fold greater affinity than non-cognate intramolecular chaperones, when added in trans. The two subtilisins also have different secondary structures, thermostability and substrate specificities. Our results indicate that an identical polypeptide can fold into an altered conformation through a mutated intramolecular chaperone and maintains memory of the folding process. Such a phenomenon, which we term 'protein memory', may be important in investigations of protein folding.

Binding Sites↗
PubMed1997

Immunocytochemical detection and spatial distribution of myosin light-chain kinase in preimplantation mouse embryos.

As a follow-up to our previous study on the role of myosin light-chain kinase (MLCK), a Ca2+/calmodulin-dependent enzyme, in the development of preimplantation mouse embryos, we examined the presence and pattern of distribution of MLCK during preimplantation development of the mouse by whole-mount, indirect immunocytochemistry and by Western blotting, using a monoclonal antibody against MLCK. At all stages of preimplantation development, the nucleus was brightly stained with an unstained region around the nucleus, and regions near the cell membrane were also brightly stained. Using the optical sectioning capability of the confocal laser scanning microscope, we found that, up to the eight-cell stage, the regions of cell contact were mostly unstained, but along with the process of compaction, cell contact regions showed a clear staining pattern along with clearing of the cytoplasm. During formation of the blastocyst, a ring of immunofluorescence was found at the margin of the blastocoel. In the blastocyst, cells of the inner cell mass were less immunofluorescent than trophectoderm cells. These staining results appear to be due to specific immunoreaction between MLCK and the antibody, because the staining patterns were abolished when the antibody was preabsorbed by MLCK purified from chicken gizzard smooth muscle. In Western blotting of blastocysts, we found a band at 130 kD. We also show by immunoblotting and immunohistochemistry of various mouse tissues that the antibody used in this study has cross-reactivity to MLCK of various muscle and non-muscle tissues of the mouse. The presence and spatial distribution of MLCK at various stages of preimplantation development of the mouse suggest that it could play a crucial role in the regulation of the contractile events involved in the initial differentiation that occurs during formation of the mouse blastocyst.

Animals↗
PubMed1997

Enhanced skin carcinogenesis in transgenic mice with high expression of glutathione peroxidase or both glutathione peroxidase and superoxide dismutase.

Female transgenic mice (C57BL/6 x CBA/J)F1 with a 1-fold increase in expression of glutathione peroxidase (GP) or with a 1-fold increase in the expression of GP and a 3-4-fold increase in the expression of superoxide dismutase (SOD) had an enhanced carcinogenic response to initiation by 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). GP- or GP+SOD-transgenic mice that were initiated by a single topical application of 200 nmol of DMBA followed by promotion with 8 nmol of TPA twice weekly for 30 weeks developed an average of 10.9 or 11.0 skin tumors per mouse and a 100% tumor incidence in comparison with the corresponding nontransgenic mice, which had 3.9 tumors per mouse and an 83% tumor incidence. After stopping TPA application, partial skin tumor regression occurred more rapidly in nontransgenic mice than in either type of transgenic mouse. At 10 weeks after termination of TPA treatment, 9-11% of the tumor-bearing transgenic mice and 26% of the tumor-bearing nontransgenic mice had complete regression of their tumors. Histopathological examination of 96 skin papillomas revealed that the area, location, degree of tumor dysplasia, bromodeoxyuridine labeling index, and p53 protein levels were closely intercorrelated. Further analysis indicated that papillomas with the same grade of dysplasia had a higher bromodeoxyuridine labeling index and a greater p53 protein level in GP- or GP+SOD-transgenic mice than those in nontransgenic mice. The data indicated that overexpression of skin antioxidant enzymes GP or GP+SOD, which are enzymes that are believed to protect cells from oxidative damage by scavenging reactive oxygen species, lead to the increased, rather than the decreased, tumorigenesis in a DMBA/TPA two-stage skin carcinogenesis model.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

Purification and characterization of the periplasmic domain of EnvZ osmosensor in Escherichia coli.

The EnvZ-OmpR histidyl-aspartyl phosphorelay system in E. coli responds to osmolarity by differentially modulating the expression of the major outer membrane porins OmpF and OmpC. To date, the natural ligand that activates EnvZ, a transmembrane histidine kinase, has not been identified and the role of the periplasmic domain of EnvZ is unclear. We now report on the purification and characterization of the periplasmic domain of EnvZ (Lys48-Arg162) which has been expressed as a soluble protein in fusion with the maltose-binding protein. Overexpression of the fusion protein did not compete for a signal that activates EnvZ. By amylose affinity chromatography and affinity blotting, interacting proteins could not be detected. The periplasmic domain was released by factor Xa and purified to homogeneity. From circular dichroism analysis, the periplasmic domain was estimated to consist of 35% alpha-helices and 16% beta-sheets.

Bacterial Outer Membrane Proteins↗
PubMed1997

Biochemical characterization of Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus, a Gram-negative developmental bacterium.

Pkn2, a protein Ser/Thr kinase, from the developmental bacterium Myxococcus xanthus was expressed under a T7 promoter in Escherichia coli and purified. Purified Pkn2 retained the autophosphorylation activity with the Km value of 177 microM for ATP and 73 nmol/min/mg for Vmax. The optimum pH and temperature were determined to be 7.5 and 35 degrees C, respectively. The autophosphorylation activity was inhibited by staurosporine with the IC50 value of 400 nM while H-7 and genistein had little effect on this kinase. Pkn2 appears to be unique for its higher manganese dependence. This is the first biochemical characterization of the prokaryotic protein Ser/Thr kinase.

Bacterial Proteins↗
PubMed1997

Demonstration of dimer formation of the cytoplasmic domain of a transmembrane osmosensor protein, EnvZ, of Escherichia coli using Ni-histidine tag affinity chromatography.

EnvZ is a transmembrane osmosensor which regulates the phosphorylation of OmpR, a transcription factor for ompF and ompC genes which encode the major outer membrane porin proteins, OmpF and OmpC in Escherichia coli. Autophosphorylation of EnvZ occurs through a transphosphorylation reaction between two EnvZ molecules. To elucidate the molecular mechanism of signal transduction by EnvZ, we examined the dimer formation of the EnvZ cytoplasmic domain [EnvZ(C)]. For this purpose, we developed a method to determine the complex formation between the purified EnvZ(C) and the purified His6-EnvZ(C) by means of Ni-6xhistidine tag affinity chromatography. When the mixture of EnvZ(C) and His6-EnvZ(C) was applied to Ni-NTA resin, both His6-EnvZ(C) and EnvZ(C) were bound to the resin, indicating that EnvZ can form an oligomer without the periplasmic and transmembrane domains. Binding experiments using the Ni-NTA resin revealed that EnvZ(C) forms a dimer with the Ka value for dimerization being approximately 10(5) M(-1) in the equilibrium state.

Bacterial Outer Membrane Proteins↗
PubMed1997

CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone.

CspA, the major cold-shock protein of Escherichia coli, is dramatically induced during the cold-shock response. The amino acid sequence of CspA shows 43% identity to the "cold-shock domain" of the eukaryotic Y-box protein family, which interacts with RNA and DNA to regulate their functions. Here, we demonstrate that CspA binds to RNA as a chaperone. First, CspA cooperatively binds to heat-denatured single-stranded RNA if it is larger than 74 bases, causing a supershift in gel electrophoresis. A minimal concentration of CspA at 2.7 x 10(-5) M is absolutely required for this cooperative binding, which is sufficiently lower than the estimated cellular concentration of CspA (10(-4) M) in cold-shocked cells. No specific RNA sequences for CspA binding were identified, indicating that it has a broad sequence specificity for its binding. When the 142-base 5'-untranslated region of the cspA mRNA was used as a substrate for ribonucleases A and T1, the addition of CspA significantly stimulated RNA hydrolysis by preventing the formation of RNase-resistant bands due to stable secondary structures in the 5'-untranslated region. These results indicate that binding of CspA to RNA destabilizes RNA secondary structures to make them susceptible to ribonucleases. We propose that CspA functions as an RNA chaperone to prevent the formation of secondary structures in RNA molecules at low temperature. Such a function may be crucial for efficient translation of mRNAs at low temperatures and may also have an effect on transcription.

Bacterial Proteins↗
PubMed1997

In vivo production of oligodeoxyribonucleotides of specific sequences: application to antisense DNA.

Retrons, bacterial retroelements found in Gram-negative bacteria, are integrated into the bacterial genome expressing a reverse transcriptase related to eukaryotic reverse transcriptase. The bacterial reverse transcriptases are responsible for the production of multicopy, single-stranded (ms) DNA consisting of a short single-stranded DNA that is attached to an internal guanosine residue of an RNA molecule by a 2',5'-phosphodiester linkage. Reverse transcriptases use an RNA transcript from the retrons, not only as primer, but also as template for msDNA synthesis. By studying the structural requirement, it was found that for msDNA synthesis an internal region of msDNA can be replaced with other sequences. msDNA can thus be used as a vector for in vivo production of an oligodeoxyribonucleotide of a specific sequence. Artificial msDNAs containing a sequence complementary to part of the mRNA for the major outer membrane lipoprotein of Escherichia coli effectively inhibited lipoprotein biosynthesis upon induction of msDNA synthesis. This is the first demonstration of in vivo synthesis of oligodeoxyribonucleotides having antisense function. Since we have previously demonstrated that bacterial retrons are functional in eukaryotes producing msDNA in yeast and in mouse NIH/3T3 fibroblasts, the present system may also be used to produce a specific oligodeoxyribonucleotide inside the cells to regulate eukaryotic gene expression artificially. We also describe a method to produce cDNA to a specific cellular mRNA using the retron system.

Animals↗