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Biomedical subjects

S Uesugi

Publications and source records attributed to S Uesugi.

At least 55 records · Page 3Linked to original sources
PubMed1996

Steady-state kinetics of Thr35 and Thr39 mutants in human adenylate kinase by site-directed mutagenesis.

Adenylate kinase (AK; EC 2.7.4.3, hAK1) catalyzes the reaction: MgATP(2-)+ AMP2- reversible MgADP-(+) ADP3-. To elucidate the catalytic and structural roles of threonine residues in human AK, Thr35 and Thr39 mutants were analyzed by steady-state kinetics. The K(m) values of T35P and T35Y were not changed for MgATP2- and AMP2-, and the kcat values were decreased by 1/39 compared to those of wild-type AK. Thr35 was suggested to be essential for catalysis. The K(m) values of T39S, T39V and T39P were increased 5.6- to 59.0-fold for AMP2-; however, the kcat values were not reduced. Although the K(m) values of T39F and T39L were unchanged, the kcat values were reduced by more than 1/57. Thr39 appears to play an important role in the binding of AMP2- and to be essential for catalysis. As noted above, a hydroxyl group of the Thr residue in human AK appears to be important.

Adenylate Kinase↗
PubMed1996

[Methicillin-resistant Staphylococcus aureus infection in the Akita University Hospital: surveillance and microbiology data].

In order to control the nosocomial infections by methicillin-resistant Staphylococcus aureus (MRSA) in the Akita University Hospital, the systematic surveillance for MRSA-infection has been done since October in 1991. We reviewed MRSA-isolation numbers, MRSA-detected patient numbers, risk factors of nosocomial infection and patient's basic diseases with the data of a total 336 cases, and then examined the microbiological characteristics of MRSA strains which had been isolated in our hospital in 1986 approximately 1994. The results obtained are as follows; 1) MRSAs were isolated at 13 per month on the average and the detection rate was 0.81 to 1,000 inpatients per day. 2) MRSAs were isolated more in the surgical ward. 3) Patients with 0 year old and more than 60 years old were more infected. 4) MRSA was isolated more from the immunocompromised patients with underlying diseases such as malignant tumors but half of the diseases were not affected by MRSA. 5) Seventy percent of patients had the risk factors of MRSA-transmission such as surgical operation and IVH-cathetering. 6) All isolates of MRSA strains in our hospital showed type II in coagulase type analysis and resistance to minocycline and ofloxacin. 7) It was suggested that one strain expanded throughout the hospital by several DNA analysis with the mecA gene in MRSA. These surveillance and microbiology data will be useful for our nosocomial mecA gene in MRSA. These surveillance and microbiology data will be useful for our nosocomial infection control.

Adolescent↗
PubMed1995

[Restriction fragment length polymorphism of coagulase gene of methicillin-resistant Staphylococcus aureus can be used as an epidemiological marker].

In order to see the restriction fragment length polymorphism (RFLP) of coagulase gene of Methicillin-resistant Staphylococcus aureus (MRSA) and the type of coagulase, a total of 118 strains of MRSA from 1986 to 1994 was collected from the Akita University Hospital, six hospitals in Akita Prefecture and a hospital in Aomori Prefecture. The RFLP analysis using the Alu I restriction enzyme brought us four kinds of electrophoretic patterns (A, B, C, D). Patterns A (87%) and B (13%) were detected in our hospital and only pattern A was detected in other hospitals in Akita. On the other hand, Patterns A (68%), B (26%), C (3%) and D (3%) were detected in Aomori. In coagulase type analysis, all strains in Akita showed type II, and the strains in Aomori also showed type II except for one strain (type IV). From the above results, the polymorphism of coagulase gene of MRSA can be useful as an epidemiological marker to examine the extent of MRSA infection in a prefecture unit or in a larger area.

Coagulase↗
PubMed1995

Soluble expression of a synthetic gene for human translation initiation factor 4E in Escherichia coli.

In order to obtain the active form of recombinant human initiation factor (eIF) 4E effectively, an artificial synthetic gene was cloned into an expression vector (pMAL-p2) and the soluble expression was attempted in Escherichia coli under the control of a tac promoter. Two expression systems were finally constructed as a fusion protein with maltose-binding protein, which contain a recognition sequence for the site specific protease alpha-thrombin and factor Xa, respectively. Most of the fusion protein was induced as a soluble form. The soluble human eIF-4E digested from the fusion protein showed binding specificity for the m7GTP affinity column.

Amino Acid Sequence↗
PubMed1995

Activity of artificial mutant variants of human growth hormone changes in charged residues around 62-67.

Our previous work has shown that the amino acid residues around 62-67 located in the connecting loop between helix I and II of human growth hormone (hGH) are important in eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes. In this study, we evaluated the role of the charged residues around 62-67 in receptor binding and biological activity. Eight artificial mutant variants of hGH were prepared in Escherichia coli by site-directed mutagenesis. Replacement of Arg64 with Tyr (R64Y variant) resulted in a significant loss of binding to the somatogenic receptors on 3T3-F442A cells, but retained full adipose conversion activity on these cells. Replacement of Arg64 with Glu (R64E) produced a considerable loss in receptor binding and a significant loss in biological activity. hGH variants in which either Glu65 or Glu66 was replaced with Asp (E65D and E66D) and with Gln (E65Q and E66Q) showed a slight loss in binding activity and retained almost a full adipogenic activity. An E65P variant (replacement of Glu65 with Pro) possessed the same binding activity as hGH, although it failed to induce full biological activity. The insertion of Ala between Asn63 and Arg64 (63NAR) caused a marked loss in both activities. These results indicate that the positively charged Arg64 is important for receptor binding and thereby in eliciting the biological activity of hGH, while negatively charged Glu65 and Glu66 are less important. In addition, our findings confirm that the conformation and size of the loop region around Arg64 is important for the adipose conversion activity of hGH.

Adipose Tissue↗
PubMed1995

[Significance of clinical examination concerning arteriosclerosis].

According to the number of deaths by major cause, Malignant neoplasma takes the first rank as the cause of death and then heart disease as a second and next rank to cerebrovascular disease since 1985, instead of top rank of cerebrovascular by that time. However, heart disease and cerebrovascular could be supposed as a arteriosclerosis disease fundamentally, arteriosclerosis disease (heart disease plus cerebrovascular) is usually top of rank in the cause of death since the Showa era. Therefore, it is great significance to make studies of risk factor on the arteriosclerosis in the laboratory medicine.

Arteriosclerosis↗
PubMed1995

[The role of hypertension as a risk factor of atherosclerosis].

Epidemiologic studies have demonstrated hypertension is one of the risk factors of atherosclerosis, but the underlying mechanism is complex and still controversial. Salt-sensitivity is an important characteristic demonstrated in a subgroup of hypertension, since the factors relating to salt-sensitivity also influence smooth muscle hypertrophy and proliferation which are essential processes of atherosclerosis. Insulin resistance is also involved in the causal relationship between hypertension and atherosclerosis, because accumulating data indicate a central role of insulin resistance in patients with hypertension, glucose-intolerance and dyslipidemia. Vasoacting substances give direct effects on not only the tension but also the growth of smooth muscle cells, namely vasodilators, such as nitric oxide and atrial natriuretic peptides inhibit the proliferation of smooth muscle cells. On the other hand, vasoconstrictors such as angiotensin II, vasopressin and endothelin promote the proliferation of smooth muscle cells. The factors which influence both tension and proliferation of smooth muscle cells may play a central role in the relationship between hypertension and atherosclerosis.

Arteriosclerosis↗
PubMed1995

Comparative CD and thermodynamic studies between sheared A:G and Watson-Crick A:U(T) base pairs in RNA and DNA.

Thermodynamic parameters for duplex formation were determined from CD melting curves for r(GGACGAGUCC)2 and d(GGACGAGTCC)2, both of which have been demonstrated to form two consecutive 'sheared' A:G base pairs at the center by our previous studies. The parameters were determined also for r(GGACUAGUCC)2 and d(GGACTAGTCC)2, where the A:G mismatches are replaced by Watson-Crick A:U(T) base pairs. Thermodynamic properties for duplex formation are compared between the sheared and the Watson-Crick base pairs, and between RNA and DNA. Difference in the thermodynamic stability is analyzed and discussed in terms of enthalpy and entropy changes.

Base Composition↗
PubMed1995

Characterization of an HDV ribozyme which consists of three RNA oligomer strands.

Properties of a mutant hepatitis delta virus (HDV) ribozyme which consists of three RNA oligomer strands (Fig. 1c) are described. Effects of the MgCl2 concentration, pH and temperature on the cleavage reaction were examined. The dependence on these conditions did not resemble those of the published data for other HDV ribozymes (the parent) which consist of single-strand RNA of about 90 nt. The highest cleavage for the mutant was observed at 35-40 degrees C, while the parent ribozymes show higher activity at around 60 degrees C than at 40 degrees C. Thus we suppose that the most of the parent ribozymes are in inactive conformation at 40 degrees C and high temperature is needed to change the equilibrium between the inactive and active conformations. Since the Tm of the mutant was around 50 degrees C, these results suggest that the most of the mutant ribozyme complex molecules are in active conformation at 40 degrees C.

Base Sequence↗
PubMed1995

Structure of translation initiation region of c-myc mRNA and thermostability of the c-myc mRNA/antisense ODN complex.

We enzymatically synthesized c-myc mRNA fragment containing the translation initiation (TI) region (36mer) and examined the structure by CD, 1H-NMR and RNaseV1 digestion. These study demonstrated that the c-myc mRNA fragment forms a hairpin structure at 37 degrees C. Six 15-mer antisense oligodeoxynucleotides(ODNs), which hybridize to the c-myc mRNA fragment were designed, and synhesized. Thermostabilities of the antisense ODNs-mRNA fragment complexes were examined. Antisense A (complementary to the c-myc mRNA from -9 to +6)-c-myc mRNA fragment complex had the highest melting temperature(Tm), and Antisense D (complementary to the c-myc mRNA from +1 to +15)-c-myc mRNA fragment complex had the most different Tm from a predicted Tm. Furthermore, Antisense D exhibited high inhibitory effect on HL60 cell proliferation, whereas Antisense A did not. The results suggest that the difference in the antisense effect is not simply due to the complex thermostability.

Cell Line↗
PubMed1995

RNA quadruplex containing G and A.

The CD study of r(GGAGGAA) has indicated that it forms a higher order structure upon either increase of the strand concentration or addition of K+. Gel electrophoresis showed that a high molecular weight species is formed in the presence of K+. The NMR study revealed a nearly 1:1 equilibrium of two kinds of higher order structures at the strand concentration of 2.1 mM and 100 mM Na+. This equilibrium shifted drastically upon addition of K+, and only one structure remained at 40 mM K+. It is suggested that the structure formed by r(GGAGGAA) in the presence of K+ is a new type of RNA quadruplex, where an A-quartet stabilized by mutual hydrogen bonding among the four adenine bases is sandwiched by conventional G-quartets.

Adenine↗
PubMed1995

Mechanism of mutagenesis induced by cytosine analogs bearing N(4)-substitutions.

The mechanism of mutagenesis induced by dihydropyrimido [4,5-c][1,2]oxazin-7-one deoxyriboside, P-nucleoside, was studied. This analog is highly mutagenic toward Escherichia coli and Salmonella typhimurium. In E. coli, it induces GC-to-AT and AT-to-GC transitions specifically. No transversions are inducible. P-nucleoside was highly mutagenic to a wild-type E. coli, but little mutagenic in a strain lacking thymidine kinase. This indicates that P-nucleoside may be phosphorylated by thymidine kinase after its uptake into bacteria. The mutagenesis induced by P-nucleoside was efficiently inhibited by the addition of thymidine. This inhibition further confirmed the involvement of thymidine kinase in the first step of the metabolism of P-nucleoside in the bacterial cells. These findings indicate that P-nucleoside is a mutagen of a nucleoside-analog type, causing mutations by the erroneous incorporation and replication. The experiments to prove its ambiguous nature in DNA synthesis is now under way.

Adenine↗
PubMed1994

Structures of heat-stable and unstable homologues of the sweet protein mabinlin. The difference in the heat stability is due to replacement of a single amino acid residue.

There are several analogues of the sweet protein mabinlin. In previous studies, we purified the heat-stable analogue, mabinlin II, from the seeds of Capparis masaikai Lévl. and determined its amino acid sequence [Liu, X., Maeda, S., Hu, Z., Aiuchi, T., Nakaya, K. & Kurihara, Y. (1993) Eur. J. Biochem. 211, 281-287] and the disulfide structure [Nirasawa, S., Liu, X., Nishino, T. & Kurihara, Y. (1993) Biochim. Biophys. Acta 1202, 277-280]. We have now purified four additional homologues of mabinlin. The sweet activities of mabinlin III and mabinlin IV were unchanged by incubation for 1 h at 80 degrees C, as was found previously for mabinlin II, while the sweet activity of mabinlin I-1 was completely abolished by a 1-h incubation at 80 degrees C. The circular dichroic spectrum showed that alpha-helical structures of mabinlins II-IV were unchanged by the 1-h incubation at 80 degrees C, while the alpha-helical structures of mabinlin I-1 were completely destroyed by the 1-h incubation in parallel with the decrease of the sweet activity. To compare the structures of the heat-stable and unstable homologues, we determined their amino acid sequences and the disulfide array. The positions of four disulfide bridges of mabinlin I-1 were the same as those of mabinlin II, suggesting that the disulfide bridges do not contribute to the difference in the heat stability among the homologues. There was a high similarity among amino acid sequences of the homologoues. Only three amino acid residues (A-chain residues at positions 22 and 32 and B-chain residue at position 47) were different between mabinlin I-1 and mabinlin III. A-chain residue at position 32 was lacking in mabinlin IV and the A-chain residue at position 22 was identical in both mabinlin I-1 and mabinlin II. The B-chain residue at position 47 was the only residue present in all three heat-stable homologues (mabinlins II-IV) and is not present in the unstable homologue (mabinlin I-1). This suggests that the difference in the heat stability of mabinlin is due to the difference in a B-chain residue at position 47; the difference in the heat-stable homologues is due to the presence of an arginine residue and the difference of the unstable homologue is due to the presence of glutamine.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Formation of sheared G:A base pairs in an RNA duplex modelled after ribozymes, as revealed by NMR.

The thermal stability and structure of an RNA duplex, r(GGACGAGUCC)2, the base sequence of which was modelled after both a hammerhead ribozyme and a lead ribozyme, were studied by CD and NMR. We previously demonstrated that the corresponding DNA duplex, d(GGACGAGTCC)2, formed unique 'sheared' G:A base pairs, where an amino proton, instead of an imino proton, of G is involved in the hydrogen bonding, and G and A bases are arranged 'side by side' instead of 'head to head' (Nucleic Acids Res. (1993) 21, 5418-5424). CD melting profiles showed that the RNA duplex is thermally more stable than the corresponding DNA duplex. NMR studies revealed that sheared G:A base pairs are formed in the RNA duplex, too, although the overall structure of the RNA is the A form, which differs from the B form taken on by the corresponding DNA. A model building study confirmed that sheared G:A base pairs can be accommodated in the double helical structure of the A form. A difference between the RNA and DNA duplexes in the stacking interaction involving G:A mismatch bases is also suggested. The demonstration that sheared G:A base pairs can be formed not only in DNA but also in RNA suggests that this base pairing plays an important role regarding the RNA structure.

Adenine↗
PubMed1994

Molecular characterization of a human group A rotavirus isolated from an adult with severe dehydrating diarrhea and its relationship to strains concurrently circulating among children.

BACKGROUND: While group A rotavirus is widely accepted as an important etiology of acute gastroenteritis in children, this agent rarely causes severe diarrhea in adults and, thus, is not considered by physicians to be an etiological agent for such diseases. OBJECTIVES: None of the reports describing such rare cases in adults has examined the causative strains genetically in detail. STUDY DESIGN: We determined the G type, the gene 4 genotype, the electropherotype, and the genomic RNA constellation (genogroup) of a group A rotavirus strain isolated from an adult with severe diarrhea. This patient, the first documented case of rotavirus diarrhea in adult in Japan, suffered from severe dehydrating diarrhea with 'rice-water' appearance, vomiting and little abdominal pain, presenting a clinical picture typical of cholera. RESULTS: Rotavirus antigen and genomic RNA were detected in the stool but other enteric pathogens including Vibrio cholerae responsible for the disease were not isolated. Molecular characterization revealed that the patient was infected with a strain of the DS-1 genogroup with G2 and gene 4 genotype 4 which was circulating among children during the same period. CONCLUSIONS: The result that the same group A rotavirus strain was isolated from children and an adult underlines the necessity of examining rotavirus in adults with acute diarrhea.

Journal Article↗
PubMed1994

Direct expression of a synthetic gene in Escherichia coli: purification and physicochemical properties of human initiation factor 4E.

An artificial synthetic gene coding for human eIF-4E was cloned into an expression vector and direct expression was attempted in Escherichia coli [BL21(DE3) strain] under the control of T7 promoter. The active gene product which was induced in high yield (ca. 4 mg/100 ml) by isopropyl-beta-D-thiogalactopyranoside was purified to homogeneity by a two-step chromatographic procedure with a good yield (ca. 74%), and was confirmed to be recombinant human eIF-4E by amino acid composition and sequence analyses, isoelectric focusing, and absorption spectral measurements. The identity of three-dimensional structures between the recombinant and native human eIF-4Es was confirmed by CD and fluorescence measurements.

Amino Acid Sequence↗