Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 55 records · Page 3Linked to original sources

PubMed2000

A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium.

Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.

Amino Acid Sequence↗

PubMed2000

Derangement of Purkinje cells in the rat cerebellum following prenatal exposure to X-irradiation: decreased Reelin level is a possible cause.

It has been reported that prenatal X-irradiation of rats during the late gestation period causes heterotopic Purkinje cells in the internal granular layer (IGL) of the abnormally foliated cerebellum. The present study was designed to demonstrate the process of X-ray-induced derangement of Purkinje cells and their surrounding cells. In addition, the expression of some morphoregulatory molecules was examined to determine which molecules are involved in the abnormal pattern of Purkinje cells. Pregnant rats (n = 22) were exposed to 2.5 Gy X-radiation on gestation day 21 and the cerebellum of progeny was examined histologically and by immunohistochemistry to identify Purkinje and Bergmann cells. At 12 h after exposure, extensive cell death was observed in the external granular layer (EGL). By postnatal day (P) 9, while Purkinje cells with well-developed dendrites aligned underneath the EGL in the control cerebellum, Purkinje cells with shorter and abnormally oriented dendrites failed to align and remained in the heterotopic location in the IGL. Bergmann cells and their fibers were also disoriented but later recovered in their proper position. Abnormal folia developed in the irradiated rats. Using immunohistochemistry, we next examined the levels of the neural cell adhesion molecule (NCAM), fibronectin, tenascin, and Reelin. Among them, only the level of Reelin was affected significantly. Reelin decreased strikingly in the premigratory zone of the EGL and IGL in the irradiated cerebellum on P1, and the decrease continued until P9. Decreased Reelin expression was demonstrated quantitatively by Northern blot analysis and the correlation between the mRNA and protein levels was well presented. The expression of reelin mRNA decreased significantly by irradiation from P0, being almost one third of the level in controls on P4, and tended to recover up to P9. It is thus indicated that X-irradiation causes a marked decrease in the level of Reelin at the critical stage for the alignment of Purkinje cells. Since Reelin has been shown to play an important role in the migration of neural cells, it is suggested that the decrease in Reelin by X-irradiation is an important factor for the derangement of Purkinje cells.

Animals↗

PubMed2000

Characterization of GTPase activity of TrmE, a member of a novel GTPase superfamily, from Thermotoga maritima.

A gene encoding a putative GTP-binding protein, a TrmE homologue that is highly conserved in both prokaryotes and eukaryotes, was cloned from Thermotoga maritima, a hyperthermophilic bacterium. T. maritima TrmE was overexpressed in Escherichia coli and purified. TrmE has a GTPase activity but no ATPase activity. The GTPase activity can be competed with GTP, GDP, and dGTP but not with GMP, ATP, CTP, or UTP. K(m) and k(cat) at 70 degrees C were 833 microM and 9.3 min(-1), respectively. Our results indicate that TrmE is a GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.

Amino Acid Sequence↗

PubMed2000

Hydrocephalus in mice following X-irradiation at early gestational stage: possibly due to persistent deceleration of cell proliferation.

The pathogenesis of X-ray-induced congenital hydrocephalus was studied. Pregnant mice were irradiated at 1.4 Gy on gestational day 7 (G7). Four hours after irradiation, extensive cell death was evident in the neuroepithelium and underlying mesoderm of the head region, and proliferating cell nuclear antigen (PCNA)-immunoreactive cells almost disappeared. Embryos with thinner lamina terminalis of the telencephalon, when compared with that of the control, were found in the irradiated group on G9. As early as G11 in some irradiated embryos the telencephalic wall was thinner and lateral ventricles were larger than those of the control. The choroid invagination from the lamina terminalis began on G11 in the control brain, but not in the affected brain. During the following development, fetuses with readily apparent hydrocephalus were consistently found among irradiated fetuses. In these brains the brain mantle was thinner, the corpus striatum and thalamic regions were smaller, and lateral ventricles were larger than those of the control. Even on G11 and G13 the frequencies of PCNA-positive cells in the brain mantle and other brain regions were lower in the hydrocephalic brain than those of the control, suggesting a decelerated proliferation of successive cell generations following exposure to X-rays. The cerebral aqueduct was open in the hydrocephalic brain during the fetal period when the lateral ventricles were dilated. The head was vaulted after birth but the cerebral aqueduct was not completely occluded even in these animals. These findings suggested that cell death in the neuroepithelium followed by a persistent deceleration of neural cell proliferation, resulting in the hypoplasia of brain parenchyma with compensatory ventricular dilatation, is important for the establishment of hydrocephalus.

Animals↗

PubMed2000

Similarity between the effects of carbon-ion irradiation and X-irradiation on the development of rat brain.

The effects of carbon-ion irradiation and X-irradiation on the development of rat brain were compared. Twenty pregnant rats were injected with bromodeoxyuridine (BrdU) at 9 pm on day 18 of pregnancy and divided into five groups. Three hours after injection (day 19.0) one group was exposed to 290 MeV/u carbon-ion radiation by a single dose of 1.5 Gy. Other groups were exposed to X-radiation by 1.5, 2.0 or 2.5 Gy, or sham-treated, respectively. Fetuses were removed from one dam in each group 8 h after exposure and examined histologically. Extensive cell death was observed in the brain mantle from the irradiated groups. The cell death after 1.5 Gy carbon-ion irradiation was remarkably more extensive than that after 1.5 Gy X-irradiation, but comparable to that after 2.0 Gy or 2.5 Gy X-irradiation. The remaining rats were allowed to give birth and the offspring were sacrificed at 6 weeks of age. All of the irradiated offspring manifested microcephaly. The size of the brain mantle exposed to 1.5 Gy carbon-ion radiation was significantly smaller than that exposed to 1.5 Gy X-radiation and larger than that exposed to 2.5 Gy X-radiation. A histological examination of the cerebral cortex revealed that cortical layers II-IV were malformed. The defect by 1.5 Gy carbon-ion irradiation was more severe than that by the same dose of X-irradiation. Although the BrdU-incorporated neurons were greatly reduced in number in all irradiated groups, these cells reached the superficial area of the cortex. These findings indicated that the effects of both carbon-ion irradiation and X-irradiation on the development of rat brain are similar in character, and the effect of 1.5 Gy carbon-ion irradiation compares to that of 2.0-2.5 Gy X-irradiation.

Animals↗

PubMed2000

ATPase and GTPase activities copurifying with GTP-binding proteins in E. coli.

Intrinsic GTPase activity of GTP-binding proteins plays the vital role in regulating the downstream activation pathway. We examined the GTP and ATP hydrolyzing (NTPase) abilities of various bacterial and human GTP-binding proteins under different metabolic conditions. Two metabolic components, acetate and 3-phosphoglyceric acid (3-PG), have shown significant stimulatory action on NTPase activity of G-protein preparations. Acetyl phosphate and 2,3-bisphosphoglyceric acid (2,3-BPG) blocked these stimulations. From gel filtration analyses, we have determined two fractions containing metabolite-inducible NTPase activities which are independent of GTP-binding protein enzymatic actions. Therefore, one should be cautious when NTPase activity is examined in a buffer containing acetate often used for NTPase assay.

Acetates↗

PubMed2000

Identification of a substrate for Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus by a novel method for substrate identification.

Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play important roles in signal transduction required for cell proliferation, differentiation, and stress response and adaptation. It is also known that some prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in the characterization of these kinases is how to identify their specific substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly expressed in E. coli, cells are unable to form colonies on agar plates. This lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the highly conserved Lys residue was changed to Asn, indicating that phosphorylation of a cellular protein(s) in E. coli resulted in growth arrest. Several clones from an E. coli genomic library were found to suppress the lethal effect when co-expressed with pkn2. Four out of seven multi-copy suppressors were identified to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part to the lethal effect in E. coli. The present method termed the STEK method (Suppressors of Toxic Effects of Kinases) may be widely used for the substrate identification not only for prokaryotic protein Ser/Thr kinases but also for eukaryotic kinases.

Bacterial Proteins↗

PubMed1999

Modulation of chemokine expression during ischemia/reperfusion in transgenic mice overproducing human glutathione peroxidases.

Renal ischemia/reperfusion (I/R) injury is a major cause of kidney damage. There is accumulating evidence that inflammatory reactions are involved in the pathogenesis of this process. Our studies demonstrate that transgenic mice overexpressing human extracellular and intracellular glutathione peroxidases (GP) are protected against kidney I/R injury. Importantly, significant reduction in neutrophil migration was observed in GP mice compared with nontransgenic mice. Analysis of signaling molecules mediating neutrophil activation and recruitment indicates reduction in the level of KC and macrophage inflammatory protein-2 chemokine expression in transgenic animals. The molecular mechanism mediating this effect appears to involve repression of NF-kappaB activation at the level of IkappaBalpha and IkappaBbeta degradation. In the case of IkappaBalpha, no apparent phosphorylation was detected. These results suggest that IkappaBalpha proteolysis is triggered during the renal I/R pro-oxidant state by a still unknown mechanism, which might be different from other stimuli. A central role of NF-kappaB in CXC chemokine activation was demonstrated in cell culture anoxia/ATP repletion experiments as a model of I/R. The data presented indicate the important role of GP-sensitive signal transduction pathways in the development of inflammatory response and tissue injury during I/R.

Adenosine Triphosphate↗

PubMed1999

Highly specific recognition of primer RNA structures for 2'-OH priming reaction by bacterial reverse transcriptases.

A minor population of Escherichia coli contains retro-elements called retrons, which encode reverse transcriptases (RT) to synthesize peculiar satellite DNAs called multicopy single-stranded DNA (msDNA). These RTs recognize specific RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2'-OH group of a specific internal G residue (branching G residue). The resulting products (msDNA) consist of RNA and single-stranded DNA, sharing hardly any sequence homology. Here, we investigated how RT-Ec86 recognizes the specific RNA structure in its primer-template RNA. On the basis of structural comparison with HIV-1 RT, domain exchanges were carried out between two E. coli RTs, RT-Ec86 and RT-Ec73. RT-Ec86 (320 residues) and RT-Ec73 (316 residues) share only 71 identical residues (22%). From the analysis of 10 such constructs, the C-terminal 91-residue sequence of RT-Ec86 was found to be essential for the recognition of the unique stem-loop structure and the branching G residue in the primer-template RNA for retron-Ec86. Using the SELEX (systematic evolution of ligands by exponential enrichment) method with RT-Ec86 and primer RNAs containing random sequences, the identical stem-loop structure (including the 3-U loop) to that found in the retron-Ec86 primer-template RNA was enriched. In addition, the highly conserved 4-base sequence (UAGC), including the branching G residue, was also enriched. These results indicate that the highly diverse C-terminal region recognizes specific stem-loop structures and the branching G residue located upstream of the stem-loop structure. The present results with seemingly primitive RNA-dependent DNA polymerases provide insight into the mechanisms for specific protein RNA recognition.

Amino Acid Sequence↗

PubMed1999

Identification and characterization of five cspA homologous genes from Myxococcus xanthus.

Escherichia coli contains a large CspA family consisting of nine homologues, in which four are cold-shock inducible and one is stationary-phase inducible. Here, we demonstrate that Myxococcus xanthus possesses at least five CspA homologues, CspA to CspE. Hydrophobic residues forming a hydrophobic core, and aromatic residues, which are included in functional motifs RNP-1 and RNP-2 involved in binding to RNA and ssDNA, are well conserved. These facts suggest that M. xanthus CspA homologues have a similar structure and function as E. coli CspA. However, in contrast to the E. coli CspA family, the expression of M. xanthus csp genes as judged by primer extension analysis is not significantly regulated by temperature changes, except for cspB of which expression was reduced to less than 10% upon heat shock at 42 degrees C. Such constitutive expression of the csp genes may be important for M. xanthus, a soil-dwelling bacterium, to survive under conditions of exposure to various environmental changes in nature.

Amino Acid Sequence↗

PubMed1999

Era, an essential Escherichia coli small G-protein, binds to the 30S ribosomal subunit.

Era is an essential G-protein in Escherichia coli identified originally as a homologue protein to Ras (E. coli Ras-like protein). It binds to GTP/GDP and contains a low intrinsic GTPase activity. Its function remains elusive, although it has been suggested that Era is associated with the cytoplasmic membrane, cell division, energy metabolism, and cell-cycle check point. Recently, a cold-sensitive phenotype was found to be suppressed by the overexpression of 16S rRNA methyltransferase, suggesting Era association with the ribosome. Here we demonstrate that Era specifically binds to 16S rRNA and the 30S ribosomal subunit. Both GTP and GDP, but not GMP, inhibit Era binding to ribosomal component. Involvement of Era in protein synthesis is suggested by the fact that Era depletion results in the translation defect both in vitro and in vivo.

Bacterial Proteins↗

PubMed1999

Transcription of cspA, the gene for the major cold-shock protein of Escherichia coli, is negatively regulated at 37 degrees C by the 5'-untranslated region of its mRNA.

The gene for CspA, the major cold-shock protein in Escherichia coli, is tightly regulated at both optimal and low temperatures. While CspA is drastically induced after temperature downshift, it is hardly detectable at 37 degrees C. Here we demonstrate that the deletion of parts of the 5'-untranslated region (5'-UTR) of the cspA mRNA results in constitutive expression of CspA at 37 degrees C. By analyzing the amounts and the stabilities of the mRNAs produced from the deletion constructs, we rule out the possibility that the CspA production is due to the stabilization of the mutant mRNAs. We propose that significant premature termination or pausing occurs during the transcription of the unusually long 5'-UTR of the cspA mRNA at 37 degrees C, which represents a new mechanism that contributes to the tight repression of CspA production at higher temperature.

5' Untranslated Regions↗

PubMed1999

A pathway for conformational diversity in proteins mediated by intramolecular chaperones.

Conformational diversity within unique amino acid sequences is observed in diseases like scrapie and Alzheimer's disease. The molecular basis of such diversity is unknown. Similar phenomena occur in subtilisin, a serine protease homologous with eukaryotic pro-hormone convertases. The subtilisin propeptide functions as an intramolecular chaperone (IMC) that imparts steric information during folding but is not required for enzymatic activity. Point mutations within IMCs alter folding, resulting in structural conformers that specifically interact with their cognate IMCs in a process termed "protein memory." Here, we show a mechanism that mediates conformational diversity in subtilisin. During maturation, while the IMC is autocleaved and subsequently degraded by the active site of subtilisin, enzymatic properties of this site differ significantly before and after cleavage. Although subtilisin folded by Ile-48 --> Thr IMC (IMCI-48T) acquires an "altered" enzymatically active conformation (SubI-48T) significantly different from wild-type subtilisin (SubWT), both precursors undergo autocleavage at similar rates. IMC cleavage initiates conformational changes during which the IMC continues its chaperoning function subsequent to its cleavage from subtilisin. Structural imprinting resulting in conformational diversity originates during this reorganization stage and is a late folding event catalyzed by autocleavage of the IMC.

Amino Acid Sequence↗

PubMed1999

Formation of 9-hydroxy linoleic acid as a product of phospholipid peroxidation in diabetic erythrocyte membranes.

The increased production of oxygen-derived free radicals (OFR) and lipid peroxidation may contribute to vascular complications in diabetes. Some lipid peroxidation products have already been reported to be formed via glucose-induced oxidative stress. We have identified 9-hydroxy linoleic acid (9-OH-C18:2) in the red cell membrane phospholipid of diabetic subjects. We hypothesized that 9-OH-C18:2 would be formed in hydroxyl radical reactions to linoleic acid (C18:2) during glucose-induced oxidative stress, and confirmed that the formation of 9-OH-C18:2 was induced by ultraviolet (UV)-C irradiation to the synthetic C18:2. UV-C light generates highly reactive hydroxy radicals. C18:2 is confirmed to be the precursor of 9-OH-C18:2. To estimate the degree of oxidative damage to red cell membrane phospholipids, we developed a selective ion monitoring gas chromatography-mass spectrometric measurement for C18:2 and 9-OH-C18:2, following methanolysis of red cell membrane phospholipids. The relative peak height ratio of C18:2 to 9-OH-C18:2 (9-OH-C18:2/C18:2) was measured in phospholipid extracts of red cell membranes from healthy (n=29, 3.1+/-1.9%) and diabetic (n=27, 20. 9+/-16.1%) subjects. It was confirmed that 9-OH-C18:2/C18:2 is significantly (P<0.001) elevated in patients with diabetes. The measurement of 9-OH-C18:2/C18:2 in red cell membranes should be useful for assessing oxidative damage to membrane phospholipids in diabetes.

Biomarkers↗

PubMed1999

Different patterns of abnormal neuronal migration in the cerebral cortex of mice prenatally exposed to irradiation.

A characteristic abnormal cortical architecture in the adult brain was produced in mice subjected to 1.5 Gy of X-irradiation on embryonic day 14. Neurons in the lateral regions were organized into an essentially six-layered structure, while neurons in the dorsal regions formed a unique four-layered cortex. The patterns of neuronal migration in these different cortical regions were examined with immunohistochemistry for anti-bromodeoxyuridine (BrdU), anti-midkine (MK), and anti-glial fibrillary acidic protein (GFAP) antibodies. In the cortical lateral region, BrdU-labeled cells in the upper layers were fewer, and those in lower layers more numerous in prenatally irradiated mice than in control, while in the dorsal region (four-layered region), BrdU-labeled cells were very few in layer 2, and a large number of labeled-cells remained in layer 4. These results indicated that some neuroblasts in the lateral cortical region could not migrate to the upper layers, and that most neuroblasts in the dorsal cortical region failed to pass through the earlier migration zone. MK- and GFAP-stained radial glial fibers showed that the radial fibers were consistently oriented in the direction of neuronal migration in the control brains. However, in the irradiated brain, such radial fibers were crumpled in the lateral region, or were reduced markedly in number in some parts of the dorsal region. These results revealed that neuronal migratory pathways (radial glial fibers) were destroyed differently in different regions, and that X-rays killed some cells including radial glial cells or their precursors during the embryonic stage. These effects of radiation on the developing brain may result from the possibility that neurogenetic time is different or there are cellular mechanisms involved in the radiosensitivity among different regions.

Animals↗

PubMed1999

Translational enhancement by an element downstream of the initiation codon in Escherichia coli.

The translation initiation of Escherichia coli mRNAs is known to be facilitated by a cis element upstream of the initiation codon, called the Shine-Dalgarno (SD) sequence. This sequence complementary to the 3' end of 16 S rRNA enhances the formation of the translation initiation complex of the 30 S ribosomal subunit with mRNAs. It has been debated that a cis element called the downstream box downstream of the initiation codon, in addition to the SD sequence, facilitates formation of the translation initiation complex; however, conclusive evidence remains elusive. Here, we show evidence that the downstream box plays a major role in the enhancement of translation initiation in concert with SD.

Base Sequence↗

PubMed1999

Acetaminophen toxicity. Opposite effects of two forms of glutathione peroxidase.

Acetaminophen is one of the most extensively used analgesics/antipyretics worldwide, and overdose or idiopathic reaction causes major morbidity and mortality in its victims. Research into the mechanisms of toxicity and possible therapeutic intervention is therefore essential. In this study, the response of transgenic mice overexpressing human antioxidant enzymes to acute acetaminophen overdose was investigated. Animals overexpressing superoxide dismutase or plasma glutathione peroxidase demonstrated dramatic resistance to acetaminophen toxicity. Intravenous injection of glutathione peroxidase provided normal mice with nearly complete protection against a lethal dose of acetaminophen. Surprisingly, animals overexpressing intracellular glutathione peroxidase in the liver were significantly more sensitive to acetaminophen toxicity compared with nontransgenic littermates. This sensitivity appears to be due to the inability of these animals to efficiently recover glutathione depleted as a result of acetaminophen metabolism. Finally, the results suggest that glutathione peroxidase overexpression modulates the synthesis of several acetaminophen metabolites. Our results demonstrate the ability of glutathione peroxidase levels to influence the outcome of acetaminophen toxicity.

Acetaminophen↗

PubMed1999

Link between glycation and lipoxidation in red blood cells in diabetes.

Oxidative stress is postulated to be increased in patients with diabetes mellitus. Glycation enhanced by elevated glucose concentrations may induce the formation of oxygen-derived free radicals (OFRs). OFRs would cause oxidative damage to endogenous molecules, including cholesterol. Accumulating evidence suggests that oxidative cell injury caused by OFRs contributes to the development of both macroangiopathy and microangiopathy in diabetes. Our previous studies have shown that 7-keto cholestadien is one of the major products of cholesterol peroxidation in diabetic erythrocyte membrane and its levels correlate with hemoglobin Alc (HbAlc) values. We have newly identified 3-cholesten-6-one, one of the minor products of cholesterol peroxidation, in it. The aim of our study is to investigate whether 3-cholesten-6-one levels also correlate with HbAlc values. Levels of 3-cholesten-6-one were assessed in erythrocyte membrane lipid by monitoring peak areas of 3-cholesten-6-one to cholesterol with gas chromatography-mass spectrometry. The peak area ratio of 3-cholesten-6-one to cholesterol was used as a marker of cholesterol peroxidation. The HbAlc value, an index of both glycemic stress and glycation, was measured by high-performance liquid chromatography. In this study, we evaluated 33 diabetic and 29 healthy subjects, matched for age (59.3+/-14.5 vs. 57.3+/-13.7 years, mean+/-S.D.) and sex (15 males and 14 females vs. 16 males and 17 females). There were both significantly raised HbAlc levels (4.6+/-0.8 vs. 8.3+/-2.4%, P<0.001) and significantly increased ratios of 3-cholesten-6-one to cholesterol (0.2+/-0.4 vs. 21+/-1.8, P<0.001) in diabetic patients compared to control subjects. A good correlation between HbAlc levels and ratios of 3-cholesten-6-one to cholesterol was found in participants (r = 0.75, P<0.001, y = 0.46x-1.8). This suggests that an oxidative stress exists in diabetes and the link between glycation and lipoxidation is found in diabetic red blood cell.

Adult↗