Biomedical subjects

S Uesugi

Publications and source records attributed to S Uesugi.

At least 19 recordsLinked to original sources

PubMed2001

Structure of the C-terminal RNA-binding domain of hnRNP D0 (AUF1), its interactions with RNA and DNA, and change in backbone dynamics upon complex formation with DNA.

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP) -type RNA-binding domains (RBDs), each of which can specifically bind to the UUAG-sequence. hnRNP D0 also binds specifically to single-stranded d(TTAGGG)(n), the human telomeric DNA repeat. We have already reported the structure and interactions with RNA of the N-terminal RBD (RBD1). Here, the structure of the C-terminal RBD (RBD2) determined by NMR is presented. It folds into a compact alpha beta structure comprising an antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. In addition to the four beta-strands commonly found in RNP-type RBDs, an extra beta-strand, termed beta 4(-), was found just before the fourth beta-strand, yielding a five-stranded beta-sheet. Candidate residues of RBD2 involved in the interactions with RNA were identified by chemical shift perturbation analysis. Perturbation was detected on the beta-sheet side, not on the opposite alpha-helix side, as observed for RBD1. It is notable that the beta 4(-) to beta 4 region of RBD2 is involved in the interactions in contrast to the case of RBD1. The chemical shift perturbation analysis also showed that RBD2 interacts with DNA in essentially the same way as with RNA. Changes in the backbone dynamics upon complex formation with DNA were examined by means of model free analysis of relaxation data. In free RBD2, the beta 4(-) to beta 4 region exhibits slow conformational exchange on the milli- to microsecond time scale. The exchange is quenched upon complex formation. The flexibility of free RBD2 may be utilized in the recognition process by allowing different conformational states to be accessed and facilitating induced fit. Additionally, faster flexibility on the nano- to picosecond time scale was observed for loop 3 located between beta 2 and beta 3 in free RBD2, which is retained by the complex as well.

Amino Acid Sequence↗

PubMed2001

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.

Basic-Leucine Zipper Transcription Factors↗

PubMed2001

Functional magnetic resonance imaging-controlled neuronavigator-guided brain surgery: a case report.

The effectiveness of functional magnetic resonance imaging (f-MRI)-controlled and navigator-guided brain surgery for a patient with a recurrent astrocytoma is demonstrated. Preoperative f-MRI was performed in order to identify the motor area and ensure that the tumour was in the left prefrontal area. A more aggressive operation was planned for the recurrent tumour. The f-MRI data were input to the MKM navigation system and during the operation the contours of the tumour and motor area were visualised b y the microscope of the navigation system. The tumour and surrounding gliotic brain tissue were removed completely. The diagnosis was a grade III astrocytoma. The combination of the navigation system and f-MRI was useful for preoperative design of the surgical strategy, and tumour orientation during the operation, enabling aggressive surgery to be performed without functional deficits ensuing.

Adult↗

PubMed1999

Structure, backbone dynamics and interactions with RNA of the C-terminal RNA-binding domain of a mouse neural RNA-binding protein, Musashi1.

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in the regulation of asymmetric cell division. Musashi1 contains two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2. Our previous studies showed that RBD1 alone binds to RNA, while the binding of RBD2 is not detected under the same conditions. Joining of RBD2 to RBD1, however, increases the affinity to greater than that of RBD1 alone, indicating that RBD2 contributes to RNA-binding. We have determined the three-dimensional solution structure of the C-terminal RBD (RBD2) of Musashi1 by NMR. It folds into a compact alpha beta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. Special structural features of RBD2 include a beta-bulge in beta2 and a shallow twist of the beta-sheet. The smaller 1H-15N nuclear Overhauser enhancement values for the residues of loop 3 between beta2 and beta3 suggest that this loop is flexible in the time-scale of nano- to picosecond order. The smaller 15N T2 values for the residues around the border between alpha2 and the following loop (loop 5) suggest this region undergoes conformational exchange in the milli- to microsecond time-scale. Chemical shift perturbation analysis indicated that RBD2 binds to an RNA oligomer obtained by in vitro selection under the conditions for NMR measurements, and thus the nature of the weak RNA-binding of RBD2 was successfully characterized by NMR, which is otherwise difficult to assess. Mainly the residues of the surface composed of the four-stranded beta-sheet, loops and C-terminal region are involved in the interaction. The appearance of side-chain NH proton resonances of arginine residues of loop 3 and imino proton resonances of RNA bases upon complex formation suggests the formation of intermolecular hydrogen bonds. The structural arrangement of the rings of the conserved aromatic residues of beta2 and beta3 is suitable for stacking interaction with RNA bases, known to be one of the major protein-RNA interactions, but a survey of the perturbation data suggested that the stacking interaction is not ideally achieved in the complex, which may be related to the weaker RNA-binding of RBD2.

Amino Acid Sequence↗

PubMed1999

Structure and interactions with RNA of the N-terminal UUAG-specific RNA-binding domain of hnRNP D0.

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), each of which can bind solely to the UUAG sequence specifically. The structure of the N-terminal RBD (RBD1) determined by NMR is presented here. It folds into a compact alphabeta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of the RNP-type RBDs. Special structural features of RBD1 include N-capping boxes for both alpha-helices, a beta-bulge in the second beta-strand, and an additional short antiparallel beta-sheet coupled with a beta-turn-like structure in a loop. Two hydrogen bonds which restrict the positions of loops were identified. Backbone resonance assignments for RBD1 complexed with r(UUAGGG) revealed that the overall folding is maintained in the complex. The candidate residues involved in the interactions with RNA were identified by chemical shift perturbation analysis. They are located in the central and peripheral regions of the RNA-binding surface composed of the four-stranded beta-sheet, loops, and the C-terminal region. It is suggested that non-specific interactions with RNA are performed by the residues in the central region of the RNA-binding surface, while specific interactions are performed by those in the peripheral regions. It was also found that RBD1 has the ability to inhibit the formation of the quadruplex structure.

Amino Acid Sequence↗

PubMed1999

A'-form RNA double helix in the single crystal structure of r(UGAGCUUCGGCUC).

Here we demonstrate the presence of the A'-RNA conformation using the single crystal structure of a tridecamer: r(UGAGCUUCGGCUC). The average A'-RNA conformation deduced from X-ray fiber diffraction data had only been available previously, but now the presence of the A'-RNA conformation has been found in a single crystal structure for the first time. Statistical analysis showed that the A'-RNA conformation is distinguishable from the A-RNA conformation in a plot of the major groove width against the base pair inclination angle. The major groove of the A'-RNA conformation is wide enough to accommodate a protein or peptide while that of the A-RNA conformation is too narrow to do so. The presence of the A'-RNA conformation is significant for protein-RNA interaction.

Crystallization↗

PubMed1999

Temporal and spatial profile of apoptotic cell death in transient intracerebral mass lesion of the rat.

Apoptosis is involved in the pathogenesis of cerebral ischemia. Previous studies have confirmed that the brain surrounding an intracerebral hematoma develops ischemia. We investigated the number and distribution of cells exhibiting DNA fragmentation with apoptotic morphology in the transient intracerebral mass lesion to determine whether apoptosis contributed to the lesion progress after intracerebral hemorrhage (ICH). Transient intracerebral mass was created by inflation of a microballoon for 10 min (group A) or 2 h (group B) in the caudoputamen in rats, and brains were examined 1, 3, 6, 24, and 48 h after microballoon deflation. The lesion volume was calculated using parallel coronal sections with cresyl violet staining. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine (dUTP)-biotin nick end labeling (TUNEL) was used to detect cells undergoing DNA fragmentation. Immunohistochemistry for Fas antigen was also done to ascertain molecular mechanisms of apoptosis. Histological examination of hematoxylin and eosin-stained sections showed the typical appearance of neuronal necrosis in the caudoputaminal lesion. Lesion volume in the caudoputamen gradually increased as time advanced from 1 to 48 h. Cells stained heavily by TUNEL with apoptotic morphology were detected in the lesion, but not in the inner boundary zone of the lesion. The number of these cells significantly increased from 6 to 24 h in each experimental group (p < 0.05). The cells with positive immunoreactivity for Fas antigen was prominently observed in the lesion at 6 h. The distribution of apoptotic cells and the rapid increase in the number of apoptotic cells after 24 h propose that apoptotic cell death may contribute to lesion core formation but not to gradual development of the lesion.

Analysis of Variance↗

PubMed1999

Design and NMR analysis of HDV ribozymes for structural investigation.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems (Rz-1 to approximately Rz-3) (Fig. 1) were designed on the basis of the "pseudoknot" structure model and synthesized. Rz-1 is a cis-acting ribozyme system (a cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. The 2D-NOESY and 2D-HSQC data for Rz-1 suggest that Rz-1 forms the pseudoknot structure and G38 which is opposite to the cleavage site makes a base-pair. Rz-2 is a trans-acting ribozyme system which consists of three RNA oligomer strands (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-3 is a ribozyme in which the three RNA strands of Rz-2 are connected. It turns out that Rz-3 forms an inactive structure with low cleavage activity (k(obs) = 0.009) and final cleavage yield (6%). Rz-3 has the highest cleavage activity at pH 5.5. The optimal activity at acidic pH is similar to that of the wild type ribozyme. We also synthesized and examined the activity and structure of Rz-4 (designed by Perrotta and Been) which consists of two RNA strands (1).

Base Pairing↗

PubMed1999

Structural study of an RNA aptamer for a Tat protein complexed with ligands.

An RNA aptamer for an HIV Tat protein has been isolated by the in vitro SELEX method. The RNA aptamer binds to the Tat protein 50-100 times more strongly than native TAR RNA does. Here, we have investigated the structure of the RNA aptamer complexed with ligands, partial peptide fragments of the Tat protein or argininamide, by multidimensional 1H/13C/15N NMR. It is strongly suggested that two U:A:U base triples are formed in the RNA aptamer upon binding of ligands. Specific hydrogen bonds between arginine side chains of ligands and guanine bases located adjacent to the base triples are identified. On the basis of many intramolecular and intermolecular NOEs, a structural model of the complex has been constructed.

Base Sequence↗

PubMed1999

Seroepidemiologic studies of hepatitis C virus infection in a population of Okayama Prefecture screened for liver disease.

To better understand the spread of hepatitis C virus (HCV) infection, we studied the association of HCV infection with similarly transmissible hepatitis B virus (HBV) infection and with hepatitis A virus (HAV) infection, which is supposed to be related to a nosocomial transmission of HCV. This was done by studying the presence or absence of antibodies to these viruses, as well as hepatitis B surface antigen, in a population of 1,398 inhabitants with abnormal liver function tests or history of liver disease and/or blood transfusion. This group was drawn from a group of 7,905 examinees screened for liver disease in 26 districts of Okayama prefecture, Japan. The prevalence of antibody-positive cases increased with age for those viruses. Small but significantly increased odds ratios were obtained among anti-HCV antibodies (HCVAb), anti-hepatitis B core antibodies (HBcAb) and anti-hepatitis A antibodies (HAVAb). After adjusting odds ratios by logistic regression analysis, a significant association was present only between HCVAb and HBcAb. The distribution of age-adjusted prevalences (AAP) of HCVAb in 26 districts was significantly wider than those of HBcAb or HAVAb. The district-based AAP of HCVAb, but not of HBcAb and HAVAb, correlated significantly with the district-based prevalence of infectious hepatitis having a tendency of chronicity reported in 1953-1955. Adjusted odds ratios calculated by logistic regression analysis of the virus markers showed that HCVAb was significantly associated with a past history of blood transfusion. Thus, the spread of HCV infection is speculated to have been triggered by blood transfusion, particularly from paid donors initially, followed by transmission by nosocomial or close person-to-person contact.

Adult↗

PubMed1998

Two metal-binding sites in a lead ribozyme bound to competitively by Pb2+ and Mg2+--induced structural changes as revealed by NMR.

We reported recently that a lead ribozyme with modified bases cleaved at an additional site at high Pb2+ concentrations (>0.1 mM), and that the cleavage at a canonical site was enhanced nearly fourfold at the optimum combination of Pb2+ and Mg2+ concentrations [Kim, M. H., Katahira, M., Sugiyama, T. & Uesugi, S. (1997) J. Biochem. (Tokyo) 122, 1062-1067]. Here we have identified two metal-binding sites (sites 1 and 2) of the lead ribozyme at the residue level by NMR. Both sites are located in an asymmetric internal loop of the lead ribozyme. Site 1 is composed of residues of an enzyme strand and site 2 of residues of a substrate strand. The two sites are bound to competitively by Pb2+ and Mg2+. It was revealed that at certain Pb2+ and Mg2+ concentrations, site 1 is occupied by Pb2+ and site 2 is occupied by Mg2+. The dependency of the cleavage at the canonical and other sites on the Pb2+ and Mg2+ concentrations is interpreted by considering the species of metal ions bound to the two sites. It is suggested that the addition of the two metal ions produces similar and different effects on the structure of the lead ribozyme, and the two metal ions have a synergistic effect on the structure.

Binding Sites↗

PubMed1998

Apoptotic cell death induced by local brain hyperthermia in a rat glioma model.

Hyperthermia has been shown to inhibit glioma growth both in vitro and in vivo, and has been reported to induce apoptosis of a variety of cells. We investigated the role of apoptosis in tumor cell death following hyperthermia in a rat glioma model representing human glioblastoma. Apoptotic cell death was evaluated by terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and hematoxylin and eosin (H & E) staining. We also examined c-Jun expression immunohistochemically. Apoptotic cell death in rat brain tumors that grew after implantation of C6 glioma cells showed regional differences. In all rats, apoptotic cells, characterized by extreme chromatin condensation and fragmented nuclei with apoptotic bodies in H & E-stained sections, were observed in the gliomas' necrotic cores. TUNEL-positive cells were observed in the border zones between necrotic and vital tumor cells. Before hyperthermia, TUNEL-positive cells were sporadically distributed in the vital tumor tissue. After hyperthermia, the number of TUNEL-positive cells in the peripheral region of the tumor mass increased significantly, reached a peak after 6 h and returned to the basal level within 24 h (P < 0.01). C-Jun protein immunoreactivity was not observed in the cells at the tumor periphery. These data indicate that significantly apoptotic cell death unrelated to c-Jun expression occurs after hyperthermia, and that this form of cell death may be the mechanism of tumor regression following hyperthermia treatment of intracranial gliomas.

Animals↗

PubMed1998

Structural characterization of a DNA duplex modeled on a DNA:RNA hybrid of the polypurine tract recognized by a reverse transcriptase.

The structure of d(TTAAAAGAAAAGGG):d(CCCTTTTCTTTTAA) has been characterized by NMR. The minor grooves of the two dA-tracts are suggested to be rather narrow, and the portion linking the two dA tracts exhibits a slightly deviated structure from a standard B DNA, in order to maintain the narrowness of the minor groove. The structure of the dG-tract is also slightly deviated. Additionally, specific broadening of resonances is observed for the residues at or near the junction between the dA-tract and the dG-tract, suggesting local structural polymorphology.

Base Sequence↗

PubMed1998

Serum alpha-fetoprotein levels in healthy Japanese adults.

With advances in lectin affinity electrophoresis of alpha-fetoprotein (AFP), the detection of significant changes in serum AFP at low levels in cirrhotics has become important for early detection of hepatocellular carcinoma. Serum AFP levels of 616 healthy individuals without abnormal liver function tests or virus markers of hepatitis B and C were determined by enzyme immunoassay with IMx-AFP Dainapack using automated IMx apparatus set at twice the ordinary sensitivity and compared with those of 241 individuals with abnormal liver function tests and/or positive hepatitis virus markers. The coefficient of variation in this assay was less than 10% at AFP levels as low as 0.2 ng/ml with a lower detection limit of 0.1 ng/ml. The AFP level of healthy population showed a Gaussian distribution curve after logarithmic transformation with a median and 2.5-97.5 percentile reference range of 2.2 (0.6-5.6) ng/ml. There was no significant difference in the AFP level between males and females. Individuals with abnormal liver function tests alone showed no significant increase in serum AFP unless they were associated with positive hepatitis virus markers.

Adult↗

PubMed1998

[Spinal cord stimulation therapy at an early stage for unresponsive patients with hypoxic encephalopathy].

Recently, spinal cord stimulation (SCS) has been used for the treatment of patients in prolonged coma. However, the results of SCS in unresponsive patients with hypoxic encephalopathy at the chronic stage have not been satisfactory. Considering these circumstances, we began SCS from one month after the onset of hypoxic encephalopathy and evaluated its effect. Twelve patients (5 males and 7 females) with hypoxic encephalopathy, ranging in age from 7 to 72 years, were treated with SCS. The causes of hypoxia were acute cardiac failure in 4, automobile exhaust gas poisoning in 2, and asthma, pneumothorax, anaphylaxis, asphyxia, drowning and hypotension during aortic surgery in one patient each. One month after the onset, an electrode for electrical stimulation was implanted in the epidural space at the C2-C4 level under general anesthesia. The spinal cord was stimulated for 8 hours each day, starting on the day after implantation, and was continued for 3 months. Magnetic resonance imaging (MRI), cerebral blood flow (CBF) measurement using xenon-computed tomography (Xe-CT), and measurement of auditory evoked potential (AEP) and somatosensory evoked potential (SEP) were carried out 3 weeks after the onset for presurgical evaluation. Among the 12 patients, 7 (58%) showed clinical improvement, beginning within two weeks after starting stimulation. They were able to communicate with others and to express their emotion. However, disturbance of writing, picture drawing and calculation were not improved by stimulation. From presurgical evaluation, cases in which SCS therapy was effective had the following features: 1) No hemorrhagic infarction in the basal ganglia was demonstrable by MRI. 2) Mean hemispheric CBF measured by the Xe-CT method exceeded 25 ml/100 g per min. 3) The mean increase in hemispheric CBF 20 min after acetazolamide administration exceeded 5 ml/100 g per min. 4) An N20 peak was evident on the median nerve SEP, SCS appears to be an effective supplementary for unresponsive patients with hypoxic encephalopathy at the subacute stage, in addition to rehabilitation and drug therapy.

Adolescent↗

PubMed1997

Essential lysine residues in the N-terminal and the C-terminal domain of human adenylate kinase interact with adenine nucleotides as found by site-directed random mutagenesis.

To elucidate the minimum requirement of amino acid residues for the active center in human adenylate kinase (hAK1), we carried out random site-directed mutagenesis of key lysine residues (K9, K21, K27, K31, K63, K131, and K194), which were conserved in mammalian AK1 species, with the pMEX8-hAK1 plasmid [Ayabe, T., et al. (1996) Biochem. Mol. Biol. Int. 38, 373-381]. Twenty different mutants were obtained and analyzed by steady-state kinetics, and all mutants showed activity loss by Km and/or k(cat) effects on MgATP2-, AMP2-, or both. The results have led to the following conclusions. (1) Lys9 would appear to interact with both MgATP2- and AMP2- but to a larger extent than with AMP2-. (2) Lys21 is likely to play a role in substrate binding of both MgATP2- and AMP2- but more strongly affects MgATP2-. (3) Lys27 and Lys131 would appear to play a functional role in catalysis by interacting strongly with MgATP2-. (4) Lys31 would appear to interact with MgATP2- and AMP2- at the MgATP2- site. (5) Lys63 would be more likely to interact with MgATP2- than with AMP2-. (6) Lys194 in the flanking C-terminal domain would appear to interact not only with MgATP2- but also with AMP2- at the MgATP2- site by stabilizing substrate binding. The loss of the positively charged epsilon-amino group of lysine affects both the affinity for the substrate and the catalytic efficiency. Hence, hydrophilic lysine residues in hAK1 would appear to be essential for substrate-enzyme interaction with the coordination of some arginine residues, reported previously [Kim, H. J., et al. (1990) Biochemistry 29, 1107-1111].

Adenine Nucleotides↗

PubMed1997

Structural properties and RNA-binding activities of two RNA recognition motifs of a mouse neural RNA-binding protein, mouse-Musashi-1.

mouse-Musashi-1 (m-Msi-1) is an RNA-binding protein, abundantly expressed in the developing mammalian central nervous system (CNS). m-Msi-1 contains two RNA recognition motifs (RRMs). In this study, we found that the N-terminal RRM of m-Msi-1 (MMA) binds strongly to poly(G) and weakly to poly(U) in a way similar to that of the full-length m-Msi-1 protein characterized previously. The C-terminal RRM of m-Msi-1 (MMB), however, does not bind to RNA. In addition, the circular dichroism (CD) spectra of the two RRMs showed that the alpha-helical content of MMA is significantly higher than that of MMB, indicating that some differences in the secondary structure may be responsible for the distinct RNA binding properties of MMA and MMB.

Amino Acid Sequence↗

PubMed1997

Substrate-binding and catalytic roles of Lys194 in the C-terminus in human adenylate kinase by site-directed random mutagenesis.

Site-directed random mutagenesis of Lys194 residue in the C-terminus of human adenylate kinase (AK) was performed, and six mutants were analyzed by steady-state kinetics. K194-mutants variously affected the apparent Michaelis constants (K(m) values) for ATP and AMP, although the kcat values strikingly decreased. The Lys194 residue appears to interact not only with MgATP2- but also with the AMP2- substrates by salt bridge formation with a nucleotide and to play a functional role in stabilizing the phosphate-transfer during catalysis. Lys194 could be essential for substrate-holdings and in catalysis and not replaceable to the other amino acids.

Adenylate Kinase↗