Biomedical subjects

S Aono

Publications and source records attributed to S Aono.

At least 19 recordsLinked to original sources

PubMed2002

Transient expression of juvenile-type neurocan by reactive astrocytes in adult rat brains injured by kainate-induced seizures as well as surgical incision.

Neurocan is one of the major chondroitin sulfate proteoglycans expressed in nervous tissues. The expression of neurocan is developmentally regulated, and full-length neurocan is detected in juvenile brains but not in adult brains. In the present study, we demonstrated by western blot analysis that full-length neurocan transiently appeared in adult rat hippocampus when it was lesioned by kainate-induced seizures. Immunohistochemical studies showed that neurocan was detected mainly around the CA1 region although the seizure resulted in neuronal cell degeneration in both the CA1 and CA3 regions of the hippocampus. Double-labeling for neurocan mRNA and glial fibrillary acidic protein demonstrated that many reactive astrocytes expressed neurocan mRNA. The re-expression of full-length neurocan was also observed in the surgically injured adult rat brain. In contrast, the expression of other nervous tissue chondroitin sulfate proteoglycans, such as phosphacan and neuroglycan C, was not intensified but rather was either reduced in the kainate-lesioned hippocampus or in the surgically injured cerebral cortex. These observations indicate that induction of neurocan expression by reactive astrocytes is a common phenomenon in the repair process of adult brain injury, and therefore, it can be postulated that juvenile-type neurocan plays some roles in brain repair.

Animals↗

PubMed2001

Conformational dynamics of the transcriptional regulator CooA protein studied by subpicosecond mid-infrared vibrational spectroscopy.

CooA, which is a transcriptional regulator heme protein allosterically triggered by CO, is studied by femtosecond visible-pump mid-IR-probe spectroscopy. Transient bleaching upon excitation of the heme in the Soret band is detected at approximately 1979 cm(-1), which is the absorption region of the CO bound to the heme. The bleach signal shows a nonexponential decay with time constants of 56 and 290 ps, caused by the rebinding of the CO to the heme. About 98% of dissociated CO recombines geminately. The geminate recombination rate in CooA is significantly faster than those in myoglobin and hemoglobin. The angle of the bound CO with respect to the porphyrin plane is calculated to be about 78 degrees on the basis of the anisotropy measurements. A shift of the bleached mid-IR spectrum of the bound CO is detected and has a characteristic time of 160 ps. It is suggested that the spectral shift is caused by a difference in the frequency of the bound CO in different protein conformations, particularly in an active conformation and in an intermediate one, which is on the way toward an inactive conformation. Thus, the biologically relevant conformation change in CooA was traced. Possible assignment of the observed conformation change is discussed.

Bacterial Proteins↗

PubMed2001

Ligand-switching intermediates for the CO-sensing transcriptional activator CooA measured by pulse radiolysis.

CooA is a heme-containing and CO-sensing transcriptional activator whose activity is regulated by CO. The protoheme that acts as a CO sensor in CooA shows unique properties for its coordination structure. The Cys75 axial ligand of the ferric heme is replaced by His77 upon the reduction of the heme iron and vice versa. In this work, the ligand-switching process induced by the reduction of the heme was investigated by the technique of pulse radiolysis. Hydrated electron reduced the heme iron in ferric CooA within 1 micros to form the first intermediate with the Soret peak at 440 nm, suggesting that a six-coordinate ferrous heme with a thiolate axial ligand was formed initially. The first intermediate was converted into the second intermediate with the time constant of 40 micros (k = 2.5 x 10(4) x s(-1)). In the second intermediate, the thiolate from Cys75 was thought to be protonated and/or the Fe-S bond was thought to be elongated. The second intermediate was converted into the final reduced form with the time constant of 2.9 ms (k = 3.5 x 10(2) x s(-1)) for wild-type CooA. The ligand exchange between Cys75 and His77 took place during the conversion of the second intermediate into the final reduced form.

Bacterial Proteins↗

PubMed2001

Binding of CO at the Pro2 side is crucial for the activation of CO-sensing transcriptional activator CooA. (1)H NMR spectroscopic studies.

CooA is a heme-containing transcriptional activator that anaerobically binds to DNA at CO atmosphere. To obtain information on the conformational transition of CooA induced by CO binding to the heme, we assigned ring current-shifted (1)H NMR signals of CooA using two mutants whose axial ligands of the heme were replaced. In the absence of CO, the NMR spectral pattern of H77Y CooA, in which the axial histidine (His(77)) was replaced with tyrosine, was similar to that of wild-type CooA. In contrast, the spectra of CooADeltaN5, in which the NH(2) termini including the other axial ligand (Pro(2)) were deleted, were drastically modulated. We assigned three signals of wild-type CooA at -4.5, -3.6, and -2.8 ppm to delta(1)-, alpha-, and delta(2)-protons of Pro(2), respectively. The Pro(2) signals were undetectable in the upfield region of the spectrum of the CO-bound state, which confirms that CO displaces Pro(2). Interestingly, the Pro(2) signals were observed for CO-bound H77Y CooA, implying that CO binds to the trans position of Pro(2) in H77Y CooA. The abolished CO-dependent transcriptional activity of H77Y CooA is therefore the consequence of Pro(2) ligation. These observations are consistent with the view that the movement of the NH(2) terminus triggers the conformational transition to the DNA binding form.

Bacterial Proteins↗

PubMed2001

Regulation of neuregulin expression in the injured rat brain and cultured astrocytes.

In this report, we investigated whether reactive astrocytes produce neuregulins (glial growth factor 2/heregulin/acetylcholine receptor-inducing activity or neu differentiation factor) and its putative receptors, ErbB2 and ErbB3 tyrosine kinases, in the injured CNS in vivo. Significant immunoreactivities with anti-neuregulin, anti-ErbB2, and anti-ErbB3 antibodies were detected on astrocytes at the injured site 4 d after injury to the adult rat cerebral cortex. To elucidate the mechanisms for the upregulation of neuregulin expression in astrocytes, primary cultured astrocytes were treated with certain reagents, including forskolin, that are known to elevate the intracellular level of cAMP and induce marked morphological changes in astrocytes. Western blot analysis showed that the expression of a 52 kDa membrane-spanning form of a neuregulin protein was enhanced in cultured astrocytes after administration of forskolin. The upregulation of glial fibrillary acidic protein was also observed in astrocytes treated with forskolin. In contrast, inactivation of protein kinase C because of chronic treatment with phorbol ester 12-O-tetradecanoyl phorbol 13-acetate downregulated the expression of the 52 kDa isoform, although other splice variants with apparent molecular sizes of 65 and 60 kDa were upregulated. These results suggest that the enhancement of neuregulin expression at injured sites is induced, at least in part, by elevation in intracellular cAMP levels and/or a protein kinase C signaling pathway. The neuregulin expressed on reactive astrocytes may stimulate their proliferation and support the survival of neurons surrounding cortical brain wounds in vivo.

Animals↗

PubMed2000

Dissociation and recombination between ligands and heme in a CO-sensing transcriptional activator CooA. A flash photolysis study.

CooA from Rhodospirillum rubrum is a transcriptional activator in which a heme prosthetic group acts as a CO sensor and regulates the activity of the protein. In this study, the electronic relaxation of the heme, and the concurrent recombination between ligands and the heme at approximately 280 K were examined in an effort to understand the environment around the heme and the dynamics of the ligands. Upon photoexcitation of the reduced CooA at 400 nm, electronic relaxation of the heme occurred with time constants of 0.8 and 1.7 ps. The ligand rebinding was substantially completed with a time constant of 6.5 ps, followed by a slow relaxation process with a time constant of 173 ps. In the case of CO-bound CooA, relaxation of the excited heme occurred with two time constants, 1.1 and 2.4 ps, which were largely similar to those with reduced CooA. The subsequent CO recombination process was remarkably fast compared with that of other CO-bound heme proteins. It was well described as a biphasic geminate recombination process with time constants of 78 ps (60%) and 386 ps (30%). About 10% of the excited heme remained unligated at 1.9 ns. The dynamics of rebinding of CO thus will help us to understand how the physiologically relevant diatomic molecule approaches the heme binding site in CooA with picosecond resolution.

Bacterial Proteins↗

PubMed2000

Redox properties and coordination structure of the heme in the co-sensing transcriptional activator CooA.

The CO-sensing transcriptional activator CooA contains a six-coordinate protoheme as a CO sensor. Cys(75) and His(77) are assigned to the fifth ligand of the ferric and ferrous hemes, respectively. In this study, we carried out alanine-scanning mutagenesis and EXAFS analyses to determine the coordination structure of the heme in CooA. Pro(2) is thought to be the sixth ligand of the ferric and ferrous hemes in CooA, which is consistent with the crystal structure of ferrous CooA (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876-880). CooA exhibited anomalous redox chemistry, i.e. hysteresis was observed in electrochemical redox titrations in which the observed reduction and oxidation midpoint potentials were -320 mV and -260 mV, respectively. The redox-controlled ligand exchange of the heme between Cys(75) and His(77) is thought to cause the difference between the reduction and oxidation midpoint potentials.

Alanine↗

PubMed2000

Identification of histidine 77 as the axial heme ligand of carbonmonoxy CooA by picosecond time-resolved resonance Raman spectroscopy.

The heme proximal ligand of carbonmonoxy CooA, a CO-sensing transcriptional activator, in the CO-bound form was identified to be His77 by using picosecond time-resolved resonance Raman spectroscopy. On the basis of the inverse correlation between Fe-CO and C-O stretching frequencies, we proposed previously that His77 is the axial ligand trans to CO [Uchida et al. (1998) J. Biol. Chem. 273, 19988-19992], whereas later a possibility of displacement of His77 by CO with retention of another unidentified axial ligand was reported [Vogel et al. (1999) Biochemistry 38, 2679-2687]. Although our previous resonance Raman study failed to detect the Fe-His stretching [nu(Fe-His)] mode of CO-photodissociated CooA of the carbonmonoxy adduct due to the rapid recombination, application of the picosecond time-resolved resonance Raman technique enabled us to observe a new intense line assignable to nu(Fe-His) at 211 cm(-)(1) immediately after photolysis, while it became nondiscernible after 100-ps delay. The low nu(Fe-His) frequency of photodissociated CooA indicates the presence of some strain in the Fe-His bond in CO-bound CooA. This and the rapid recombination of CO characterize the heme pocket of CooA. The 211 cm(-)(1) band was completely absent in the spectrum of the CO-photodissociated form of the His77-substituted mutant but the Fe-Im stretching band was observed in the presence of exogenous imidazole (Im). Thus, we conclude that His77 is the axial ligand of CO-bound CooA and CO displaces the axial ligand trans to His77 with retention of ligated His77 to activate CooA as the transcriptional activator.

Bacterial Proteins↗

PubMed2000

Patterning of cell assemblies regulated by adhesion receptors of the cadherin superfamily.

During morphogenesis, cell-cell association patterns are dynamically altered. We are interested in how cell adhesion molecules can regulate the patterning of cellular assemblies. Cadherins, a group of cell-cell adhesion receptors, are crucial for the organized assembly of many cell types, but they also regulate dynamic aspects of cell association. For example, during neural crest emigration from the neural tube, the cadherin subtypes expressed by crest cells are switched from one subtype to another. Artificial perturbation of this switch results in blocking of their escape from the neural tube. Intracellular modulations of cadherin activity also seem to play a role in regulation of cell adhesion. We identified p120ctn as a regulator of cadherin function in carcinoma cells. With such regulators, cells may make a choice as to whether they should maintain stable cell contacts or disrupt their association. Finally, we found another type of cadherin-mediated cell patterning: Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity in Drosophila imaginal discs. Thus, the cadherin superfamily receptors control the patterning of cell assemblies through a variety of mechanisms.

Animals↗

PubMed2000

Molecular interactions of neural chondroitin sulfate proteoglycans in the brain development.

Aggrecan family proteoglycans, phosphacan/RPTPzeta/beta, and neuroglycan C (NGC) are the major classes of chondroitin sulfate proteoglycan in the developing mammalian brain. A multidomain is a common structural feature of these proteoglycans which can interact with various molecules including growth factors, cell adhesion molecules, and extracellular matrix molecules. Individual proteoglycans are distributed in the developing brain in a distinct temporal and spatial pattern, suggesting that they are involved in distinct phases of the brain development through multiple molecular interactions. This review mainly summarizes recent studies on the involvement of these three classes of proteoglycan in cell-cell and cell-substratum interactions during the brain development. Their expressions and proposed functional roles in injured brains are also mentioned. In addition, this review briefly covers potential functions of other neural chondroitin sulfate proteoglycans such as decorin, testican, NG2 proteoglycan, and amyloid precursor protein (APP) in developing and injured brains.

Animals↗

PubMed2000

Control of CooA activity by the mutation at the C-terminal end of the heme-binding domain.

A constitutively active mutant of a CooA, in which Met131 was replaced by Leu, was isolated by random mutagenesis. Site-directed mutagenesis at position 131 revealed that M131R-CooA was also constitutively active even in the absence of CO and that M131P-, M131D-, and M131E-CooA were constitutively inactive regardless of the presence or absence of CO. While M131L- and M131E-CooA showed almost the same electronic absorption spectra as those of the wild type in the ferric, ferrous, and CO-bound forms, M131D-CooA showed the typical spectrum of a five-coordinate heme protein in the ferric form. The conformational change around the heme induced by CO binding, which triggers the activation of CooA, is thought to be linked to the rearrangement of the conformation around the hinge region between the heme-binding and DNA-binding domains and/or of the relative orientation of the two domains to activate CooA.

Amino Acid Sequence↗

PubMed2000

Genomic organization and expression pattern of mouse neuroglycan C in the cerebellar development.

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate proteoglycan with an epidermal growth factor module that is expressed predominantly in the brain. Cloning studies with mouse NGC cDNA revealed the expression of three distinct isoforms (NGC-I, -II, and -III) in the brain and revealed that the major isoform showed 94. 3% homology with the rat counterpart. The NGC gene comprised six exons, was approximately 17 kilobases in size, and was assigned to mouse chromosome band 9F1 by fluorescence in situ hybridization. Western blot analysis demonstrated that, although NGC in the immature cerebellum existed in a proteoglycan form, most NGC in the mature cerebellum did not bear chondroitin sulfate chain(s), indicating that NGC is a typical part-time proteoglycan. Immunohistochemical studies showed that only the Purkinje cells were immunopositive in the cerebellum. In the immature Purkinje cells, NGC, probably the proteoglycan form, was immunolocalized to the soma and thick dendrites on which the climbing fibers formed synapses, not to the thin branches on which the parallel fibers formed synapses. This finding suggests the involvement of NGC in the differential adhesion and synaptogenesis of the climbing and parallel fibers with the Purkinje cell dendrites.

Alternative Splicing↗

PubMed2000

CO sensing and regulation of gene expression by the transcriptional activator CooA.

The transcriptional activator CooA from Rhodospirillum rubrum contains a six-coordinate protoheme that acts as a CO sensor in vivo. CO is a physiological effector of CooA and replaces one of the axial ligands of the ferrous heme to form the CO-bound CooA that is active as the transcriptional activator. Cys75 or His77 is coordinated to the ferric and ferrous hemes in CooA, respectively. The redox-controlled ligand exchange between Cys75 and His77 proceeds during the change in the redox state of the heme. The reduction and oxidation midpoint potentials of CooA have been determined to be -320 and -260 mV, respectively. The properties of a functional chimera derived from CRP and CooA suggest that CooA activates the transcription by a similar mechanism to that for CRP at Class II CRP-dependent promoters. Alanine-scanning mutagenesis has revealed that Arg24 and Arg53 of CooA, which will be concerned with the protein-protein interaction with RNA polymerase, are critical amino acid residues for the transcriptional activator activity of CooA, and that Lys26 and Asp94 modulate the activity of CooA.

Bacterial Proteins↗

PubMed2000

Marriage rate and number of children among young adults with insulin-dependent diabetes mellitus in Japan.

The main purpose of our study was to identify the social circumstances and lifestyle of IDDM patients in Japan. The present study focused on the marriage status of both men and women with IDDM as well as the number of children of women with IDDM. A questionnaire was sent to hospitals across the country. Doctors handed it or mailed it to IDDM patients aged 18 years or older. Unsigned answer sheets were returned directly by the patients. Data on the marriage rate and number of children were obtained, and possible factors affecting these indices were assessed. One thousand and thirteen patients (354 men and 659 women) answered the questionnaire. Both men and women with IDDM were less likely to be married in comparison with age-matched Japanese. The number of children of married IDDM women in various age groups was also lower in comparison with the general Japanese female population. Several factors other than diabetes complications including job discrimination, high medical costs, and psychological pressures, were thought to be responsible for these results.

Adult↗

PubMed2000

Antibodies against hepatitis C virus-like particles and viral clearance in acute and chronic hepatitis C.

We recently described the efficient assembly of hepatitis C virus (HCV) structural proteins into HCV-like particles (HCV-LPs) in insect cells. These noninfectious HCV-LPs have similar morphologic and biophysical properties as putative virions isolated from HCV-infected humans and can induce a broadly directed immune response in animal models. The HCV envelope proteins of HCV-LPs are presumably presented in a native, virion-like conformation and may therefore interact with antienvelope antibodies directed against conformational epitopes. In this study, HCV-LPs were used as capture antigens in an enzyme-linked immunosorbent assay (ELISA) to detect and quantify antibodies against HCV structural proteins in patients with acute and chronic hepatitis C. High titers of anti-HCV-LP antibodies were detected in patients chronically infected with HCV genotypes 1 to 6. In contrast to individuals with chronic hepatitis C, patients with acute self-limited hepatitis C displayed only a transient and weak seroreactivity against HCV-LPs. Patients with chronic HCV infection successfully treated with interferon demonstrated a gradual decline of anti-HCV-LP titers during or subsequent to viral clearance. Sustained interferon responders were characterized by significantly higher pretreatment levels of anti-HCV-LP antibodies as compared with nonresponders (P =.0001). In conclusion, HCV infection is associated with limited humoral immunity against the envelope proteins present on the HCV-LPs. An HCV-LP-based ELISA may be a useful diagnostic tool to distinguish acute hepatitis C from chronic HCV infection with exacerbation, and to predict viral clearance in response to interferon.

Acute Disease↗

PubMed2000

The gene encoding a novel halorhodopsin-like protein of extremely halophilic archaeon Haloarcula japonica strain TR-1.

The gene encoding the halorhodopsin (hR)-like protein was cloned and sequenced from Haloarcula japonica strain TR-1. The structural gene consisted of an open reading frame of 828 nucleotides encoding 276 amino acids. The deduced amino acid sequence of the Ha. japonica hR-like protein showed the highest homology to those of cruxhalorhodopsins. Furthermore, we have demonstrated that the Ha. japonica chR gene is transcriptionally induced by light.

Amino Acid Sequence↗

PubMed2000

Comparative mapping of seven genes in mouse, rat and Chinese hamster chromosomes by fluorescence in situ hybridization.

By fluorescence in situ hybridization (FISH) using mouse probes, we assigned homologues for cathepsin E (Ctse), protocadherin 10 (Pcdh10, alias OL-protocadherin, Ol-pc), protocadherin 13 (Pcdh13, alias protocadherin 2c, Pcdh2c), neuroglycan C (Cspg5) and myosin X (Myo10) genes to rat chromosomes (RNO) 13q13, 2q24-->q25, 18p12-->p11, 8q32.1 and 2q22.1-->q22.3, respectively. Similarly, homologues for mouse Ctse, Pcdh13, Cspg5 and Myo10 genes and homologues for rat Smad2 (Madh2) and Smad4 (Madh4) genes were assigned to Chinese hamster chromosomes (CGR) 5q28, 2q17, 4q26, 2p29-->p27, 2q112-->q113 and 2q112-->q113, respectively. The chromosome assignments of homologues of Ctse and Cspg5 reinforced well-known homologous relationships among mouse chromosome (MMU) 1, RNO 13 and CGR 5q, and among MMU 9, RNO 8 and CGR 4q, respectively. The chromosome locations of homologues for Madh2, Madh4 and Pcdh13 genes suggested that inversion events were involved in chromosomal rearrangements in the differentiation of MMU 18 and RNO 18, whereas most of MMU 18 is conserved as a continuous segment in CGR 2q. Furthermore, the mapping result of Myo10 and homologues suggested an orthologous segment of MMU 15, RNO 2 and CGR 2.

Animals↗

PubMed1999

Recognition of target DNA and transcription activation by the CO-sensing transcriptional activator CooA.

CooA from Rhodospirillum rubrum is a heme-based CO-sensing transcriptional activator, in which CO acts as a physiological effector. In this study, we examined the mechanism of site-specific recognition and transcriptional activation by CooA by elucidating the transcriptional activator activity of the mutant CooA proteins and the chimeric proteins derived from CRP and CooA and the promoter activity of the mutant promoters. Site-directed mutagenesis has revealed that Arg(177), Gln(178), and Ser(181) on the recognition helix of the helix-turn-helix motif in CooA are responsible for the site-specific recognition. The side chains of these amino acid residues at positions 177, 178, and 181 are believed to be hydrogen bonding to the G:A, T:A, and C:G pairs at positions 2/15, 3/14, and 4/13 in the CooA-dependent promoters to recognize the DNA site for CooA. The properties of the CRP/CooA chimeric proteins constructed in this work suggest that CooA activates transcription by a similar mechanism to that of CRP at Class II CRP-dependent promoters.

Amino Acid Sequence↗