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Biomedical subjects

S Uesugi

Publications and source records attributed to S Uesugi.

At least 109 records · Page 6Linked to original sources
PubMed1990

An active site of growth hormone for eliciting the differentiation of preadipose 3T3-F442A cells to adipose cells.

In order to elucidate the active sites of growth hormone for eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes, four artificial mutant variants of human growth hormone (hGH) modified in the loop region of amino acid residues 54-74 were prepared in Escherichia coli by site-directed mutagenesis. Although the P59A (replacement of Pro59 with Ala) variant retained almost the same biological- and receptor binding-activity as hGH, the P61A (replacement of Pro61 with Ala) and the P59A-P61A (replacement of both Pro59 and Pro61 with Ala) both exhibited about half the activity, and the delta (62-67) variant (deletion of the residues 62-67) exhibited only about 0.1% the activity of those of intact hGH. The results suggest that Pro61 may be involved in formation of the active conformation of hGH, but Pro59 may not, and that the amino acid residues around 62-67 may be critical for the specific biological features of hGH.

Adipose Tissue↗
PubMed1990

Studies on the structure and stabilizing factor of the CUUCGG hairpin RNA using chemically synthesized oligonucleotides.

A tridecaribonucleotide, r-UGAGCUUCGGCUC, and two analogues r(UGAGC)d(UUCG)r(GCUC) and r-UGAGCUUCIGCUC, which form a hairpin structure with a four-base-paired stem and a UUCG loop, were synthesized by the solid-phase phosphoramidite method. Properties of these three oligomers and d-TGAGCTTCGGCTC, the DNA analogue, were studied by UV, CD and NMR spectroscopy. The melting temperature (Tm) data suggest that the 2'-hydroxy1 groups and the 2-amino group of guanosine in the loop (9G) stabilize the CUUCGG hairpin which is known to have an unusually high Tm. NMR studies show that this 9G takes a syn conformation and the phosphodiester backbone has a turn at 9G-10G which is a junction of the stem and loop.

Base Sequence↗
PubMed1990

The role of a conserved guanosine residue in the hammerhead-type RNA enzyme.

We have designed a hammerhead-type RNA system which consists of three RNA fragments for normal and modified complexes which contain a non-cleavable substrate with 2'-O-methylcytidine and a guanosine-to-inosine replaced enzyme. Examination of the RNA-cleaving activity and conformational properties of the complexes suggests that the 2-amino group of a conserved guanosine residue in the loop region plays an important role for maintaining both the activity and loop conformation.

Base Sequence↗
PubMed1990

Secretion of recombinant ribonuclease T1 into the periplasmic space of Escherichia coli with the aid of the signal peptide of alkaline phosphatase.

The ribonuclease T1 (RNase T1) gene was ligated to a synthetic gene for the signal peptide of Escherichia coli alkaline phosphatase. When this fusion gene was expressed in E. coli under the control of the trp promoter, active RNase T1 having the correct N-terminal sequence was secreted into the periplasmic space, indicating that the heterologous signal peptide had been cleaved off correctly. The enzyme could be readily purified from the periplasmic fraction with a yield of 1.8 mg from 1 liter culture. Adopting the same strategy, it was possible to produce a labile mutant of RNase T1 (Glu-58----Ala mutant) in E. coli, the yield of the purified mutant enzyme being 2.0 mg from 1 liter culture.

Alkaline Phosphatase↗
PubMed1990

In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model.

Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.

Adenosine Monophosphate↗
PubMed1990

27 amino acid residues can be deleted from the N-terminus of human lymphotoxin without impairment of its cytotoxic activity.

In order to study the relationship between activity and structure of human lymphotoxin (hLT, 171 aa), we synthesized the gene (519 bp) for hLT and expressed it in Escherichia coli. Purification of the recombinant hLT from crude extracts was difficult because of the low level of expression of the gene. To improve the yield of the recombinant protein, we prepared five truncated genes for mutant proteins in which 25, 26, 27, 28 and 37 amino acid residues, respectively, were missing from the N-terminus. All of the genes were efficiently expressed and adequate amounts of mutant proteins were synthesized. The proteins were recovered mainly in the supernatant fractions after disruption of cells, with the exception of LT delta 37N, in which 37 residues were absent from the N-terminal region. Cytotoxic activities against mouse fibroblast L929 cells were detected in supernatant fractions that contained these mutant proteins, except in the case of LT delta 28N, which lacks the first amino acid residue conserved in both hLT and human tumour necrosis factor (hTNF). LT delta 27N, which is the smallest of the active proteins, was purified to homogeneity, and its cytotoxic activity was found to be similar to that of recombinant hTNF.

Amino Acid Sequence↗
PubMed1990

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds.

Molecular-dynamic calculations predict that, if Tyr24 and Asn84 are each replaced by a Cys residue, it should be possible to form a third disulfide bond in ribonuclease T1 (RNase T1) between these residues, with only minimal conformational changes at the catalytic site. The gene encoding such a mutant variant of RNase T1 (Tyr24----Cys24, Asn84----Cys84) was constructed by the cassette mutagenesis method using a chemically synthesized gene. In order to reduce the toxic effect of the mutant enzyme (RNase T1S) on an Escherichia coli host, we arranged for the protein to be secreted into the periplasmic space by using a vector that harbors a gene for an alkaline phosphatase signal peptide under the control of the trp promoter. The nucleolytic activity of RNase T1S toward pGpC was approximately the same as that of RNase T1 at 37 degrees C (pH 7.5). Moreover, at 55 degrees C, RNase T1S retained nearly 70% of its activity while the activity of the wild-type enzyme was reduced to less than 10%. RNase T1S was also more resistant to denaturation by urea than the wild-type enzyme. However, unlike RNase T1, RNase T1S was irreversibly and almost totally inactivated by boiling at 100 degrees C for 15 min.

Base Sequence↗
PubMed1990

Base recognition mechanism of bleomycin: solution structure of d(GGGGAGCTCCCC)2 based on 1H-NMR and restrained molecular dynamics.

The self-complementary d(GGGGAGCTCCCC) dodecanucleotide cleavage by bleomycin occurs at G(4)pA(5) site rather than G(6)pC(7) site which is known as the preferential sequence. The molecular structure of this dodecamer is observed as the distorted B-DNA by the CD spectra and proton NMR measurements. Three-dimensional structures in solution evaluated by molecular dynamics calculations with inter proton distance restraints have the distorted form of the minor groove structure at G(6)pC(7) site. These evidences offer the recognition mechanism model of the DNA base sequence by bleomycin.

Base Sequence↗
PubMed1990

[An autopsy case of glioblastoma occurred in the region after lobotomy].

The authors describe an autopsy case of glioblastoma occurred after 38 years received lobotomy. The patient was a 72 year-old male, who received lobotomy at 34 year old against schizophrenia. CT scan taken at 72 year old showed irregular low density areas without mass effect in the bilateral frontal white matter adjacent to the anterior horn. After 4 months, the signs of intracranial hypertension were observed and his consciousness was disturbed abruptly. CT scan revealed ring enhancement with marked mass effect in the left frontal lobe. A biopsy specimen from the tumor showed a picture of anaplastic astrocytoma. Family rejected the remission maintenance treatment. The patient died 3 months later the onset. At autopsy, a large tumor occupied in the left frontal lobe was recognized. The tumor demarcated poorly from the cerebral tissue and invaded into the left anterior cingulate gyrus and the corpus callosum. Histologically, tumor cells composed of fibrillary, gemistocytic and multinucleated astrocytes. GFAP, NSE and vimentin were found in large cells. Histological diagnosis was glioblastoma. It was suggested that the tumor occurred from the region around a cyst of prefrontal lobotomy in the left frontal lobe. In the right frontal lobe, a large cyst in size of 30-18 mm was present in the centrum semiovale. The wall of cyst was composed of layer of glial scar tissue. The origin of the cyst was discussed.

Aged↗
PubMed1990

Identification of DNA degradation products by antitumor antibiotic, esperamicin.

Esperamicin A1 is a DNA-damaging agent characterized by a unique ten-membered ene-diyne core. We studied the detailed reaction mechanism by using synthetic DNA oligomers. The cleavage site and activity depend on the sequence of the oligomers. d(GGATCC) and d(GGTACC) were cleaved by the drug while d(CCATGG) and d(CCTAGG) were not cleaved under the present conditions. d(GGTACC) gave two major 5'-fragments. The result of partial nuclease digestion experiments suggests that these products are trimer and pentamer with a modified 3'-end.

Aminoglycosides↗
PubMed1990

Conformation and properties of hammerhead-type RNA enzymes.

We designed a hammerhead-type RNA enzyme system which consists of three ribooligonucleotide strands (1-3), synthesized these oligomers and their analogues, and examined conformation and properties of the RNA complexes. Imino proton NMR spectra of the complexes were measured and the signals were assigned by comparison with the spectra of some model duplexes. Examination of a complex containing a G----I mutation (Y = I) revealed that the 2-amino group of the third guanosine residue in the loop 1 (L1) plays an important role for maintaining both the activity and loop conformation.

Base Sequence↗
PubMed1989

A 2-deoxyribonolactone-containing nucleotide: isolation and characterization of the alkali-sensitive photoproduct of the trideoxyribonucleotide d(ApCpA).

It has been reported that ACA sequences in DNA are mutagenic hot spots in UV-induced mutagenesis and are sites of an alkali-sensitive lesion produced by UV irradiation. In order to characterize the UV-induced lesion of an ACA site, chemically synthesized trideoxyribonucleotide d(ApCpA) was irradiated with UV light and the alkali-sensitive photoproduct was isolated. The structure of this photoproduct was characterized as the trinucleotide containing 2-deoxyribonolactone at the internal residue by 2D DQF-COSY, FT-IR, FAB-MS, and chemical properties. It is known that this lesion is also produced by gamma-irradiation, neocarzinostatin, the 1,10-phenanthroline-copper complex, and hydrogen peroxide and is highly mutagenic because of its resistance to cleavage by certain apurinic/apyrimidinic (AP) endonucleases. Thus, 2-deoxyribonolactone may be one of the lethal DNA lesions induced by UV irradiation to organisms and one of the intermediates of UV-induced DNA strand breaks because the DNA strand is cleaved at this site with beta- and subsequent delta-elimination mechanisms.

Base Composition↗
PubMed1989

Novel 1H-31P heteronuclear coherence transfer spectroscopy based on spin locking MLEV-17 and its application to signal assignment for a short DNA fragment.

We have applied the MLEV-17 proton spin locking pulse to 1H-31P 2D heteronuclear correlation spectroscopy. Using this technique, long-range correlations between a proton and phosphorus that is up to 5 bonds distant are observed. Thus, the 3'-, 4'- and 5'-proton resonances can be traced 'sequentially' along the chain on the 2D NMR chart. Application of this technique to the complete assignment of the sugar proton and phosphorus resonances for d(ApGpA) is reported. This technique can also be used for convenient assignment of the phosphorus resonances of oligonucleotides as correlations between the 1'-proton and its 3'-side phosphorus are observed.

DNA↗
PubMed1989

Synthetic genes for human muscle-type adenylate kinase in Escherichia coli.

An artificial gene coding for the human muscle-type cytosolic adenylate kinase (hAK1) was chemically synthesized and directly expressed in Escherichia coli under the control of trp promoter. The DNA duplex of 596 bp was designed and constructed from 40 oligonucleotide fragments of typically 30 nucleotides in length. Twelve unique restriction sites were fairly evenly spaced in the synthetic gene to facilitate site-specific mutagenesis at any part of this recombinant protein. The genes for mutant hAK1 (Tyr 95----Phe 95, Y95F hAK1; Arg 97----Ala 97, R97A hAK1) were constructed by cassette mutagenesis and utilized restriction sites incorporated in the hAK1 gene. The recombinant hAK1 was purified to homogeneity by a two-step chromatographic procedure with a good yield, and showed the same adenylate kinase activity as that of authentic hAK1. Preliminary kinetic studies show that the enzymatic activity (Vmax app,cor/Et) of Y95F hAK1 was slightly greater than that of recombinant hAK1, whereas R97A hAK1 still possessed approximately 4% of recombinant hAK1 activity. These results suggest that the Arg-97 residue is important but not essential for catalytic activity, and that Tyr-95 can be replaced by phenylalanine without substantial effects on the enzymatic activity. Moreover, preliminary estimates of the apparent kinetic parameters suggest that these residues are not required for MgATP binding, and therefore they do not appear to be part of the MgATP binding site.

Adenylate Kinase↗
PubMed1989

Structure and activity of artificial mutant variants of human growth hormone.

Four artificial mutant variants of human growth hormone (hGH) with the following characteristics were prepared in Escherichia coli by in vitro mutagenesis: (i) replacement of Trp86 with Tyr (W86Y hGH); (ii) deletion of Trp86 (delta W86 hGH); (iii) deletion of residues 32-46 (20-kd-hGH); and (iv) deletion of residues 32-71 (17.5-kd-hGH). Both W86Y hGH and delta W86 hGH have a point mutation at Trp86 which is the only Trp residue in hGH and is conserved among members of the growth hormone family. 20-kd-hGH is a minor component of hGH in the pituitary gland and plasma and is the result of an alternative splicing of the primary gene transcript, while only the corresponding mRNA and not the protein has been found in the case of 17.5-kd-hGH. The biological activities (adipogenic activity and potentiation of gain in body weight) and structures (as analyzed by circular dichroism) were mostly retained by the W86Y, delta W86 and 20-kd-hGH variants but not by 17.5-kd-hGH.

Adipose Tissue↗
PubMed1989

[Nosocomial infections and clinical microbiology].

Hospital-acquired infection with strains of methicillin-resistant Staphylococcus aureus (MRSA) have considerably increased in recent years. In addition to being resistant to methicillin, these strains are resistant to practically all forms beta-lactams, aminoglycosides and many other antibiotics. There appears no cost-effective control and preventive measures for this common but also potentially life-threatening disease. Although not clearly presented, the overall cost for the treatment of patients infected with MRSA should be enormous. Can laboratory medicine (or clinical microbiology) contribute to this global medical problem? Multiple strains of MRSA circulate within a hospital and some strains are even localized within specific wards. These facts suggest yet undisclosed routes of transmission and/or foci of infection. Triumph over these versatile organisms may have to await the development of new antibiotics effective for MRSA.

Adult↗