[Molecular epidemiology of rotaviruses--a perspective from the study of the genetic diversity of rotaviruses].
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Genes, Viral↗
Biomedical subjects
S Uesugi
Publications and source records attributed to S Uesugi.
[Quantitative assays for C-reactive protein: a review].
C-reactive protein (CRP) is a single most important test among various serological tests which are routinely performed in clinical laboratories. While qualitative assays for CRP are being replaced by quantitative assays, standardization among various laboratories has become an urgent issue. The results of the past three-year surveys conducted by the Japanese Medical Association showed that the coefficients of variation (CV) for CRP assays ranged between 12.6% and 46.9% (1990), although these figures gradually changed for the better. These results clearly indicate that a CRP value obtained in one laboratory cannot be compared directory with that obtained in another laboratory. Every assay for CRP is considered to show good reproducibility, however, which is predicted by the results of within-run precision tests (CV:3.9-7.8%). It is very important to establish standardization for CRP assays but one has to conclude that the path toward this goal is very difficult when one takes into consideration various factors inherent to immunological reactions of macromolecules including possible microheterogeneity of CRP molecules.
C-Reactive Protein↗
NMR studies for identification of dI:dG mismatch base-pairing structure in DNA.
One- and two-dimensional NMR experiments have been undertaken to investigate deoxyinosine:deoxyguanosine (dI:dG) base pairing in a self-complementary dodecadeoxyribonucleotide, d(C1-G2-C3-I4-A5-A6-T7-T8-G9-G10-G11-G12) (designated IG-12), duplex. The NMR data indicate formation of a dI(syn):dG(anti) base pair in a B-DNA helix. This unusual base pairing results in altered NOE patterns between the base protons (H8 and H2) of the I4 residue and the sugar protons of its own and the 5'-flanking C3 residues. The dI(syn):dG(anti) base pair is accommodated in the B-DNA duplex with only a subtle distortion of the local conformation. Identification of the dI:dG base pairing in this study confirms that a hypoxanthine base can form hydrogen-bonded base pairs with all of the four normal bases, C, A, T, and G, in DNA.
Base Composition↗
Non-cognizable ribonucleotide, 2'AMP, binds to a mutant ribonuclease T1 (Y45W) at a new base-binding site but not at the guanine-recognition site.
Complex of a mutant ribonuclease T1 (Y45W) with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined by X-ray diffraction at 1.7 A resolution. The 2'AMP molecule locates at a new base-binding site which is remote from the guanine-recognition site, where 2'GMP was found to be bound. The nucleotide adopts the anti conformation of the glycosidic bond and C3'-exo sugar pucker. There exists a single hydrogen bond between the adenine base and the enzyme, and, therefore, the site found is apparently a non-specific binding site. The results indicate that the binding of 2'AMP to the guanine-recognition site is weaker than that to the new binding site.
Adenosine Monophosphate↗
Plasma endothelin-1 levels in patients with disseminated intravascular coagulation.
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Adult↗
NMR studies of a DNA containing 8-hydroxydeoxyguanosine.
The effects of hydroxylation at the C8 of a deoxyguanosine residue in DNA were studied by NMR analysis of a self-complementary dodecanucleotide, d(C1-G2-C3-oh8G4-A5-A6-T7-T8-C9-G10-C11-G12), which has an 8-hydroxy-2'-deoxyguanosine (oh8dG) residue at the 4th position. NMR data indicate that the 8-hydroxyguanine (oh8G) base takes a 6,8-diketo tautomeric form and is base-paired to C with Watson-Crick type hydrogen bonds in a B-form structure. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(CGCGAATTCGCG) duplex. The structural changes caused by 8-hydroxylation of the deoxyguanosine, if any, are localized near the modification site.
8-Hydroxy-2'-Deoxyguanosine↗
Combination of Trp and Glu residues for recognition of mRNA cap structure. Analysis of m7G base recognition site of human cap binding protein (IF-4E) by site-directed mutagenesis.
Four mutants of the human cap binding protein (hCBP), in which Trp-102, Glu-103, Asp-104 or Glu-105 was changed to the aliphatic Leu or Ala, were prepared, and their cap binding abilities were examined. Cap binding abilities of two mutants, W102L (Trp-102----Leu) and E105A (Glu-105----Ala), were significantly decreased in comparison with the wild-type hCBP. This result suggests that Trp-102 and Glu-105 are both necessary for the cap binding, and the most probable binding mode with the m7G of cap structure is the combination of the stacking by Trp-102 and the hydrogen-bond pairing by Glu-105, as was already proposed from the model studies.
Amino Acid Sequence↗
Expression of a synthetic gene for human cap binding protein (human IF-4E) in Escherichia coli and fluorescence studies on interaction with mRNA cap structure analogues.
An artificial gene coding for the human cap binding protein (hCBP: human IF-4E) was chemically synthesized and expressed in Escherichia coli under the control of a trp promoter. The DNA duplex of 662 bp was designed and constructed from 44 oligodeoxynucleotide fragments of typically 30 nucleotides in length. Although the hCBP gene was not directly expressed in E. coli HB101, we succeeded in its high-level expression as a fusion protein connected with a portion of human growth hormone through a tetradecapeptide (Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) that contains the recognition sequence for a site-specific protease alpha-thrombin. Upon induction with 3-indoleacrylic acid, the fusion protein accumulated with a yield of about 20% of the total proteins of the host cell. Upon the treatment of the fusion protein with alpha-thrombin, which recognizes the sequence "Val-Pro-Arg," specific proteolysis at the fused junction occurred efficiently. In this system, nonspecific digestion by alpha-thrombin was not marked. About 15 mg of recombinant hCBP was obtained from a 1-liter culture. Association constants between the recombinant hCBP and mRNA cap structure analogues were determined by fluorescence spectroscopy. The values obtained for the m7GpppA, m7GTP, and m7GMP were almost the same as those reported for the IF-4E isolated from human erythrocyte cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Conformation of 2'GMP bound to a mutant ribonuclease T1 (Y45W) determined by X-ray diffraction and NMR methods.
The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a specific inhibitor, 2'GMP, has been determined by X-ray diffraction and refined at 1.9 A resolution to a conventional R-factor of 0.164. The mode of recognition of the guanine base by the enzyme is similar to that found for the wild-type ribonuclease T1 complexed with 2'GMP. The binding of the guanine base is clearly enhanced by maximum overlapping of the indole ring of Trp45 and the base. The glycosyl torsion angle of the inhibitor is in the syn conformation and the sugar exhibits a C3'-endo type pucker, which differs from that observed in the crystal of the complex between the wild-type ribonuclease T1 and 2'GMP. Analysis of 500-MHZ NMR spectra has also indicated that the 2'GMP molecule as bound to the mutant enzyme in solution exhibits a C3'-endo type pucker, similar to that bound to the wild-type enzyme in solution [Inagaki, Shimada, & Miyazawa (1985) Biochemistry 24, 1013-1020].
Carbohydrate Conformation↗
Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease T1.
Hydrophobic effects on binding of ribonuclease T1 to guanine bases of several ribonucleotides have been proved by mutating a hydrophobic residue at the recognition site and by measuring the effect on binding. Mutation of a hydrophobic surface residue to a more hydrophobic residue (Tyr45----Trp) enhances the binding to ribonucleotides, including mononucleotide inhibitor and product, and a synthetic substrate-analog trinucleotide as well as the binding to dinucleotide substrates and RNA. Enhancements on binding to non-substrate ribonucleotides by the mutation have been observed with free energy changes ranging from -2.2 to -3.9 kJ/mol. These changes are in good agreement with that of substrate binding, -2.3 kJ/mol, which is calculated from Michaelis constants obtained from kinetic studies. It is shown, by comparing the observed and calculated changes in binding free energy with differences in the observed transfer free energy changes of the amino acid side chains from organic solvents to water, that the enhancement observed on guanine binding comes from the difference in the hydrophobic effects of the side chains of tyrosine and tryptophan. Furthermore, a linear relationship between nucleolytic activities and hydrophobicity of the residues (Ala, Phe, Tyr, Trp) at position 45 is observed. The mutation could not change substantially the base specificity of RNase T1, which exhibits a prime requirement for guanine bases of substrates.
Binding Sites↗
10-year follow-up study of epilepsy with psychosis.
Thirty-three cases of psychosis associated with epilepsy were followed in our clinic for 10 years. In 12 cases, the psychotic symptom appeared episodically and 9 of them had more than one relapse and 7 of them more than 2 relapses. Only 3 cases had no relapse. In 21 cases, the psychotic symptoms were continuous, lasting more than 2 years. In 8 of them, the symptoms were stable and remained unchanged but in 13 of them, the symptom showed exacerbation and partial remissions. In 11 cases, a decrease or discontinuation of antipsychotic drugs (mostly haloperidol) significantly provoked or aggravated the psychotic symptoms.
Adult↗
[Microbiological characterization of clinical isolates of Staphylococcus epidermidis].
As a result of recent development in medical practice including use of new antimicrobial agents, coagulase-negative Staphylococci (CNS) that were once considered nonpathogenic contaminants have captured attention as causes of disease. The 43 clinical isolates of Staphylococcus epidermidis sensu stricto and 7 isolates of S. epidermidis from the medical staffs were characterized with regard to (1) their biochemical profiles, (2) slime productivity, (3) beta-lactamase productivity and (4) plasmid patterns. Most of the isolates have an identical biochemical profile code. The slime production was observed in the 9 strains isolated from pleural effusions or the tubes used for pleural drainage. These strains had a similar antimicrobial susceptibility profile that are commonly seen in multi-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA). In addition, most of these strains produced beta-lactamase. For the plasmid profiling, we selected 12 isolates that possessed 5 distinct antimicrobial susceptibility profiles. Upon agarose gel electrophoresis, 8 isolates were shown to possess a 1.9 kb plasmid. These 8 isolates had resistance against tobramycin (TOB) and erythromycin (EM). Emergence of S. epidermidis as causes of disease will increasingly necessitate the detailed microbiological characterization of the clinical isolates.
DNA, Bacterial↗
Activity of artificial mutant variants of human growth hormone deficient in a disulfide bond between Cys53 and Cys165.
In order to understand the role of Cys53 and Cys165 of human growth hormone (hGH) in receptor-binding and biological activity, artificial mutant variants of hGH were prepared in Escherichia coli by in vitro mutagenesis. Variants of hGH were constructed by replacement of Cys165 with Ala ([Ala165]hGH) or Ser ([Ser165]hGH), by replacement of Cys53 with Ala ([Ala53]hGH), by replacement of Cys53 and Cys165 with Ala ([Ala53, Ala165]hGH), or by replacement of Cys53 with Ala and Cys165 with Ser ([Ala53,Ser165]hGH). All of the variants constructed as well as reduced hGH exhibited less biological activity than that of intact hGH, and the decreases in biological activity were almost equal, as measured by a sensitive biological assay for growth hormone: adipose conversion assay using 3T3-F442A cells. These variants also showed less receptor-binding activity than that of intact hGH. These results suggest that it is possible neither the residue Cys53 nor Cys165 is directly involved in the receptor binding, and that the disulfide bridge between Cys53 and Cys165 in hGH may not always be crucial for the biological activity, though necessary to express full hGH activity.
Animals↗
Interaction of bleomycin with deoxyribonucleic acid oligomer: proton nuclear magnetic resonance titration study using novel bleomycin complexes with Ni2+ and VO3+.
Two metal complexes of bleomycin (BLM), BLM-Ni2+ and BLM-VO3+ are used for studying interactions between BLM and deoxyribonucleic acid (DNA) by nuclear magnetic resonance. Although these BLMs do not mediate DNA strand scission under the usual conditions, they bind to DNA in the same manner as the active metal complexes of bleomycin (BLM-Fe2+ and BLM-Co3+). A self-complementary dodecanucleotide, d(CCCCAGCTGGGG), having a single site for cleavage was synthesized. d(CCCCAATTGGGG), which contains no -GpC- sequence, was also synthesized. The BLM-metal complexes were shown to bind specifically to the GpC site by circular dichroism and fluorescence titration studies. We assigned all the resonances for imino protons and phosphorus, and most of the nonexchangeable proton resonances of d(CCCCAGCTGGGG). No substantial change in the chemical shifts of these signals was observed upon titration with either BLM-Ni2+ or BLM-VO3+. This result is not consistent with a model of the strong intercalation of the BLMs between the base-pairs. The BLMs bind to DNA in a different manner, and DNA does not change its conformation upon binding with BLMs.
Base Sequence↗
The molecular recognition mechanism of DNA by bleomycin.
The interaction between the antibiotic bleomycin (BLM) and an oligonucleotide d(CCACCTAGGTGG) was studied by 1H NMR. The DNA oligomer was titrated with BLM-VO(III), which is an 'inactive analogue' of BLM-Fe(II). A marked upfield shift was observed for the T10N3H signal, but no other imino proton signals either shifted or broadened. When BLM-VO(III) was titrated with d(CCACCTAGGTGG), a large upfield shift (0.27 ppm) was observed for the delta NH signal of butylguanidinium residue (G). Analysis of NOESY spectra of a 1:1 complex of DNA:BLM-VO(III) strongly suggests that the connectivities of the sequential NOEs of the DNA duplex are the same as those of free DNA. These results indicate that BLM does not intercalate between the base-pairs but binds to DNA in a different manner. A model of 'minor-groove binding' is more likely to explain our NMR data.
Base Sequence↗
Molecular structure of extraordinarily stable RNA: model building and X-ray analysis.
A tridecaribonucleotide, rUGAGCUUCGGCUC which adopts the extraordinarily thermally stable hairpin structure, was chemically synthesized. The crystallographic study of this RNA molecule was done. Crystals grew as plates belonging to space group C2 with a = 38.49A b = 32.30A c = 38.76A and beta = 117.56. and one molecule per asymmetric unit. Intensity data were collected at a resolution 2.1A. Structure determination using molecular replacement method has been underway. The favorable three-dimensional model of hairpin was built by molecular dynamics and molecular mechanics calculations.
Base Sequence↗
Crystallization and preliminary X-ray investigation of non-specific complexes of a mutant ribonuclease T1 (Y45W) with 2'AMP and 2'UMP.
We have succeeded in crystallizing complexes of a mutant ribonuclease T1 (Y45W) with the non-cognizable ribonucleotides 2'AMP and 2'UMP by macroscopic seeding of microcrystals of the mutant enzyme complexed with 2'GMP, which is the cognizable nucleotide inhibitor. The mutant enzyme has a tryptophan residue instead of Tyr45 of the wild-type enzyme and thus this mutation enhances the binding of ribonucleotides to the enzyme. The space group is P212121 with unit cell dimensions a = 49.40 A, b = 46.71 A, c = 41.02 A for the complex with 2'AMP and a = 48.97, b = 46.58 A, c = 40.97 A for the complex with 2'UMP, both of which are poorly isomorphous to the mother crystals. Diffraction data for the complexes with 2'AMP and 2'UMP were collected on a diffractometer at 1.7 A and 2.4 A resolution, respectively. The present studies show that crystallization of non-specific complexes of other protein-ligand systems with the dissociation constants around 10(-3) M, or even larger, could be feasible by application of the seeding technique. A comparison of the crystal structures of the complexes with that with 2'GMP may serve as a structural basis for the determination of differences between the specific and non-specific interactions of the enzyme.
Adenosine Monophosphate↗
Synthesis and NMR study of ribo-oligonucleotides forming a hammerhead-type RNA enzyme system.
We designed a hammerhead-type ribozyme system which consists of three RNA fragments and synthesized the component and related ribo-oligonucleotides by the solid-phase phosphoramidite method using o-nitrobenzyl groups for 2'-hydroxyl protection. Improved conditions for photolytic removal of the o-nitrobenzyl groups are described. Imino proton NMR spectra of the ribozyme complex were measured. The resonances for the hydrogen-bonded imino protons were assigned by comparison with the spectra of model complexes which contain the base sequences of one or two of the stem regions. The results suggest that the complex indeed forms a hammerhead structure and the loop regions take an ordered conformation in the absence of magnesium ions.
Base Composition↗