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Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 145 records · Page 8Linked to original sources
PubMed1996

Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase.

Adenosine Diphosphate↗
PubMed1996

Endoplasmic reticulum is missing in dendritic spines of Purkinje cells of the ataxic mutant rat.

Dilute-opisthotonus (dop) is a spontaneous ataxic mutation in the rat, regulated by an autosomal recessive gene. Immunohistochemical staining with anti-inositol 1,4,5-trisphosphate receptor antibody and electron microscopic examinations revealed that the endoplasmic reticulum in dendritic spines of Purkinje cell was missing in the ataxic rat. This could impair the intracellular signal transduction in the parallel fiber-Purkinje cell synapse, and be a cause of the severe ataxic movement.

Animals↗
PubMed1996

Effect of overexpression of human Cu,Zn superoxide dismutase in transgenic mice on macrophage functions.

Properties of macrophages from transgenic mice with the human Cu,Zn superoxide dismutase (SOD) gene under the control of the mouse hydroxyl-methyl coenzyme A reductase (HMGCR) promoter were studied. In these mice, a twofold overproduction of Cu,Zn SOD in intraperitoneal macrophages resulted in the significant reduction of their microbicidal and fungicidal activity. Intracellular production and release of H2O2 in macrophages from transgenic mice activated by PMA was found to be significantly increased, whereas extracellular release of O2- was inhibited. When treated with LPS or LPS plus IFN-gamma, macrophages from transgenic mice were found to produce less nitric oxide (NO) than normal mice, suggesting that the nitrocompound metabolism in macrophages overproducing Cu,Zn SOD was also affected. Analysis of NF-kappa B DNA-binding activity and antiphosphotyrosine immunoblotting experiments suggest that impairment of macrophage functions may be attributed to the inhibition of signal transduction pathways as well as to changes in oxygen radical metabolism. The present data support the notion that antioxidant enzymes play important roles in the function of macrophages.

Animals↗
PubMed1996

Regulation of preimplantation development of mouse embryos: effects of inhibition of myosin light-chain kinase, a Ca2+/calmodulin-dependent enzyme.

We have examined the effects of ML-9 and wortmannin, which are, respectively, specific reversible and irreversible inhibitors of myosin light-chain kinase, a Ca2+/calmodulin-dependent enzyme, on preimplantation development of the mouse in an attempt to establish a regulatory role for this enzyme in preimplantation development. When late two-cell stage embryos were treated continuously with ML-9 or wortmannin at a concentration of 0, 1, 5, 10, or 15 microM, compaction and formation of the blastocyst were inhibited in a dose-dependent manner. Stage-specific treatment with ML-9 at 25 microM induced stage-specific responses of embryos after the eight-cell stage during the processes of compaction and cavitation. These morphological responses included aborted compaction, decompaction of compacted embryos, and the inability of embryos to form a cavity. These morphological effects were reversible, but, since cell proliferation was inhibited, the "recovered" embryos were small. Counting of cells on day 4 of culture, in both continuously treated and stage-specifically treated embryos, showed that the effect of ML-9 on cell proliferation was also dose-dependent. Wortmannin also had stage-specific effects at 15 microM, but these effects were irreversible and were more deleterious than those of ML-9. With neither inhibitor was there any apparent effect at the two-cell or the four-cell stage, although wortmannin inhibited cell division when applied stage-specifically at the four-cell stage. These results indicate that myosin light-chain kinase may be an important enzyme in the first steps of differentiation and in the maintenance of the differentiated state during preimplantation development of the mouse.

Androstadienes↗
PubMed1996

Mutational analysis of Era, an essential GTP-binding protein of Escherichia coli.

Era is an essential GTP-binding protein of an unknown function in Escherichia coli. On the basis of its sequence similarities to other GTP-binding proteins such as E. coli EF-Tu, EF-G, IF2 and eukaryotic Ras proteins, it has been suggested that the Era function is activated by GTP binding, and that subsequent conversion of bound GTP to GDP by the intrinsic GTPase activity modulates its function. Two Era mutants, one dominant negative mutant (dE), which has a deletion mutation from Ala40 to Gly49, and the other non-functional mutant (T42A/T43A), which has two substitution mutations, Thr42 to Ala and Thr43 to Ala, were analyzed for their abilities of GTP-binding and GTPase activity. It was found that the dE mutant lost the GTP-binding ability, while it still retained the GTPase activity. On the other hand, the T42A/T43A mutant retained both the GTP-crosslinking and GTPase activities. However, the Km values for GTPase activity increased 5- and 12-fold for dE and T42A/T43A mutants, respectively. These results indicate that both the GTP-binding and GTPase activities are important for the Era function.

Amino Acid Sequence↗
PubMed1996

Reverse phosphotransfer from OmpR to EnvZ in a kinase-/phosphatase+ mutant of EnvZ (EnvZ.N347D), a bifunctional signal transducer of Escherichia coli.

EnvZ of Escherichia coli is a transmembrane histidine kinase belonging to the family of two-component signal transducing systems prevalent in prokaryotes and recently discovered in eukaryotes. In response to changes in medium osmolarity EnvZ regulates the level of phosphorylated OmpR, its conjugate response-regulating transcription factor for ompF and ompC genes. EnvZ has dual opposing enzymatic activities; OmpR-phosphorylase (kinase) and phospho-OmpR-dephosphorylase (phosphatase). The osmotic signal is proposed to regulate the ratio of the kinase to the phosphatase activities of EnvZ to modulate the level of OmpR phosphorylation. In this work we used a COOH-terminal fragment of a previously identified kinase-/phosphatase+ EnvZ mutant (EnvZ-N347D) to demonstrate that the phosphoryl group on phospho-OmpR is transferred back to EnvZ to the same histidine residue (His243) that is utilized for the autokinase reaction by the wild type protein. Phospho-EnvZ-N347D thus formed could also transfer its phosphoryl group back to OmpR. The phosphotransfer reaction from phospho-OmpR to EnvZ.N347D was inhibited by ADP while Mg2+ ions stimulated the dephosphorylation reaction, resulting in release of inorganic phosphate. These results indicate that the energy levels of phosphoryl groups on OmpR and EnvZ are very similar and that the phosphatase reaction in the EnvZ.N347D mutant involves a reversal of the phosphotransfer reaction from EnvZ to OmpR using the identical His243 residue.

Bacterial Outer Membrane Proteins↗
PubMed1996

Cold shock induces a major ribosomal-associated protein that unwinds double-stranded RNA in Escherichia coli.

A 70-kDa protein was specifically induced in Escherichia coli when the culture temperature was shifted from 37 to 15 degrees C. The protein was identified to be the product of the deaD gene (reassigned csdA) encoding a DEAD-box protein. Furthermore, after the shift from 37 to 15 degrees C, CsdA was exclusively localized in the ribosomal fraction and became a major ribosomal-associated protein in cells grown at 15 degrees C. The csdA deletion significantly impaired cell growth and the synthesis of a number of proteins, specifically the derepression of heat-shock proteins, at low temperature. Purified CsdA was found to unwind double-stranded RNA in the absence of ATP. Therefore, the requirement for CsdA in derepression of heat-shock protein synthesis is a cold shock-induced function possibly mediated by destabilization of secondary structures previously identified in the rpoH mRNA.

Amino Acid Sequence↗
PubMed1996

Crystallization, X-ray studies, and site-directed cysteine mutagenesis of the DNA-binding domain of OmpR.

A C-terminal fragment of the transcription factor OmpR has been crystallized using the sitting drop vapor-diffusion method. Crystals belong to the trigonal space-group P3n12 with cell dimensions a = b = 54.4 A, c = 135.5 A, and gamma = 120.00 degrees. A second crystal form has been obtained by soaking this crystal form in a cryo-buffer and flash-cooling to 108 K in a cold nitrogen stream. Crystals belong to the trigonal space-group P3n12 with cell dimensions a = b = 108.07 A, c = 131.81 A, and gamma = 120.00 degrees. Both crystal forms diffract to at least 2.3 A at a synchrotron light source. Single-site cysteine mutations have been introduced to provide mercury-binding sites for multiple isomorphous replacement.

Amino Acid Sequence↗
PubMed1996

Complete growth inhibition of Escherichia coli by ribosome trapping with truncated cspA mRNA at low temperature.

BACKGROUND: CspA, the major cold-shock protein of Escherichia coli, is transiently induced upon temperature downshift and considered to play an important role in low-temperature adaptation. RESULTS: Overproduction of truncated cspA mRNAs retaining translational ability was found to completely block cell growth at low temperatures. This effect was termed 'low-temperature antibiotic effect of truncated cspA expression (LACE)'. In contrast to the significant reduction of polysomes in normal cells upon cold shock, cells under LACE maintained a high polysome profile, producing only truncated cspA products. Growth inhibition of cells under LACE was suppressed when CspA was overproduced together with the truncated cspA mRNA. CONCLUSION: LACE is caused by the overproduction of a truncated cspA mRNA in the absence of CspA production, which in turn traps all the cellular ribosomes in a non-adaptive form incapable of forming initiation complexes with other cellular mRNAs. LACE may provide a novel approach to the development of a new antibiotic.

Bacterial Proteins↗
PubMed1996

Differential thermoregulation of two highly homologous cold-shock genes, cspA and cspB, of Escherichia coli.

BACKGROUND: The major cold-shock protein in Escherichia coli is CspA, a 7.4 kDa protein. A CspA family has been found which consists of four additional proteins, CspB, CspC, CspD and CspE. The expression of cspB, unlike the other homologues, is cold-shock inducible like cspA. RESULTS: We examined the cold-shock induction of CspA and CspB at various temperatures. The cspA induction is observed by temperature shift from 37 to 30 degrees C and high levels of CspA production are observed between 24 and 10 degrees C. In contrast, CspB production occurs only by temperature shift to below 20 C, with maximum induction at 15 degrees C. Both cspA and cspB expressions were found to be induced at the level of transcription as determined by primer extension. CONCLUSIONS: These results show that cspA and cspB expressions are differentially regulated at low temperature indicating that E. coli contains at least two different biothermostats or thermoregulators that are likely to play important roles in cellular adaptation to low temperature. The cspB promoter shows sequence similarity to the cspA promoter. Furthermore, both cspA and cspB mRNAs have unusually long 5' untranslated regions (159 and 161 bases, respectively), both of which are able to form similar extensive secondary structures. These features are considered to contribute to the nature of the thermostats for cspA and cspB.

Bacterial Proteins↗
PubMed1996

The 19-residue pro-peptide of staphylococcal nuclease has a profound secretion-enhancing ability in Escherichia coli.

Staphylococcus aureus secretes two forms of extracellular nuclease, nuclease A and nuclease B. Nuclease A, consisting of 149 residues, is a proteolytic product of nuclease B, which is a processing intermediate that has a 19-residue N-terminal pro-peptide between the signal peptide and nuclease A. It has been shown that nuclease A can be secreted by Escherichia coli by fusing it to the OmpA signal peptide. We now demonstrate that the addition of the pro-peptide between the OmpA signal peptide and nuclease A leads to a significantly enhanced secretion rate in E. coli. The processing and secretion rates of nuclease B at 37 degrees C were at least 10 times faster than those of nuclease A. Nuclease B was also secreted efficiently under conditions which blocked the secretion of nuclease A, such as secA mutations and the addition of phenethyl alcohol or sodium azide. This enhancing effect of the pro-peptide was not as striking when it was attached to beta-lactamase, indicating that the pro-peptide acts as a specific secretion enhancer for nuclease A. Equilibrium circular dichroism on purified nuclease A and nuclease B indicated that the pro-peptide itself had no significant destabilizing effect on the mature protein. The existence of similar pro-peptides in Gram-positive bacterial secretory proteins indicates that they may also serve as secretion enhancers for individual proteins.

Adenosine Triphosphatases↗
PubMed1996

Follow-up study on histogenesis of microcephaly associated with ectopic gray matter induced by prenatal gamma-irradiation in the mouse.

Brain malformation with ectopic gray matter was visualized with magnetic resonance imaging in small-sized heads of prenatally exposed atomic bomb survivors. The identical brain malformation was reproduced in mice and its histogenesis was studied in the present experiment. Pregnant mice were exposed to 60Co gamma-irradiation at a single dose of 1.5 Gy on embryonic day 13 (E13), and then injected intraperitoneally with 30 mg/kg BrdU on E15. The extensive dead cells appeared throughout the brain mantle at 6 hours (h) after exposure. On E16 cell aggregations formed rosettes. On E18 a high proportion of BrdU-labeled cells reached the superficial layers of the cortical plate with the remaining cells located in the ectopic neuronal masses. The quantitative study showed that labeled cells in layers II to III were fewer and those in layers IV to VI more numerous in the prenatally irradiated adult mice than in controls. The anti-GFAP immunostaining revealed that the glial fibers in the irradiated mice were preserved, but disorganized. These findings suggested that the majority of migrating neurons were able to arrive at their normal layers, but some neurons remained due to the interrupted migratory pathway and eventually formed ectopic neuronal masses beneath the subcortical white matter.

Age Factors↗
PubMed1996

RbfA, a 30S ribosomal binding factor, is a cold-shock protein whose absence triggers the cold-shock response.

The cold-shock response, characterized by a specific pattern of gene expression, is induced upon a downshift in temperature and in the presence of inhibitors of ribosomal function. Here, we demonstrate that RbfA of Escherichia coli, considered to be involved in ribosomal maturation and/or initiation of translation, is a cold-shock protein. Shifting the rbfA mutant to a lower temperature resulted in a constitutive induction of the cold-shock response accompanied by slower growth at low temperatures, while shifting the rbfA mutant that overproduces wild-type RbfA resulted in an increase in total protein synthesis accompanied by faster growth adaptation to the lower temperature. Furthermore, the cold-shock response was also constitutively induced in a cold-sensitive 16S rRNA mutant at low temperatures. Accompanying the transient induction of the cold-shock response, we also report that shifting E. coli from 37 degrees C to 15 degrees C resulted in a temporary inhibition of initiation of translation, as evidenced by the transient decrease in polysomes accompanied by the transient increase in 70S monosomes. The accumulative data indicate that the inducing signal for the cold-unadapted non-translatable ribosomes which are converted to cold-adapted translatable ribosomes by the association of cold-shock proteins such as RbfA. Therefore, the expression of the cold-shock response, and thus cellular adaptation to low temperature, is regulated at the level of translation. The data also indicate that cold-shock proteins can be translated by ribosomes under conditions that are not translatable for most mRNAs.

Bacterial Proteins↗
PubMed1996

Reciprocal regulation of the differentiation of Myxococcus xanthus by Pkn5 and Pkn6, eukaryotic-like Ser/Thr protein kinases.

Myxococcus xanthus contains a large family of genes encoding eukaryotic-like serine/threonine kinases. Among them, two genes, pkn5 and pkn6, are divergently located on the chromosome and share a 46 bp promoter region between their transcription initiation sites, as determined by RNA protection. Pkn5, consisting of 380 amino acid residues, is a soluble protein in the cytoplasm, while Pkn6, consisting of 710 amino acid residues, is a transmembrane protein. Its membrane topology was determined using the Pkn6-PhoA fusion protein in Escherichia coli, which has a single transmembrane domain with the N-terminal domain in the cytoplasm and the C-terminal domain outside the cytoplasmic membrane. Both proteins, when expressed in E. coli, were autophosphorylated: Pkn5 only at Ser, and Pkn6 at both Ser and Thr. In M. xanthus, both genes are expressed constitutively throughout the life cycle, with slight increases at an early stage of development. Most strikingly, a pkn5-deletion strain forms fruiting bodies much faster than the wild-type strain, while a pkn6-deletion strain develops slower than the wild-type strain. These results, together with the fact that the pkn5-deletion strain is able to form fruiting bodies on semi-rich media, suggest that Pkn5 and Pkn6 have reciprocal roles in M. xanthus growth and development. Furthermore, Pkn6 may be a transmembrane sensor of external signals for development, while Pkn5 is a kinase that negatively regulates M. xanthus development.

Amino Acid Sequence↗
PubMed1996

Effects of T4 phage infection and anaerobiosis upon nucleotide pools and mutagenesis in nucleoside diphosphokinase-defective Escherichia coli strains.

Bacteriophage T4 encodes nearly all of its own enzymes for synthesizing DNA and its precursors. An exception is nucleoside diphosphokinase (ndk gene product), which catalyzes the synthesis of ribonucleoside triphosphates and deoxyribonucleoside triphosphates (dNTPs) from the corresponding diphosphates. Surprisingly, an Escherichia coli ndk deletion strain grows normally and supports T4 infection. As shown elsewhere, these ndk mutant cells display both a mutator phenotype and deoxyribonucleotide pool abnormalities. However, after T4 infection, both dNTP pools and spontaneous mutation frequencies are near normal. An E. coli strain carrying deletions in ndk and pyrA and pyrF, the structural genes for both pyruvate kinases, also grows and supports T4 infection. We examined anaerobic E. coli cultures because of reports that in anaerobiosis, pyruvate kinase represents the major route for nucleoside triphosphate synthesis in the absence of nucleoside diphosphokinase. The dNTP pool imbalances and the mutator phenotype are less pronounced in the anaerobic than in the corresponding aerobic ndk mutant strains. Anaerobic dNTP pool data, which have not been reported before, reveal a disproportionate reduction in dGTP, relative to the other pools, when aerobic and anaerobic conditions are compared. The finding that mutagenesis and pool imbalances are mitigated in both anaerobic and T4-infected cultures provides strong, if circumstantial, evidence that the mutator phenotype of ndk mutant cells is a result of the dNTP imbalance. Also, the viability of these cells indicates the existence of a second enzyme system in addition to nucleoside diphosphokinase for nucleoside triphosphate synthesis.

Adenosine Triphosphate↗
PubMed1996

The role of the 5'-end untranslated region of the mRNA for CspA, the major cold-shock protein of Escherichia coli, in cold-shock adaptation.

During cellular adaptation to low temperature, Escherichia coli transiently synthesizes the major cold-shock protein CspA. It was found that adaptation to cold shock is blocked when the 143-base sequence of the 5' untranslated region (5' UTR) of the cspA mRNA is overproduced. The overproduction of this UTR at 15 degrees C caused the synthesis of not only CspA but also other cold-shock proteins such as CspB and CsdA to be no longer transient but rather prolonged. In addition, inhibition of both the synthesis of cellular proteins other than cold-shock proteins and cell growth was observed. Interestingly, when CspA was also overproduced together with the 5' UTR, normal cold-shock adaptive response was resumed without a prolonged lag period of cell growth. This indicates that the 5' UTR of the cspA mRNA and its gene product CspA play a critical role in the regulation of the expression of cold-shock genes and cold-shock adaptation. An 11-base common sequence (cold box) was found in the 5' UTRs of cspA, cspB, and csdA mRNAs. Indeed, the 25-base sequence within the 5' UTR of the cspA mRNA containing the cold-box sequence was able to prolong CspA production at 15 degrees C. We propose that a putative repressor binds to the cold-box sequence of the cold-shock mRNAs during the adaptive process and this binding in turn blocks the transcription of the cold-shock genes or destabilizes their mRNAs. CspA appears to promote either directly or indirectly the repressor function.

Adaptation, Physiological↗