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Biomedical subjects

S Kuwata

Publications and source records attributed to S Kuwata.

At least 109 records · Page 6Linked to original sources
PubMed1992

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the mixed anhydride and related methods.

In segment couplings by the mixed anhydride method using isobutyloxycarbonyl chloride, the use of copper(II) chloride as an additive suppressed racemization completely in the same manner as in the carbodiimide method reported previously. This was confirmed by employing a number of couplings between Z-dipeptides and amino acid esters. The racemization-suppressing effect of other compounds were also evaluated by employing one of these model couplings to be at best only limitedly effective. Copper(II) chloride was effective also in the related method using EEDQ. Thus, in the couplings where a low level of racemization was observed without an additive, the addition of copper(II) chloride eliminated racemization even at ambient temperature where EEDQ is usually used. The effectiveness of copper(II) chloride was confirmed also in the BOP-Cl method. In the presence of HOBt racemization was reduced to a low but still detectable level, while it was suppressed completely by the addition of copper(II) chloride.

Carbon Dioxide↗
PubMed1992

Racemization studies in peptide synthesis through the separation of protected epimeric peptides by reversed-phase high performance liquid chromatography.

Separation of protected epimeric peptides, Z-Gly-Xaa-Xbb-OMe (where Xaa and Xbb = chiral amino acid residues), by reversed-phase HPLC was utilized for studying racemization in peptide synthesis. Thus, the following factors which might affect the extent of racemization during the coupling by the carbodiimide method were investigated: the combination of amino acid residues to be coupled, coexisting tertiary amine salts, and the relative configuration of the amino acid residues. The following points were revealed: the combination of bulky residues at the coupling site results in extensive racemization in a polar solvent such as DMF, the amine hydrochlorides cause less racemization than the p-toluene-sulfonates in DMF, and the influence of relative configuration differs depending on the solvent and the individuality of the amino components. Furthermore, the racemization-suppressing effect of some additives in the carbodiimide method was reevaluated by employing the same procedure.

Amino Acids↗
PubMed1992

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the carbodiimide method.

Copper(II) chloride was found to be an extremely efficient racemization-suppressing additive in the DCC method as compared with the hitherto known ones, by employing the model coupling Z-Gly-L-Val-OH + H-L-Val-OMe in DMF. Although some other copper salts also had a profound effect, copper(II) chloride was the best from the viewpoint of both racemization suppression and coupling efficiency. The effectiveness of copper(II) chloride was further confirmed by employing the EDC-mediated couplings of Z-Gly-containing dipeptides with amino acid esters or dipeptide esters, and those of Z-L-Ala (or L-Val)-L-Val-OH with amino acid esters or dipeptide esters. In almost all the cases studied, no detectable amount (less than 0.1%) of epimer was observed by the HPLC analysis in the presence of copper(II) chloride. This was also the case even with an extremely stringent coupling system Z-L-Pro-L-Val-OH + H-L-Pro-OMe. With reference to the mechanism of racemization suppression, it was found that copper(II) chloride has a strong ability to suppress the racemization of the 5(4H)-oxazolone, which may be formed from an activated carboxyl component during the coupling.

Amino Acid Sequence↗
PubMed1992

Simultaneous use of 1-hydroxybenzotriazole and copper(II) chloride as additives for racemization-free and efficient peptide synthesis by the carbodiimide method.

In the carbodiimide mediated coupling of Z-Gly-L-Val-OH with H-L-Val-OMe in DMF, the simultaneous use of HOBt and copper(II) chloride as additives was found to give the desired peptide in a high yield without racemization. In the presence of HOBt, reducing the amount of copper(II) chloride produced a higher yield. Besides improving the coupling efficiency as compared with the case using copper(II) chloride alone as an additive, the present procedure offered another advantage for racemization suppression. Thus, even for the couplings where a low level of racemization was observed in the presence of copper(II) chloride, the simultaneous addition of HOBt and copper(II) chloride resulted in the elimination of racemization. The effectiveness of this new procedure using the two carbodiimide additives in the synthesis of biologically active peptides was assessed by the preparation of a protected Leu-enkephalin. In the 4 + 1 segment condensation using HOBt and copper(II) chloride simultaneously as additives, no racemization was detected and the yield was high enough. The elimination of racemization and improvement of coupling efficiency produced by the present procedure can be attributable to a reduced tendency for the activated forms of the carboxyl component to form a 5(4H)-oxazolone by the action of HOBt, and to the prevention of racemization by copper(II) chloride of the small amount of the oxazolone formed which is not eliminated by the action of HOBt alone.

Amino Acid Sequence↗
PubMed1992

Analysis by the polymerase chain reaction of histocompatibility leucocyte antigen-DR9-linked susceptibility to insulin-dependent diabetes mellitus.

DNA sequence analysis of class II HLA from Caucasian and black patients with type 1 (insulin-dependent) diabetes mellitus has suggested that aspartic acid at position 57 (Asp 57) of the DQ beta chain provides protection against insulin-dependent diabetes mellitus (IDDM). In contrast, most Japanese patients with IDDM have Asp 57-positive alleles. To determine the reason for the differences and to localize the HLA-linked diabetogenic gene in Japanese, we studied the DQA1 and DQB1 genes of Japanese patients with IDDM and control subjects by the polymerase chain reaction in combination with restriction fragment length polymorphism analysis. Associations of DQA1*0301 and DQB1*0303 with IDDM were observed. DQA1*01 was associated negatively with IDDM. The HLA-DR9 haplotype, which is associated positively with IDDM in Japanese, was associated with DQA1*0301 and DQB1*0303, indicating that the Japanese DR9 haplotype is the same as that in caucasians but different from that in blacks. Of the loci on Japanese DR9 haplotypes, the DQA1*0301 allele showed the highest association with IDDM. DQB1*0303 was also positively associated with IDDM. Since DQB1*0303 is identical to DQB1*0302 except that it contains Asp 57, the data suggests that an Asp 57-positive allele confers susceptibility to IDDM when the whole molecule of the DQ beta chain is similar to other susceptible DQ beta chains. DQA1*0301 appears to be a marker of IDDM in all these populations: Japanese, caucasian, and black.

Adult↗
PubMed1992

A young female case of polyarteritis nodosa strongly suspected by typical angiographic findings which improved rapidly after prednisolone and cyclophosphamide therapy.

A 16-year-old girl was admitted with the complaints of headache, chest pain, low abdominal pain and left hemi-numbness. Her blood pressure was high and plasma renin activity and aldosterone levels were elevated. Renal angiography revealed vascular stenoses and microaneurysms although the renal artery and its main branches were not involved. Polyarteritis nodosa (PN) was strongly suspected and oral prednisolone and intravenous pulse therapy of cyclophosphamide were started. The second renal angiography which was performed 11 days after the therapy was started, showed marked improvement of vascular lesions. This is a case which suggests that the angiographic findings of PN can improve very rapidly with therapy.

Adolescent↗
PubMed1992

[Application of PCR and RT-PCR method to molecular biology study in nephrology].

Recent advance in molecular biology and genetic engineering has made a profound influence on basic and clinical medicine. Development of PCR (polymerase chain reaction) has transformed our trend of thought in molecular biology. Applications of PCR method has extended to nephrology study. RT (reverse transcription)-PCR method was elaborated to identify mRNA even in a small number of cells. It is also important and advantageous that PCR facilitates the utilization of a non-radioisotopic detection system. Newly developed chemiluminescent procedure yields as high sensitivity just as radioactive probes. Further applications are now being investigated including in situ PCR. PCR and RT-PCR methods are of great value for studying a molecular biological background of intricate kidney functions and diverse renal disorders.

Cloning, Molecular↗
PubMed1991

Study of HLA class I, class II and complement genes (C2, C4A, C4B and BF) in Japanese psoriatics and analysis of a newly-found high-risk haplotype by pulsed field gel electrophoresis.

Genetic polymorphisms of HLA antigens and HLA-linked serum complement components (C2, C4A, C4B and BF) were investigated in 79 Japanese patients suffering from psoriasis. HLA typing revealed increased frequencies of HLA-A1, A2, B39, Bw46, Cw6, Cw7 and Cw11. Among complement components, positive associations were obtained with C4A4 and C4B2 and a negative association with BFF. The major histocompatibility complex haplotype (supratype), HLA-A2-Cw11-Bw46-C2C-BFS-C4A4-C4B2-DRw8 is purported to be a new high-risk haplotype in Japanese patients with psoriasis. Analysis of patients with this supratype via pulsed field gel electrophoresis showed the existence of specific, extensive DNA deletions near HLA-DR genes, but no disease-specific patterns could be observed by means of this technique. The newly-found high-risk haplotype indicates racial and ethnic differences among psoriatic patients.

Alleles↗
PubMed1991

Functional analysis of deletion mutants of cucumber mosaic virus RNA3 using an in vitro transcription system.

Full-length DNA copies of RNAs 1, 2, and 3 of CMV Y strain (CMV-Y) were cloned downstream of modified phage T7 promoter sequences to obtain infectious RNA transcripts. The small number of extra nonviral nucleotides at the 5' ends considerably decreased the specific infectivity of the transcripts of RNAs 1 and 2 but did not affect that of the RNA3 transcripts. Using the most infective transcripts, up to 45% of tobacco protoplasts could be infected. Various cDNA mutants were constructed from the full-length RNA3 cDNA to give RNA transcripts having deletions in the coding region of the 3a protein or the coat protein. These mutants replicated in tobacco protoplasts but did not produce systemic symptoms on tobacco when inoculated together with transcripts of RNAs 1 and 2. One of the mutants having a small in-frame deletion near the N-terminal region of the coat protein produced local lesions on cowpea and local chlorotic spots on the inoculated leaves of tobacco. These results suggest that both the 3a protein and the coat protein are involved in virus transport, and that viral assembly is associated with long-distance movement of CMV.

Base Sequence↗
PubMed1991

Reciprocal phenotype alterations between two satellite RNAs of cucumber mosaic virus.

Cucumber mosaic virus Y satellite RNA (Y-satRNA) induces distinctive yellow mosaic symptoms on tobacco, whereas S19 satellite RNA (S19-satRNA) causes an attenuated green mosaic on tobacco, although they show considerable sequence identity. Biological assays of infectious chimeric satellite RNA molecules synthesized from cDNA clones of Y-satRNA and S19-satRNA using common restriction sites showed that the determinant for the induction of yellow mosaic symptoms lies in the BstXI-NheI fragment, in which 14 nucleotide differences are found between the two satellite RNAs. To define more precisely the yellow mosaic determinant(s) in this fragment, several site-directed mutants of Y-satRNA were created. The replacement of AUU, at nucleotides 191 to 193 in Y-satRNA, with GC, which mimics the S19-satRNA sequence at the corresponding site, abolished the ability of Y-satRNA to elicit a yellow mosaic. Conversely, a mutant RNA molecule derived from S19-satRNA in which GC at nucleotides 192 and 193 was changed to AUU induced the yellow mosaic symptoms. Thus, the phenotypes of two satellite RNAs on tobacco can be altered reciprocally by changing the sequences in this limited region.

Base Sequence↗
PubMed1991

Specific nucleotide sequence of HLA-C is strongly associated with psoriasis vulgaris.

The association of specific HLA-C nucleotide sequences with psoriasis vulgaris was investigated in 75 Japanese patients by the polymerase chain reaction method, followed by slot-blot hybridization using two specific oligonucleotide probes. The synthesized nucleotide primers were C180P, 5'-GACCGGGAGACACAGAAGTACAAG-3' (coding for amino acid residues 61 to 68 of the alpha 1 domain of the HLA-C molecule) and C243PR, 5'-GCTCTGGTTGTAGTAGCCGCG-3' (residues 82 to 88), respectively. The amplified sequence detected with the probe C208A (5'-AGGCACAGGCTGACCGA-3'), including the coding region for alanine at position 73, was significantly increased in frequency in the patients compared with the healthy individuals (81% versus 48%, relative risk = 4.7, chi 2 = 15.3, p less than 0.0001). This specific nucleotide sequence is common to Cw6 and Cw7, but some other HLA-C alleles including Cw4 and C blank (Cx52) also proved to have this sequence. It is suggested that alanine at position 73 of HLA-C molecules can be a good marker for psoriasis vulgaris and that this residue may play an important role in determining susceptibility to this disease.

Alanine↗
PubMed1991

[DNA typing of HLA].

Recent advance of molecular biology and genetic engineering has made a profound influence on clinical and laboratory medicine. In this article we will review present aspects of DNA typing of HLA-class II alleles. The HLA system is characterized by its highly genetic polymorphisms. HLA -class I (A, B, C) molecule consists of alpha 1, alpha 2, alpha 3, and beta 2-microglobulin domain, and HLA-class II (DR, DQ, DP) molecule consists of alpha 1, alpha 2, beta 1, and beta 2 domain. Differences of amino acid sequences determining polymorphisms of HLA-class II molecule are located mainly on the beta 1 domain, while alpha 1 chain of DQ molecule is also polymorphic. Polymerase chain reaction (PCR) has enabled us to make great progress in DNA typing of HLA, in place of RFLP (restriction fragment length polymorphisms) analysis. One of the most important and valuable advantages is to make us non-radioisotopic detection method available. There are several methods for the further analysis of products of PCR amplification; 1) dot-blot hybridization, 2) PCR-RFLP, 3) PCR-SSCP (single strand conformation polymorphism), 4) DGGE (denaturing gradient gel electrophoresis), 5) direct sequencing. As for dot-blot hybridization, there are also several methods for detecting hybridization; 1) radioactive detection with 32P - labelled probes, 2) colorimetric reaction and 3) chemiluminescent assay. Newly developed chemiluminescent assay yields as high sensitivity just as radioactive probes. HLA-D and -DP specificities have been typed cytologically by mixed lymphocyte culture (MLC) or primed lymphocyte test (PLT) method. Cytological typing will be replaced by PCR analysis in near future.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1988

Immunoregulatory effects of interleukin 2 and interferon on syngeneic murine malignant glioma-specific cytotoxic T-lymphocytes.

The effects of interleukin 2 (IL2) and interferon (IFN) on the generation and lytic activation of syngeneic murine malignant glioma (a methylcholanthrene-induced ependymoblastoma of C57BL/6 mouse origin, 203-glioma)-specific cytotoxic T-lymphocyte (G-CTL) were investigated. The surface marker analysis showed that G-CTLs from both intracranial and s.c. tumor-bearing mice were composed of thymectomy-resistant (mature) Lyt-1-.2.3+ and thymectomy-sensitive (immature) Lyt-1+.2.3+ CTLs, which markedly decreased concurrently with increased intracranial pressure. G-CTLs were confirmed to be activated with target specificity by both factors in a different way. The CTL activation by IL2 (20 units/ml) remained for a longer time, although a lag time of 5 days after initial culture was required. IL2 influenced Lyt-1+.2.3+ CTLs to proliferate and develop the lytic potential. In contrast, even a 3-h incubation with IFN (1000 units/ml) could enhance the cytotoxicity, but the augmenting effects were observed no longer than 5 days later. IFN activated Lyt-1-.2.3+ CTLs and increased their proportion of the total cell population with a simultaneous decrease of Lyt-1+.2.3+ CTLs. Therefore, it was suggested that IL2 may provide a growth of CTL populations and that IFN can accelerate recruitment of new effectors, causing activation of the lytic process.

Animals↗
PubMed1988

Effects of extra 5' non-viral bases on the infectivity of transcripts from a cDNA clone of satellite RNA (strain Y) of cucumber mosaic virus.

Full-length cDNA of a satellite RNA (Strain Y) which induces bright yellow symptoms on tobacco plants infected with cucumber mosaic virus (CMV) was cloned and sequenced. The published sequence of the satellite RNA was revised with three possible differences (residues 161, 167, and 173) and a nucleotide insertion at residue 234. The satellite cDNA was then inserted into a commercially available transcription vector. In vitro transcription products from the recombinant plasmid harbored 24 non-viral bases at their 5' ends and had very low infectivity when coinoculated with CMV. After removal of the extra 5' sequence of the transcripts with RNase H, the infectivity of the transcripts increased markedly. Analysis of the effects of extra 5' sequences of several lengths confirmed the importance of natural 5' ends for biological activity of the satellite. Trimming down to 6-9 extra bases at the 5' end enhanced the infectivity of the transcripts by 10-fold, although the specific activity of the natural satellite is still 100-fold higher. Dideoxynucleotide sequence analysis proved that the progeny satellite RNA did not retain the 24 non-viral bases at the 5' end of the transcript from pIBI 31-MC.

Amino Acid Sequence↗
PubMed1988

Contribution of hepatic reticuloendothelial system to glomerular IgA deposition in rat liver injury.

Liver damage was induced in rats by a single dose of dimethylnitrosamine or D-galactosamine. In the dimethylnitrosamine model, marked glomerular IgA deposition occurred between Days 4 and 28, with its peak at Day 14. Serum IgA levels were significantly increased at Days 2 and 4, then gradually decreased, and normalized at Day 14. In the D-galactosamine model, however, no such deposition was observed, though serum IgA levels similarly increased on Days 2 and 4. IgA content in high molecular weight fraction from serum increased at Day 3 in both models. This increment remained at Day 7 only in the dimethylnitrosamine model, in which carbon clearance from the circulation was significantly decreased at Day 3. These data suggest that dysfunction of the hepatic reticuloendothelial system is a factor contributing to glomerular IgA deposition occurring in liver injury.

Alanine Transaminase↗
PubMed1986

Interleukin 2 induces rapid phosphorylation of cellular proteins in murine T-lymphocytes.

When quiescent murine T-lymphocyte cells were stimulated by the addition of interleukin 2 (IL-2), they reinitiated DNA synthesis after a lag period of 5 h. Under these conditions, rapid but transient phosphorylation of two cellular proteins with Mr values of 27 000 and 26 000 was detected; maximal phosphorylation occurred within 10-15 min after the addition of IL-2. The protein of Mr 27 000 contained phosphoserine, while the protein of Mr 26 000 contained phosphothreonine.

Animals↗