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Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 109 records · Page 6Linked to original sources
PubMed1998

Dose response relationship of disturbed migration of Purkinje cells in the cerebellum due to X-irradiation.

Pregnant rats were exposed to 2.0, 2.25 or 2.5 Gy X-irradiation on gestation day 21. Pups were sacrificed 12 hr after exposure, and on postnatal day 5 (P5), P7 and P9. Their cerebella were observed immunohistochemically using anti-inositol 1,4,5 triphosphate (IP3) receptor antibody to identify Purkinje cells. These cells were disturbed to migrate and remained in the internal granular layer and white matter of the cerebellum. They had short dendrites, and some showed an abnormal direction of dendrites in rats exposed to 2.25 or 2.5 Gy. Alignment of Purkinje cells was also disturbed when examined either on P5, P7 or P9 especially by doses of 2.25 and 2.5 Gy. There was a relationship between X-ray doses and the number of cells piling up in the Purkinje cell layer of the cerebellum. The dose-response relationship with the number of ectopic Purkinje cells was noted in the anterior lobes of the cerebellum.

Animals↗
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↗
PubMed1997

Signal transduction via the histidyl-aspartyl phosphorelay.

The histidyl-aspartyl phosphorelay, formerly described as the two-component system, is the predominant mode of signal transduction in bacteria. Adaptation to environmental changes occurs through a sensor histidine protein kinase and a response regulator. The histidine protein kinase is usually a transmembrane receptor and the response regulator is a cytoplasmic protein. Together the histidyl-aspartyl phosphorelay proteins mediate reversible phosphorylation events that control downstream effectors. Following autophosphorylation at a conserved histidine residue, the histidine kinase serves as a phospho-donor for the response regulator. Once phosphorylated, the response regulator mediates changes in gene expression or cellular locomotion. The EnvZ-OmpR phosphorelay system in Escherichia coli, which monitors external osmolarity and responds by differentially modulating the expression of the OmpF and OmpC major outer membrane porins, will be described as a model system. While histidine kinases were thought to be present only in prokaryotes, they have recently been identified in eukaryotic systems. Here, we review the unique and conserved features of this growing family of signal transducers.

Aspartic Acid↗
PubMed1997

Promoter-independent cold-shock induction of cspA and its derepression at 37 degrees C by mRNA stabilization.

The gene for CspA, the major cold-shock protein of Escherichia coli is known to be dramatically induced upon temperature downshift. Here, we report that three-base substitutions around the Shine-Dalgarno sequence in the 159-base 5'-untranslated region of the cspA mRNA stabilizes the mRNA 150-fold, resulting in constitutive expression of cspA at 37 degrees C. This stabilization was found to be at least partially due to resistance against RNase E degradation. The cold-shock induction of cspA was also achieved by exchanging its promoter with the non-cold-shock Ipp promoter. The results presented indicate that the cspA gene is efficiently transcribed even at 37 degrees C. However, the translation of the cspA mRNA is blocked because of its extreme instability at 37 degrees C. The presented results also demonstrate that the cspA gene is constitutively transcribed at all temperatures; however, its expression at 37 degrees C is prevented by destabilizing its mRNA.

Bacterial Proteins↗
PubMed1997

Deletion analysis of cspA of Escherichia coli: requirement of the AT-rich UP element for cspA transcription and the downstream box in the coding region for its cold shock induction.

In order to analyse the mechanism of cold shock induction of CspA, a major cold shock protein of Escherichia coli, deletion analysis of the cspA gene was carried out. It was found that (i) the AT-rich sequence (-47 to -38) upstream of the cspA -35 region may act as the UP element playing a crucial role in cspA transcription at both 37 degrees C and 15 degrees C; (ii) the unusually long 5'-UTR of the cspA mRNA has negative effects on cspA expression at 37 degrees C; and (iii) in contrast, the 5'-UTR exerts a positive effect on mRNA stabilization at low temperature. Furthermore, it was demonstrated that the 14 base downstream box (DB) locating 12 bases downstream of the initiation codon of the cspA mRNA and complementary to a region near the decoding region of 16S rRNA was essential for the mRNA translation during the growth lag acclimation phase immediately after cold shock. During this phase, translation of non-cold shock gene mRNAs is blocked, since they require cold shock-specific ribosomal factors for the formation of the translation initiation complex. It is proposed that DB in cold shock mRNAs allows the formation of a stable initiation complex at low temperature in the absence of the cold shock ribosomal factors.

Bacterial Proteins↗
PubMed1997

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus.

The Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus. This region also shows 29, 25 and 29% identify with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively. A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane. The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I-III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Two-dimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.

Amino Acid Sequence↗
PubMed1997

Effects of prenatal treatment with tritiated water on the developing brain in mouse.

Pregnant mice were injected intraperitoneally with various doses of tritiated water on embryonic day 13. The litters received total cumulative absorbed beta-irradiation to a dose of 0, 0.1, 0.2, 0.4 or 0.8 Gy. We found that 0.4 Gy beta-irradiation caused a significant reduction in brain weight but not in body weight among 8-week-old mice. The highest dose (0.8 Gy) inhibited both body and brain development. A quantitative study showed that a significant reduction in cerebral pyramidal cells was present in the groups exposed to > or = 0.2 Gy. In tritiated, water-treated groups, the pyramidal cells reduced in a dose-dependent manner, with reductions ranging from 3 to 12%. The thickness of cerebral cortex was significantly reduced in the groups exposed to > or = 0.4 Gy. Numerical density of pyramidal cells was the most sensitive indicator of developmental disturbance of cerebral cortex manifested postnatally. No significant difference in the numerical density of Purkinje cells was found between the irradiated and control groups. These results demonstrated that the time at which the insult occurred was an important factor determining the resulting abnormalities.

Animals↗
PubMed1997

Catalytic efficiency of signal peptidase I of Escherichia coli is comparable to that of members of the serine protease family.

A method for estimating the activity of bacterial signal peptidase I (SPase I) was used to determine its activation energy (E[act]). Pro-OmpA-nuclease A, a hybrid secretory precursor, was purified to homogeneity under denaturing conditions and used as a substrate. This substrate was used to determine the activity of SPase I at different temperatures. The results show that the conformation of the mature domain of the substrate pro-OmpA-nuclease A has no discernible effect on the activity of SPase I. The activity data at a range of temperatures were then used to determine the activation energy using the Arrhenius equation. We have estimated E(act) to be 10.4 +/- 0.6 kcal/mol. This work indicates that SPase I is as catalytically efficient as the His-Ser-Asp family of proteases.

Bacterial Outer Membrane Proteins↗