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M Inouye

Publications and source records attributed to M Inouye.

At least 127 records · Page 7Linked to original sources
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↗
PubMed1997

An immunohistochemical study of radial glial cells in the mouse brain prenatally exposed to gamma-irradiation.

The features of a glial cell population in the developing brain of mice prenatally exposed to 60Co gamma-irradiation at the most radiosensitive stage were studied with immunohistochemistry for anti-midkine (MK), anti-vimentin (Vim), and anti-GFAP antibodies. Anti-MK- and anti-Vim-positive radial glial fibers distributed in a similar radial fashion; these fibers were observed primarily in the embryonic period and disappeared after birth. Anti-MK- and anti-Vim-stained radial fibers ran perpendicular to the pial surface in controls, whereas such fibers were disorganized 6 hours (h) after irradiation. This finding provided new evidence that the migratory pathways of young neurons were interrupted beginning a few hours after irradiation. By E17 the ectopic cell masses formed so as to replace the parts of the ventricular zone where no anti-MK immunoreactive radial fibers were present, but where anti-GFAP-stained fibrillary astrocytes emerged in the ectopic cell masses from the early postnatal period. The results suggested a twofold source of the generated astrocytes: either directly from a separate precursor of the astrocytes, or due to the transformation of the classic radial glial cells. In the newborn, numerous protoplasmic transitional forms displaced by astrocytes in irradiated brains indicated that reactive gliosis was a powerful response of a brain exposed to irradiation.

Animals↗
PubMed1997

Mutational analysis of the linker region of EnvZ, an osmosensor in Escherichia coli.

EnvZ, a transmembrane signal transducer, is composed of a periplasmic sensor domain, transmembrane domains, and a cytoplasmic signaling domain. Between the second transmembrane domain and the cytoplasmic signaling domain there is a linker domain consisting of approximately 50 residues. In this study, we investigated the functional role of the EnvZ linker domain with respect to signal transduction. Amino acid sequence alignment of linker regions among various bacterial signal transducer proteins does not show a high sequence identity but suggests a common helix 1-loop-helix 2 structure. Among several mutations introduced in the EnvZ linker region, it was found that hydrophobic-to-charged amino acid substitutions in helix 1 and helix 2 and deletions in helix 1, loop, and helix 2 (delta14, delta8, and delta7) resulted in constitutive OmpC expression. In the linker mutant EnvZ x delta7, both kinase and phosphatase activities were significantly reduced but the ratio of kinase to phosphatase activity increased, consistent with the constitutive OmpC expression. In contrast, the purified cytoplasmic fragment of EnvZ x delta7 possessed both kinase and phosphatase activities at levels similar to those of the cytoplasmic fragment of wild-type EnvZ. In addition, the linker mutations had no direct effect on EnvZ C-terminal dimerization. These results together with previous data suggest that the linker region is not directly involved in EnvZ enzymatic activities and that it may have a crucial role in propagating a conformational change to ensure correct positioning of two EnvZ molecules within a dimer during the transmembrane signaling.

Amino Acid Sequence↗
PubMed1997

Growth-phase-dependent expression of cspD, encoding a member of the CspA family in Escherichia coli.

The cspD gene of Escherichia coli encodes a protein of high sequence similarity with the cold shock protein CspA, but cspD expression is not induced by cold shock. In this study, we analyzed the regulation of cspD gene expression. By using a cspD-lacZ fusion and primer extension analysis, the expression of cspD was found to be dramatically induced by stationary-phase growth. However, this induction does not depend on the stationary-phase sigma factor sigmaS. Moreover, the expression of cspD is inversely dependent on growth rates and induced upon glucose starvation. Using a (p)ppGpp-depleted strain, we found that (p)ppGpp is one of the positive factors for the regulation of cspD expression.

Bacterial Proteins↗
PubMed1997

CspA, the major cold shock protein of Escherichia coli, negatively regulates its own gene expression.

When the gene for CspA, the major cold shock protein of Escherichia coli, was disrupted by a novel positive/negative selection method, the deltacspA cells did not show any discernible growth defect at either 37 or 15 degrees C. By two-dimensional gel electrophoresis, total protein synthesis was analyzed after temperature downshift in the deltacspA strain. The production of the CspA homologs CspB and CspG increased, and the duration of their expression was prolonged, suggesting that both CspB and CspG compensate for the function of CspA in the absence of CspA during cold shock adaptation. Interestingly, the production of the 159-base 5'-untranslated region (5'-UTR) of cspA from the chromosomal cspA::cat gene, detected by primer extension, failed to be repressed after cold shock. When an independent system to produce CspA was added to the deltacspA strain, the 5'-UTR production for the cspA::cat gene was significantly reduced compared to that of the deltacspA strain. By examining the expression of translationally fused cspA and cspB genes to lacZ in the deltacspA strain, it was found that cspA is more strongly regulated by CspA than cspB is. We showed that the increased expression of the 5'-UTR of the cspA mRNA in the deltacspA strain occurred mainly at the level of transcription and, to a certain extent, at the level of mRNA stabilization. The mRNA stabilization in the deltacspA strain was observed for other mRNAs, supporting the notion that CspA functions as an mRNA chaperone to destabilize secondary structures in mRNAs.

Artificial Gene Fusion↗
PubMed1997

msDNA-Ec48, the smallest multicopy single-stranded DNA from Escherichia coli.

Previously we have reported a novel bacterial reverse transcriptase (RT) from Escherichia coli ECOR58 strains in which the YXDD box was replaced with LVDD (J.-R. Mao, S. Inouye, and M. Inouye, Biochem. Biophys. Res. Commun. 227:489-493, 1996). Here we determined the structure of the multicopy single-stranded DNA (msDNA) produced by the RT. The msDNA was found to consist of a single-stranded DNA of 48 nucleotides in length, the shortest msDNA thus far identified from natural sources. The msDNA, the RT, and the retron are designated msDNA-Ec48, RT-Ec48, and retron-Ec48, respectively. On the basis of the structure of the msr gene, the RNA molecule of msDNA-Ec48 is predicted to be composed of 119 ribonucleotides; it is the longest RNA among the known msDNAs. Analysis of the DNA sequences flanking the retron indicates that retron-Ec48 is associated with a prophage related to phages P2 and P4.

Amino Acid Sequence↗
PubMed1997

Intestinal ischemia and reperfusion injury in transgenic mice overexpressing copper-zinc superoxide dismutase.

Superoxide dismutase (SOD) scavenges oxygen radicals that are implicated in the pathogenesis of intestinal ischemia-reperfusion injury. The effect of intestinal ischemia and reperfusion was investigated in transgenic mice overexpressing human Cu-Zn SOD. Ischemia was induced by occluding the superior mesenteric artery. Myeloperoxidase activity was determined as an index of neutrophil infiltration, and malondialdehyde levels were measured as an indicator of lipid peroxidation. Forty-five minutes of intestinal ischemia followed by 4 h of reperfusion caused an increase in intestinal levels of malondialdehyde in both nontransgenic and transgenic mice, but the concentration of malondialdehyde was significantly greater in nontransgenic mice. Intestinal ischemia-reperfusion also caused an increase in intestinal and pulmonary myeloperoxidase activity in nontransgenic and transgenic mice, but the transgenic mice had significantly lower levels of myeloperoxidase activity than nontransgenic mice. Transgenic mice had higher levels of intestinal SOD activity than nontransgenic mice. There were no significant differences in the catalase or glutathione peroxidase activities. In conclusion, our study demonstrates that the overexpression of SOD protects tissues from neutrophil infiltration and lipid peroxidation during intestinal ischemia-reperfusion.

Animals↗
PubMed1997

Segregation analysis of animal pedigree data from inter-population crosses.

The general method of segregation analysis of pedigree data has been developed and widely used in human genetics. We modified this method to examine pedigree data coming from inter-population crosses. These kinds of pedigrees are common in laboratory and farm animal breeding. This paper describes a rationale for the method and illustrates its application to the study of inheritance of litter size and of male sterility in hybrid stock of the house musk shrew (Suncus murinus) derived from crosses of two geographically isolated populations.

Animals↗
PubMed1997

Cortical fiber distribution in the somatosensory cortex of rats following prenatal exposure to X-irradiation.

Exposure of fetal rats to X-irradiation at a dose of 1.5 Gy on gestational day 15 results in severe microcephaly. In these X-irradiated rats with microcephaly, a possible alteration of local circuits in the somatosensory cortex was examined using a fluorescent dye, DiI. The dye was placed at the superficial part of the transversely sectioned somatosensory cortex just beneath the pial surface of both the control and X-irradiated rats on postnatal day 11. In the controls, DiI-labeled fibers showed a layer-dependent distribution in the somatosensory cortex, whereas in the X-irradiated rats, this layer-dependent distribution of DiI-labeled fibers was distorted. Furthermore, the fasciculus observed beneath cortical layer VI (white matter), which was made of DiI-labeled axons originating from the cortical neurons, was well developed in the controls. On the other hand, in the X-irradiated rats, the fasciculus made of DiI-labeled fibers was split, reduced in number, and irregularly distributed. These observation suggest that prenatal exposure of fetal rats to X-irradiation may affect the development of local circuits in the cerebral cortex, and may also damage the axonal projection of cortical neurons to the thalamus.

Abnormalities, Radiation-Induced↗
PubMed1997

Fractionated-dose effect of X-irradiation on the induction of neural tube defects in mice.

This experiment examined whether mouse embryos have a radio-adaptive response to the induction of malformations. Pregnant Slc:ICR mice were exposed to 0.05 Gy X-radiation followed by 0.5 or 1.0 Gy 4 hr later on day 8 of pregnancy. To compare this with the effect of single doses, four other groups were exposed to 0.5, 0.55, 1.0 or 1.05 Gy. All dams were put to death on day 18, and fetuses were examined for malformations. The frequency of neural tube defects induced by the fractionated irradiation into 0.05 Gy and 1.0 Gy was significantly higher than that induced by single doses of 1.0 and 1.05 Gy. Among groups exposed to a dose split into 0.05 and 0.5 Gy, and single doses of 0.5 and 0.55 Gy, there was no significant difference in incidence of the malformation. The present findings suggest that mouse embryos are not adapted to X-irradiation, but become more susceptible following a low-dose exposure.

Abnormalities, Radiation-Induced↗
PubMed1996

Nucleoside-diphosphate kinase-mediated signal transduction via histidyl-aspartyl phosphorelay systems in Escherichia coli.

Nucleoside-diphosphate kinase (NDP kinase), a key enzyme in nucleotide metabolism, is also known to be involved in growth and developmental control and tumor metastasis suppression. Interestingly, we find that coexpression of NDP kinase with Taz1, a Tar/EnvZ chimera, in the absence of its native signal, can activate a porin gene ompC-lacZ expression in Escherichia coli. Further studies show that NDP kinase can act as a protein kinase to phosphorylate histidine protein kinases such as EnvZ and CheA which are members of the His-Asp phosphorelay signal transduction systems in E. coli. Instead of ATP, the exclusive phosphodonor for histidine kinases, GTP can be utilized in vitro in the presence of NDP kinase to phosphorylate EnvZ and CheA, which then transfer the phosphoryl group to OmpR and CheY, the respective response regulators. The direct involvement of GTP for the phosphorylation of EnvZ through NDP kinase was further demonstrated by the use of a mutant EnvZ, which lost ability to be autophosphorylated with ATP. Phospho-OmpR thus formed can bind specifically to an ompF promoter sequence. These results suggest that NDP kinase may play a physiological role in signal transduction.

Aspartic Acid↗
PubMed1996

Positive-negative KG cassettes for construction of multi-gene deletions using a single drug marker.

Positive-negative KG cassettes were developed in order to create a number of independent deletion mutations on the bacterial chromosome using a single drug marker. These cassettes consist of a kanamycin-resistant (KmR) gene for positive screening and a galactokinase gene (galK) for negative screening. Both genes are in an operon driven by the native KmR promoter and are flanked by identical fragments of yeast chromosomal DNA approximately one kb in size. An internal region of a cloned target gene of a bacterium is replaced with a cassette, which is then transformed into the bacterium. The intact gene on the chromosome is replaced with the mutated gene by homologous recombination. From the KmR cells thus obtained, those cells which lose both KmR and galK genes by homologous recombination between the identical yeast DNA fragments are subsequently screened on plates containing 2-deoxygalactose, a non-metabolizable analogue of galactose. This method was applied to isolate a triple-deletion mutant of pkn3, pkn1, and pkn11 from Myxococcus xanthus.

Anti-Bacterial Agents↗
PubMed1996

Enhancement of frame-shift mutation by the overproduction of msDNA in Escherichia coli.

A minor population of wild Escherichia coli strains contain retroelements called retrons, which produce a peculiar satellite DNA, multicopy single-stranded DNA (msDNA). It has been reported that mismatched base pairs in the secondary structure formed in msDNA are mutagenic in E. coli[Maas et al.(1994) Mol.Microbiol. 14,437-441; Maas et al. (1996) Mol. Microbiol, 19, 505-509]. We reexamined this proposal by converting mismatched base pairs to matched base pairs using a single msDNA species, msDNA-Ec86, or by deleting mismatched regions using msDNA-Ec73. We also examined the effect of reverse transcriptases (RT) without msDNA production on mutagenesis. All the constructs are under the lpp/lac promoter-operator control so that their mutagenic effects can be tested in the absence and the presence of a lac inducer. It was found that when the production of msDNA-Ec86 or Ec73 was induced, reversion frequencies from Lac- to Lac+ significantly increased in the case of a Lac- mutation caused by a frame-shift mutation, but much less by a substitution mutation. The removal of mismatched base pairs eliminated the high mutation frequencies, and the inducible expression of RT alone was not mutagenic. These results are consistent with the hypothesis of Maas and his associates that mismatched base pairs in msDNA sequester a cellular mismatch repair system, resulting in the increase of frame-shift mutations.

Base Composition↗
PubMed1996

An unusual bacterial reverse transcriptase having LVDD in the YXDD box from Escherichia coli.

A minor population of wild strains of Escherichia coli contains a gene for reverse transcriptase (RT) which is responsible for the synthesis of multicopy single-stranded DNA (msDNA), a branched DNA-RNA complex. A DNA fragment capable of synthesizing msDNA was cloned from strain ECOR-58, one of the 72 wild strains in the ECOR collection. The complete open reading frame of a novel reverse transcriptase, designated ECOR-58 RT, was identified. ECOR-58 RT consisted of 408 amino acid residues, and its 227-residue polymerase domain from residue 43 to 269 showed significant homologies to all the other bacterial RTs so far identified. Most significantly, its YXDD box, the most highly conserved sequence in all RTs from prokaryotes to eukaryotes, was found to be replaced with LVDD. ECOR-58 RT was found to be most distantly related in a phylogenetic tree to all 9 other bacterial RTs so far identified.

Amino Acid Sequence↗
PubMed1996

The mechanism of autoprocessing of the propeptide of prosubtilisin E: intramolecular or intermolecular event?

The propeptide of subtilisin, an alkaline serine protease, is known to be required for the folding of subtilisin, functioning as an intramolecular chaperone. Upon folding of prosubtilisin, the propeptide of 77 amino acid residues is autocatalytically cleaved. A histidine-tag was added to the N-terminal end of prothiolsubtilisin E, or prosubtilisin(S221C), in which the active site serine residue at position 221 was substituted with cysteine. The histidine-tagged prosubtilisin(S221C) was denatured and immobilized on Ni-NTA resin. The denatured protein was then refolded on the resin, and the efficiency of the renaturation was determined by the efficiency of the propeptide cleavage. It was found that the cleavage of the propeptide was independent of the concentration of prosubtilisin(S221C), indicating that the autoprocessing is an intramolecular reaction. We also showed that prosubtilsin(S221A) can be autoprocessed if it is mixed with histidine-tagged prosubtilsin(S221C). These results demonstrate that prosubtilisin is intrinsically capable of being autoprocessed in an intramolecular manner, while it can also be processed in an intermolecular manner if it exists at higher concentrations.

Catalysis↗
PubMed1996

Deletion of the putative effector region of Era, an essential GTP-binding protein in Escherichia coli, causes a dominant-negative phenotype.

Era is an essential gene in E. coli, encoding a GTP-binding protein of unknown function. In the present work, a mutant designated Era-dE, for deletion of effector region is described. This is the first and only known era allele that confers a dominant-negative phenotype. Phenotypic analysis of the mutant showed that overproduction of Era-dE caused a dominant inhibition of growth when TCA cycle intermediates such as succinate, pyruvate, malate, alpha-ketoglutarate, and fumarate were provided as the sole carbon source. Examination of the macromolecular composition of cells overexpressing the mutant showed protein, DNA, and ATP levels expected for cells growing at slow rates. The response of cells expressing Era-dE to different stress conditions was studied by examining the rates of synthesis of stress-inducible proteins. Interestingly, when subjected to succinate starvation, cells expressing Era-dE showed a defective carbon starvation response, whereas response to glucose starvation was similar to that seen in control cells. Taken together with previous results, these studies indicate that Era is perhaps involved in multiple cellular processes and Era-dE disrupts more than one of these functions. Furthermore, it appears that some possible functions of Era include regulation of the TCA cycle and response to carbon starvation.

Adenosine Triphosphate↗
PubMed1996

The dilute-lethal (dl) gene attacks a Ca2+ store in the dendritic spine of Purkinje cells in mice.

The absence of smooth endoplasmic reticulum (SER) in the dendritic spine of Purkinje cells was found in dilute-lethal (dl) mouse cerebella as detected by immunohistochemistry using anti-inositol 1,4,5-triphosphate receptor antibody and electron microscopy. Since SER in the spine has been suggested to play a crucial role for synaptic regulation as an intracellular Ca2+ store (for reviews, see [Miller, R.J., Prog. Neurobiol., 37 (1991) 255-285: Simpson, P.B., Challiss, R.A.J. and Nahorski, S.R., Trends Neurosci., 18 (1995) 299-306]), a neurological defect, characterized by clonic convulsions with opisthotonus and ataxia, in the dilute-lethal mouse with homozygous trait may be attributable to the absence of SER in the dendritic spine of Purkinje cells.

Animals↗