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Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 217 records · Page 12Linked to original sources
PubMed1994

Establishment of new inbred strains derived from Japanese wild rats (Rattus norvegicus).

Inbreeding of Japanese wild rats (Rattus norvegicus) resulted in five new strains. The conception rates of wild rat strains were lower than those of inbred laboratory strains, and maternal aggressiveness was observed in some wild strains. The body size of domesticated animals was larger than that of captured ancestors. Two strains have been established as inbred strains; the MITA strain has been tamed to the level of laboratory rats, while the nature of the MITD strain is wild. The MITB strain was derived from the MITA strain as a coat color variant. The MITC and MITE strains have been bred to the F15 and F11 generations, respectively. The new strains can provide new genetic variations against a wild type background and might be useful for physiological and psychological experiments.

Animals↗
PubMed1994

Body and brain development following exposure to 60Co gamma-irradiation during pregnancy in mice.

We studied the dose-response effects of gamma-irradiation on the weight gains of the body and the brain in ICR mice exposed to various doses of 60Co gamma-irradiation ranging from 0 to 1.5 Gy on day 13 of pregnancy (El3). We found that 0.5 Gy gamma-irradiation caused a significant reduction in brain weight but not in body weight among 6-week-old mice. Higher doses (1.0, 1.5 Gy) seriously inhibited body and brain development, resulting in significantly low weights at 6 weeks of age. A significantly lower brain weight among fetuses exposed to 1.5 Gy was found as early as 24 hours after exposure, while significant reductions in the body weight of these same fetuses appeared 3 days after exposure. The effects of radiation on brain and body development were similar for both males and females.

Animals↗
PubMed1994

Irradiation injury to the developing nervous system: mechanisms of neuronal injury.

Undifferentiated neural cells in the developing brain are particularly vulnerable to irradiation and easily involved in cell death. We investigated in mice cytological features of radiation-induced death of neuron-precursor cells and their high sensitivity. The acute cell injury in the embryonic telencephalon by doses as low as 0.1 Gy was not reversed up to 6 hours and injured cells expressed apoptotic death which began at 2 hours after exposure and peaked at 6-9 hours. Radiation-induced cell death in the cerebellar -external granular layer of newborn mice exposed to 0.24 Gy was suppressed completely by cycloheximide, a protein synthesis inhibitor. The high incidence of radiation-induced apoptosis of the telencephalic ventricular cells observed at the beginning of cortical neuron production could be attributed to the emergence of radiosensitive G1phase cells at this stage. One of the significant factors determining the period of high sensitivity for radiation-induced apoptosis could be a certain initial phase of chemical cytodifferentiation prior to their actual morphological differentiation.

Animals↗
PubMed1993

Crystal structure of Myxococcus xanthus nucleoside diphosphate kinase and its interaction with a nucleotide substrate at 2.0 A resolution.

The X-ray crystallographic structure of nucleoside diphosphate (NDP) kinase from Myxococcus xanthus has been determined using multiple isomorphous replacement techniques and refined at 2.0 A resolution to a crystallographic R-factor of 0.17. This is the first report of the structure of an enzymatically active NDP kinase and of the enzyme with a bound nucleotide. The structure has been determined in P4(3)2(1)2 and I222 crystal forms. The enzyme monomer consists of a four-stranded antiparallel beta-sheet. The surfaces of the sheet are partially covered with five helical segments. There are two protein molecules in the asymmetric unit of the tetragonal crystal form. They form a dimer with an extensive interface in which 1092 A2 per monomer is buried. The majority of the contact area in the dimer interface is between hydrophobic or aromatic residues. Two dimers are related by a crystallographic 2-fold axis to yield a tetramer. This tetramer is also present in the orthorhombic crystals; however, in this case, the 222 symmetry is entirely crystallographic. Upon tetramer formation, an additional 473 A2 of solvent-accessible surface area from each monomer becomes buried. The interface between dimers in the tetramer is stabilized by salt bridges. Equilibrium sedimentation studies are consistent with the enzyme being a tetramer in solution. The structure of a complex of adenosine diphosphate (ADP) with the enzyme was determined and reveals that most of the nucleotide interactions with the protein are with the pyrophosphate and ribose groups, while the base has no hydrogen bonds with the protein and interacts only by stacking with the side chain of Phe59. The Mg2+ interacts with the pyrophosphate of the ADP and via a solvent molecule with the side chain of the conserved Asp120 residue. The mode of interaction with the nucleotide is novel, with the nucleotide binding at the side of the beta-sheet. The structures of the nucleotide in crystals grown in the presence or absence of Mg2+ are essentially identical. In addition, the phosphotransfer reaction from adenosine triphosphate (ATP) to the enzyme can occur without Mg2+. This suggests that only the second step of the reaction in which the enzyme transfers the phosphate to a nucleoside diphosphate acceptor is significantly catalyzed by the metal.

Adenosine Diphosphate↗
PubMed1993

Effect of the relative position of the UGA codon to the unique secondary structure in the fdhF mRNA on its decoding by selenocysteinyl tRNA in Escherichia coli.

The fdhF mRNA for formate dehydrogenase H of Escherichia coli contains a UGA codon at position 140. This termination codon is decoded by selenocysteinyl tRNA (the selC product) with the aid of its own specific elongation factor, SelB. For this decoding, a unique secondary structure immediately downstream of the UGA codon has been shown to be essential (Zinoni, F., Heider, J., and Böck, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4660-4664). We examined the positional effect of the UGA codon relative to the secondary structure on its decoding using a fdhF-lacZ fusion gene. When the UGA codon was separated by one codon (position -1) from the secondary structure, the UGA decoding, as measured by the beta-galactosidase activity, dropped to approximately 76% of the normal level but was still almost as fully dependent upon selC and selenium in the culture medium as in the case of the UGA codon in the normal position (position 0). However, when the UGA codon was separated by two codons (position -2), the decoding level further dropped to 20% of the normal level, and in addition, became dependent only on selC but independent of selenium. When the UGA codon was further separated by three codons (position -3), the decoding level of UGA (-3) became higher than the decoding of UGA (-2) and was completely independent from selC and selenium, indicating that the UGA codon was nonspecifically suppressed. A similar nonspecific suppression was observed for the UGA codon at position -4, but at a lower level. When two UGA codons were tandemly placed at positions 0 and -1, they were still able to be decoded at 17% of the normal level in a selC- and selenium-dependent manner. In the absence of the SelB function, the decoding level of UGA(0) dropped to 1.6% of the normal level, whereas the UGA(-1) decoding dropped to 7.5%. These results indicate that the UGA codon at position 0 is not only most effectively decoded by selenocysteinyl tRNA but also tightly blocked from its nonspecific suppression in the absence of any components required for the decoding.

Base Sequence↗
PubMed1993

Folding pathway mediated by an intramolecular chaperone.

The N-terminal propeptide of subtilisin, a serine protease, functions as an intramolecular chaperone which is crucial for proper folding of the active enzyme. This nascent N-terminal propeptide is removed after completion of the folding process. Here we present a possible pathway by which intramolecular chaperones mediate protein folding. Using circular dichroism to analyze acid-denatured subtilisin we have identified a folding-competent state which can refold to an active conformation in the absence of the propeptide. Earlier work had shown that guanidine hydrochloride-denatured subtilisin was in a state incapable of folding in absence of its propeptide. Comparison of the folding-incompetent and folding-competent states indicates that refolding is facilitated by the presence of residual structure present only in the folding-competent state. The analysis further indicates that the propeptide is essential for inducing this state. Therefore the folding-competent state may lie on--or be in rapid equilibrium with an intermediate on--the folding pathway of subtilisin. In the absence of the propeptide, formation of such a state--and hence refolding--is extremely slow.

Amino Acid Sequence↗
PubMed1993

Ligand binding to the receptor domain regulates the ratio of kinase to phosphatase activities of the signaling domain of the hybrid Escherichia coli transmembrane receptor, Taz1.

Taz1 is a hybrid receptor, in which the periplasmic receptor domain of Tar, an aspartate chemoreceptor, is fused with the cytoplasmic signaling domain of EnvZ, an osmosensor. Taz1 is able to induce ompC-lacZ expression in response to aspartate added to the medium. We introduced amino acid substitution mutations in the highly conserved region of the signaling domain of Tar near the Tar-EnvZ junction. The same mutations in Tsr, a serine chemoreceptor, are known to lock the flagella rotation in either a clockwise (CW) or in a counter-clockwise (CCW) mode. It was found that a CW-biased mutation in Taz1 resulted in ompC-lacZ expression in the "off mode", or low ompC-lacZ expression in both the absence and presence of aspartate, while CCW-biased mutations caused ompC-lacZ expression in the "on mode", or constitutive expression regardless of aspartate. The OmpR kinase and phospho-OmpR phosphatase activities of the wild-type and mutant Taz proteins were also examined in response to aspartate. The phosphatase activity of the wild-type Taz1 was found to decrease in the presence of aspartate, while the OmpR kinase activity remained constant. This indicated that aspartate binding to the Taz1 receptor domain modulates the ratio of kinase to phosphatase activity of the signaling domain. An increased kinase to phosphatase ratio in the presence of aspartate resulted in higher levels of phospho-OmpR in the cell and therefore induced ompC-lacZ expression. In contrast to the wild-type Taz1 protein, the enzymatic activities of CW as well as CCW mutants did not change in response to aspartate, indicating that mutant Taz proteins are incapable of transducing the signal across the membrane as a result of a locked conformation of the signaling domain in either the on or off mode.

Aspartic Acid↗
PubMed1993

Ligand binding induces an asymmetrical transmembrane signal through a receptor dimer.

Two ligand (aspartate)-binding pockets are formed at the interface between the subunits of the Tar homodimer, a bacterial chemoreceptor. Using mutant heterodimers of a hybrid receptor, Taz1, which consists of the external domain of Tar and the cytoplasmic domain of EnvZ, we disrupted either one or the other of the two ligand-binding pockets. We found that occupation of only one of the ligand-binding pockets was sufficient for induction of a transmembrane signal, and that the subunit responsible for the binding of the amino group of the ligand transduces the signal.

Bacterial Outer Membrane Proteins↗
PubMed1993

Requirement of both kinase and phosphatase activities of an Escherichia coli receptor (Taz1) for ligand-dependent signal transduction.

Taz1 is a hybrid receptor in the Escherichia coli cytoplasmic membrane, consisting of the N-terminal ligand binding domain of Tar (a chemoreceptor for aspartate) and the C-terminal signaling domain of EnvZ (an osmosensor). The binding of aspartate to an extra cytoplasmic domain induces the transmembrane signal to the cytoplasmic signaling domain. The signaling domain functioning as a protein kinase evokes a response by transferring a phosphate from an intracellular histidine to OmpR. This domain also encodes an OmpR-specific phosphatase whose action is crucial in completing the OmpR phosphorylation cycle. Phosphorylated OmpR acts as a transcriptional activator for the ompC gene. A number of mutations were introduced into the signaling domain in conserved sequences of the prokaryotic histidine kinase family. All Taz1 mutants lost the ability to both autophosphorylate the histidine residue and transfer the phosphate to OmpR. These mutated receptors were unable to activate ompC-lacZ expression. However, ompC-lacZ was able to be activated by complementation of Taz1 mutants. In some combinations, two different defective Taz1 mutants could restore both OmpR kinase and phosphatase activities when co-expressed. In other combinations only kinase activity was restored. Aspartate-inducible ompC-lacZ expression was restored only in the former cases, while in the latter cases ompC-lacZ expression became constitutive. These results indicate that the kinase activity is essential to activate ompC expression while the phosphatase activity is required to regulate ompC gene expression in a ligand-dependent manner.

Aspartic Acid↗
PubMed1993

Reverse transcriptases from bacterial retrons require specific secondary structures at the 5'-end of the template for the cDNA priming reaction.

Multicopy single-stranded DNA (msDNA) is a peculiar molecule consisting of a single-stranded DNA that is branched out from an internal G residue of an RNA molecule (msdRNA) via a 2',5'-phosphodiester linkage. The genetic unit required for msDNA synthesis is designated "retron" and consists of msr (a gene for msdRNA), msd (a gene for msDNA), and a gene for reverse transcriptase (RT) in a single operon. To date, four different msDNAs have been isolated from Escherichia coli. They do not share any primary sequences in either RNA or DNA. To elucidate the specificity of bacterial RT for msDNA synthesis, the msr-msd region from retron-Ec67 was introduced into E. coli cells producing RT-Ec73, or the msr-msd region from retron-Ec73 into E. coli cells producing RT-Ec67. In both cases, msDNA was not synthesized. However, when the msdRNA coding regions (msr) for retron-Ec67 and -Ec73 were mutually exchanged and the chimeric genes were introduced into E. coli cells producing either RT-Ec67 or RT-Ec73, it was thus found that msDNA was produced only when msr and RT were from the same retron. Requirement of the msr region for msDNA synthesis by RT was further investigated by mutations in the msr region for retron-Ec67. These analyses revealed that there is a strict requirement for specific primary sequences as well as the secondary structure in msdRNA. This finding is discussed in relationship to the mechanism of the priming reaction of cDNA synthesis by eukaryotic retroviral RTs using tRNAs.

Bacterial Proteins↗
PubMed1993

Eukaryotic-like protein serine/threonine kinases in Myxococcus xanthus, a developmental bacterium exhibiting social behavior.

Myxococcus xanthus, a gram-negative bacterium exhibits a spectacular life cycle and social behavior. Its developmental cycle and multicellular morphogenesis resemble those of eukaryotic slime molds such as Dictyostelium discoideum. On the basis of this resemblance, we explored the existence of eukaryotic-like protein serine/threonine kinases which are known to play important roles in signal transduction during development of D. discoideum. It was indeed found that M. xanthus contains a large family of protein serine/threonine kinases related to the eukaryotic enzymes. This is the first unambiguous demonstration of eukaryotic-like serine/threonine kinases in the prokaryotes.

Amino Acid Sequence↗
PubMed1993

Intramolecular chaperones and protein folding.

Many proteins from both prokaryotic and eukaryotic sources are produced with amino-terminal propeptides. These propeptides, which are usually located between the signal peptide and the mature protein, are essential for the proper function of that protein. Recent research has indicated that these polypeptides are indispensible for proper folding of the proteins they are attached to. As propeptides perform a function similar to that of a large family of heat shock proteins, they had been broadly classified as molecular chaperones. However, significant differences exist between these two classes of proteins and to distinguish them from one another, propeptides have been termed intramolecular chaperones. Recent results have suggested that such intramolecular chaperones may be found in a large number of proteins.

Amino Acid Sequence↗
PubMed1993

The retron: a bacterial retroelement required for the synthesis of msDNA.

'Retrons' are bacterial retroelements responsible for the synthesis of msDNA, a hybrid nucleic acid consisting of a single-stranded DNA that is branched out from an internal guanosine of an RNA molecule via a 2',5'-phosphodiester linkage. Retrons are found in a minor population of various bacterial species and are extensively diverse. Two important questions now demanding attention are whether retrons are mobile elements and why are they so diverse?

Base Sequence↗
PubMed1993

Evaluation of the use of antisense tRNA(met) as an inhibitor for eukaryotic protein synthesis.

We attempted to explore the use of antisense RNAs against tRNA as an inhibitor of eukaryotic protein synthesis. For this purpose, antisense RNA against the 5'-end half of the initiator tRNA of wheat germ was synthesized, and its effect on translation of the Brome mosaic virus mRNA was investigated in a wheat germ cell-free system. When the antisense RNA against the 5'-end half of the initiator tRNA including the anticodon sequence was added at the concentration of 8 microM to the cell-free system, protein synthesis was completely inhibited. This inhibitory effect could be suppressed by the addition of wheat germ tRNA. In contrast, sense and control RNA showed slight inhibitory effects, which were not, however, suppressed by wheat germ tRNA. The antisense tRNA formed a double-stranded RNA duplex with the target methionine tRNA in the wheat germ extract which became resistant to ribonuclease treatment. These experiments suggest that antisense tRNA could be utilized for control of tRNA functions and to block protein synthesis.

Base Sequence↗
PubMed1993

The backbone structure of the major cold-shock protein CS7.4 of Escherichia coli in solution includes extensive beta-sheet structure.

CS7.4 is the major cold-shock protein specifically expressed to a level as high as 13% of the total cellular protein within the first hour when Escherichia coli cell culture is shifted from 37 to 15 degrees C [Goldstein et al. (1990) Proc. Natl. Acad. Sci. USA 87, 283-287]. It consists of 70 amino acid residues with a very high content of aromatic residues. CS7.4 was overproduced and purified to homogeneity. Its secondary structure was analyzed by examining circular dichroism at both the far and near-UV regions; the results suggest that the protein is largely beta-sheet in conformation. The predominance of beta-sheet structure in the protein was confirmed by using Fourier-transform infrared spectroscopy. A folded compact conformation was also verified by fluorescence emission spectroscopy. We evaluated Tm, delta H, and delta S from the thermal denaturation profile of the protein. Unusual spectral features observed in the far-UV region are attributed to the high content of aromatic residues. The protein is relatively small and contains no disulfide bonds. However, it is surprisingly stable to heat denaturation.

Bacterial Proteins↗
PubMed1993

Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli.

In Escherichia coli the OmpR and EnvZ proteins regulate the expression of the outer membrane porin proteins OmpC and OmpF. EnvZ and OmpR belong to a family of sensor/effector protein pairs that control adaptation to a variety of environmental conditions. EnvZ acts as the sensor protein that phosphorylates OmpR, which in turn regulates porin gene expression. The level of phosphorylated OmpR appears to be a determining factor for ompC and ompF regulation. Phosphorylation of OmpR is considered to occur at one or more aspartic acid residues (Asp-11, Asp-12 and/or Asp-55) that are highly conserved among the effector proteins. In this report we biochemically characterized the aspartic acid residue(s) in OmpR that were phosphorylated by EnvZ. Reduction of aspartyl phosphate residues in the amino-terminal domain of OmpR with [3H]-NaBH4 indicated that Asp-55 was a primary site of modification. We further studied the role of the highly conserved aspartate residues by creating OmpR mutants having aspartate to alanine substitutions at positions 11 (D11A), 12 (D12A) and 55 (D55A). Studies of ompF and ompC expression as well as in vivo and in vitro phosphorylation experiments also demonstrated that while Asp-55 is the primary phosphate acceptor site in OmpR, Asp-11 may also serve as a phosphorylation site, particularly in the absence of Asp-55.

Amino Acid Sequence↗