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Y Mitsuishi

Publications and source records attributed to Y Mitsuishi.

At least 19 recordsLinked to original sources
PubMed2001

Enzymatic synthesis and hydrolysis of xylogluco-oligosaccharides using the first archaeal alpha-xylosidase from Sulfolobus solfataricus.

The first, recently identified, archaeal alpha-xylosidase from Sulfolobus solfataricus (XylS) shows high specificity for hydrolysis of isoprimeverose [alpha-D-xylopyranosyl-(1,6)-D-glucopyranose, (X)], the p-nitrophenyl-beta derivative of isoprimeverose, and xyloglucan oligosaccharides and has transxylosidic activity, forming, in a retaining mode, interesting alpha-xylosides. This article describes the synthesis of isoprimeverose, the disaccharidic repeating unit of xyloglucan, of the p-nitrophenyl-beta derivative of isoprimeverose, and of a trisaccharide based on isoprimeverose that is one of the trisaccharidic building blocks of xyloglucan. A substrate structure-activity relationship is recognized for both the hydrolysis and the synthesis reactions of XylS, it being a biocatalyst (i) active hydrolytically only on X-ending substrates liberating a xylose molecule and (ii) capable of transferring xylose only on the nonreducing end glucose of p-nitrophenyl-(PNP)-beta-D-cellobioside. The compounds synthesized by this enzyme are a starting point for enzymological studies of other new enzymes (i.e., xyloglucanases) for which suitable substrates are difficult to synthesize. This study also allows us to define the chemical characteristics of the xylose-transferring activity of this new archaeal enzyme, contributing to building up a library of different glycosidases with high specific selectivity for oligosaccharide synthesis.

Archaeal Proteins↗
PubMed2001

Validation of DNA-based HLA-A and HLA-B testing of volunteers for a bone marrow registry through parallel testing with serology.

A total of 42,160 individuals were typed for HLA-A and HLA-B by both serology and PCR-based typing. The HLA assignments included all of the known serological equivalents. The majority of the individuals (99.9%) were from U.S. minority population groups. The serologic typing was performed between 1993 and 1997 at the time of recruitment for the National Bone Marrow Program (NMDP) registry. The polymerase chain reaction (PCR)-based typing was carried out in two phases. In phase I, DNA typing was performed by PCR using sequence-specific oligonucleotide probes (PCR-SSOP) or PCR using sequence-specific primers (PCR-SSP) without knowledge of the serologic assignments. Discrepancies were identified between the serologic and DNA assignments in 24% of the volunteers (8% of volunteers differed for only HLA-A assignments, 13% for HLA-B, and 3% for both HLA-A and -B) and a potential explanation was assigned each discrepant serology/DNA pair. In phase II, a random sampling scheme was used to select a statistically significant number of individuals for repeat DNA typing from each of these categories. The categories included antigens missed by serology, nonexpressed (null) alleles, PCR amplification failures, misassignment of antigens and nomenclature issues. Only a single individual was found to carry a null allele. DNA-based testing correctly typed nearly 99% of the donors at HLA-A, more than 98% at HLA-B, and more than 97% at both HLA-A and -B validating this methodology for registry typing.

Bone Marrow Examination↗
PubMed2001

An approach for protein to be completely reversible to thermal denaturation even at autoclave temperatures.

Reversibility of protein denaturation is a prerequisite for all applications that depend on reliable enzyme catalysis, particularly, for using steam to sterilize enzyme reactors or enzyme sensor tips, and for developing protein-based devices that perform on-off switching of the protein function such as enzymatic activity, ligand binding and so on. In this study, we have successfully constructed an immobilized protein that retains full enzymatic activity even after thermal treatments as high as 120 degrees C. The key for the complete reversibility was the development of a new reaction that allowed a protein to be covalently attached to a surface through its C-terminus and the protein engineering approach that was used to make the protein compatible with the new attachment chemistry.

Amino Acid Substitution↗
PubMed2000

Human tumor necrosis factor-alpha mutant RGD-V29 (F4614) shows potent antitumor activity and reduced toxicity against human tumor xenografted nude mice.

The antitumor effects of human tumor necrosis factor-alpha (TNF) mutant RGD-V29 (code no. F4614), that includes the cell adhesive sequence (4)Arg-(5)Gly-(6)Asp and (29)Arg-->Val substitution, were evaluated. The therapeutic index, a measure of the extent of the therapeutically-effective range, using three constitutive administrations of RGD-V29 in Meth A-bearing mice was 4.8, whereas that of recombinant human TNF (rhTNF) ((1)SSS(4)RTPSDK...(29)RR...(155)L) was 2.8, clearly indicating that the effective RGD-V29 dose-range was extended. Furthermore, RGD-V29 showed potent antitumor activity against human lung cancer Mqnu-1 xenografted nude mice without severe gastrointestinal and other organ toxicities, even when administered at the maximal tolerated dose (MTD). In contrast, rhTNF induced severe toxicity at the MTD. Direct cytotoxicity of RGD-V29 against Mqnu-1 cells was similar to that of rhTNF. In addition, a cytotoxicity assay using a tumor-derived endothelial-like cell (tEC)/normal endothelial cell (nEC) system used to study TNF antitumor effects on tumor-associated endothelial cells, suggested that RGD-V29 showed preferential cytotoxicity toward tumor-associated endothelial cells compared with rhTNF. Thus, RGD-V29 appears to be a low-toxicity mutant of rhTNF that shows preferential activity towards tumors, and therefore merits further investigation in pre-clinical and clinical studies.

Amino Acid Sequence↗
PubMed2000

Large-scale DNA-based typing of HLA-A and HLA-B at low resolution is highly accurate specific and reliable.

DNA-based typing of HLA class I alleles of the HLA-A and HLA-B loci using sequence-specific oligonucleotide primers and/or probes has been used for the large-scale typing of individuals for the National Marrow Donor Program unrelated donor registry. Typing was performed by 16 laboratories at a low level of resolution (e.g. A*01, B*07). The results of blinded quality control analysis for the first 12 months of the project show the typing to be highly accurate, specific and reliable. The total error rate based on 11,545 HLA-A and 11,428 HLA-B assignments was 1.1% for HLA-A and 1.9% for HLA-B. This level of accuracy is particularly remarkable because the quality control samples could not be distinguished from 64,180 donor samples tested at the same time by the laboratories.

Bone Marrow Transplantation↗
PubMed2000

Characterization of cross-links between cellulose microfibrils, and their occurrence during elongation growth in pea epicotyl.

The occurrence and chemical nature of the cross-links between cellulose microfibrils in outer epidermal cell walls in Pisum sativum cv. Alaska was investigated by rapid-freezing and deep-etching techniques coupled with chemical and enzymatic treatments. The cell wall in the elongating region of epidermal cells was characterized by the absence of the cross-links, while in the elongated region, the cell wall was characterized by the presence of cross-links. The cross-links remained in the cell wall of the elongated region after treatment with SDS electrophoresis sample buffer and treatment with 4% potassium hydroxide. After treatment with endo-1,4-beta-glucanase, which fragments xyloglucan, the cross-links were remarkably reduced from the cell wall of the elongated region. The endoglucanase treatment also reduced immunogold labeling of xyloglucan in the cell wall. The endoglucanase hydrolysate from the cell wall fraction of the elongated region gave spots of oligosaccharides in thin layer chromatography, which were identical to the spots of xyloglucan oligosaccharides produced by xyloglucanase from both the cell wall fraction and tamarind xyloglucan. These results indicate that the cross-links are made of xyloglucan. We discussed the possibility of cross-links involved in the control of mechanical properties of the cell wall.

Cell Wall↗
PubMed2000

Preferential activity of wild-type and mutant tumor necrosis factor-alpha against tumor-derived endothelial-like cells.

Tumor-derived endothelial-like cells (tEC) were prepared by culturing human umbilical vein endothelial cells (HUVEC) in the presence of HT1080 human fibrosarcoma-conditioned medium. tEC showed higher permeability and less cell-adhesion activity than normal HUVEC (nEC). Tumor necrosis factor-alpha (TNF) is known to have tumor-vasculature disrupting activity. tEC showed higher cytotoxicity to recombinant human TNF (rhTNF) than nEC, and was not observed using HUVEC cultured with WI38 human diploid cell-conditioned medium as a medium-control. These results demonstrate that tEC acquire physiological properties of tumor-associated vasculature, and may be a useful model system for the study of the mechanisms of TNF antitumor action. The TNF-mutant RGD-V29 (code No. F4614), which has an inserted 4Arg-Gly-Asp sequence and an 29Arg-->Val replacement, was found to induce greater preferential destruction of tEC compared to rhTNF. When the preferential activities were evaluated in terms of 30% cytotoxicity (IC30) ratio (nEC/tEC), the ratio was 460 for RGD-V29 compared to 4.2 for rhTNF. RGD-V29 also exhibited cell-adhesive function and bound preferentially to the p55 TNF-receptor. Both these properties of RGD-V29 contributed to the tEC selective cytotoxicity, indicating that the RGD ligands and selective p55 receptor binding on the cells, although uncharacterized, are involved in tEC targeting. Therefore, the TNF mutant RGD-V29 may show greater selectivity toward tumor vasculature than wild-type TNF.

Binding, Competitive↗
PubMed1999

Analysis of p53 gene mutations in Helicobacter pylori-associated gastritis mucosa in endoscopic biopsy specimens.

BACKGROUND: No previous report has shown the relationship between Helicobacter pylori infection and a direct sequence analysis of p53 gene mutation in a biopsy sample of human gastric mucosa. METHODS: A total of 60 endoscopic biopsies samples (21 patients with H. pylori-positive gastritis and 9 patients with H. pylori-negative gastritis), including antral mucosa and corpus mucosa, were used in this study. Direct DNA sequencing of exons 5, 6, 7, and 8 of the p53 gene was performed by the dyedeoxy terminator method. RESULTS: Mutations in the p53 gene were identified in non-hot spot codons in exon 7 and 8 in 11 of 21 samples (52.4%) from H. pylori-positive gastritis patients. There was no mutation in H. pylori-negative gastritis patients. CONCLUSIONS: This finding shows that H. pylori infection can induce p53 point mutations and appears to be involved in the pathway leading to dysplasia or carcinoma.

Adult↗
PubMed1998

Influence of different glycosidic linkages on relative ion intensities in post-source decay fragmentation of a xyloglucan heptaoligosaccharide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Post-source decay fragment analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been applied to a highly branched xyloglucan heptasaccharide from tamarind seed. All fragment ions were produced by cleavage of the glycosidic linkages, including multi-site cleavages. The relative intensities of fragment ions that originated from one-site cleavages of the glycosidic linkages were much higher than those arising from two-site cleavages of the same kind of glycosidic linkage, which were in turn higher than those from three-site cleavages. The types of glycosidic linkages were an important factor which influenced the relative intensities of the MALDI-PSD (post-source decay) fragment ions. In the MALDI-PSD fragment spectrum of the xyloglucan heptasaccharide, the relative intensities of the ions produced by the cleavage of an alpha 1-6 glycosidic linkage were much higher than those arising from cleavage of the beta 1-4 glycosidic linkage.

Carbohydrate Sequence↗
PubMed1998

Analysis of the oligosaccharide units of xyloglucans by digestion with isoprimeverose-producing oligoxyloglucan hydrolase followed by anion-exchange chromatography.

A method for rapidly identifying six of the most commonly found xyloglucan oligosaccharide units, XXXG, XLXG, XXLG, XLLG, XXFG, and XLFG was developed by high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) before and after digestion with purified isoprimeverose-producing oligoxyloglucan hydrolase (IPase). Using this method, the compositions of oligosaccharide units of soybean and mung bean xyloglucans were re-examined. Significant amounts of oligosaccharides that have not previously been reported to be oligosaccharide units of soybean and mung bean xyloglucans were found.

Chromatography, Ion Exchange↗
PubMed1998

Structural analysis of xyloglucan oligosaccharides by the post-source decay fragmentation method of MALDI-TOF mass spectrometry: influence of the degree of substitution by branched galactose, xylose, and fucose on the fragment ion intensities.

Highly branched xyloglucan oligosaccharides were analyzed by the post-source decay (PSD) fragmentation method of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The ratio of [M-Xyl]+ and [M-Gal]+ fragment ion intensities could be used to characterize the degree of Gal substitution at the non-reducing end, because the number of possible chemical species was directly related to their relative ion intensity. The intensity of the [M-Fuc]+ ion was predominantly strong in the fragment spectrum of fucosyl oligosaccharides as the first fragmentation, indicating the fucosyl linkage to be much weaker than the other glycosidic linkages in the MALDI-PSD fragmentation. Setting fragment ion [M-Fuc]+ to the pseudo precursor ion [MF]+, the second fragmentation ions were produced from [MF]+ in the drift region in PSD fragmentation of fucosyl oligosaccharides.

Fucose↗
PubMed1997

Comprehensive method for the typing of HLA-A, B, and C alleles by direct sequencing of PCR products obtained from genomic DNA.

Molecular testing is gradually replacing standard typing techniques in the field of HLA because it allows higher resolution, which has significant functional implications. Although several techniques have been so far described for this purpose, the definitive means to determine which alleles are present in a particular sample is to identify their sequence. We describe a simplified method for typing HLA-A, B, and C alleles by direct sequencing of polymerase chain reaction (PCR) products amplified from genomic DNA that could allow large-scale handling of samples for clinical use. The template is the product of a nested PCR. A first round of PCR amplifications from genomic DNA is performed with three different sets of primers, one pair specific for each locus. The PCR products encompass exons 2 and 3, the regions of interest to determine the allele present. These fragments are a mixture of both alleles present in one locus. In a second round of PCRs using the first fragment as template, exons 2 and 3 are separately amplified and simultaneously tailed with sequences corresponding to fluorescent-labeled commercial primers. The sense and antisense sequence of each exon is obtained and compared with a database of all known HLA-A, B, or C alleles. Heterozygous positions are determined and the most probable alleles assigned. This simplified procedure has the practical advantage of allowing high-resolution typing of clinical material by utilizing the same genomic DNA used for standard molecular typing of HLA class I.

Alleles↗
PubMed1997

Structural analyses of xyloglucan heptasaccharide by the post-source decay fragment method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In the post-source decay (PSD) fragment spectrum of a reduced xyloglucan heptasaccharide (XXXGol) from tamarind seeds, eleven sodium-adduct fragment ions and a precursor ion [M + Na]+ were clearly observed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Each fragment ion interval corresponded to the absence of unhydroxylose, unhydroglucose, and glucitol residues, indicating that PSD fragmentation cleavage in the sugar compound occurred only at glycosidic linkages close to the oxygen atom of saccharide ring members, and not in inner sugar ring bonds. The PSD fragment ions were classified into two series, one involving the reducing end and the other involving the non-reducing end. Structural information from both the reducing and non-reducing ends could therefore be simultaneously obtained from the measurement of the positive ion mode. Almost all the fragment ions from species larger than trisaccharide residues could be detected in this PSD fragment experiment. Such fragmentation information will enable the structural determination of xyloglucan oligosaccharides.

Carbohydrate Sequence↗
PubMed1996

Xyloglucan endotransglycosylation in suspension-cultured poplar cells.

Xyloglucan endotransglycosylase activity was identified and defined by transfer of a part of xyloglucan to reduced xyloglucan heptasaccharide ([3H]XXXGol) in an enzyme preparations from suspension-cultured poplar cells. Although the activity was distributed in buffer-soluble and buffer-insoluble fractions associated with cells and in the extracellular fraction, it was mostly recovered in the buffer-insoluble fraction, suggesting that the enzyme was bound to the cell wall. The affinity for acceptor XXXGol was increased at a higher concentration of donor xyloglucan with a constant Vmax. The Vmax for donor xyloglucan was increased at a higher concentration of the oligosaccharide without any change in affinity. These kinetic data suggest that the acceptor acts by combining with the enzyme independently of the donor. The velocity of the reaction decreased gradually as the heptasaccharide units was increased from two to four, suggesting that the xyloglucan endotransglycosylase reaction caused donor xyloglucan substantially to decrease in molecular size. The activity in buffer-soluble fraction was increased by ABA in auxin-starved cells, when cultured in MS medium containing various plant hormones. Nevertheless, the activity increased markedly at the exponential growth and decreased immediately at the stationary phase of cells in the presence of 2,4-D. The activity of xyloglucan endotransglycosylase is developmentally regulated during the growth but is not directly induced by plant hormones.

Carbohydrate Sequence↗
PubMed1996

Purification and characterization of an intracellular alpha-D-xylosidase from Penicillium wortmannii IFO 7237.

Intracellular alpha-D-xylosidase from Penicillium wortmannii IFO 7237 was obtained by grinding the mold with almina in phosphate buffer, and the cell-free extract was purified to a homogeneous state on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was estimated to be 290,000 by gel filtration chromatography (Superdex 200) and 73,000 was obtained by SDS-PAGE. The purified alpha-D-xylosidase had an isoelectric point at pH 5.0. The optimum activity for the enzyme was found to be at pH 6.5 and 45 degrees C. The enzyme showed a hydrolytic activity on p-NO2-phenyl-alpha-D-xylopyranoside (alpha-p-NPX) while methyl-alpha-D-xylopyranoside (alpha-MX) was not hydrolyzed at all. It also showed lower activity for xyloglucan oligosaccharides. The apparent Km values of the enzyme for alpha-p-NPX and isoprimeverose were 1.9 mM and 50 mM, respectively.

Glucans↗
PubMed1995

Overcoming the metastasis-enhancing potential of human tumor necrosis factor alpha by introducing the cell-adhesive Arg-Gly-Asp sequence.

A mutein, F4168, of human tumor necrosis factor alpha (hTNF-alpha) containing the cell-adhesive Arg-Gly-Asp (RGD) sequence near the N terminus was constructed. In contrast to hTNF-alpha, the mutein had binding activity to B16F10/L5 melanoma cells similar to that of fibronectin or laminin, indicating that the adhesive nature of the RGD sequence is conferred upon hTNF-alpha. Introduction of the RGD sequence did not alter the antitumor potential of hTNF-alpha. Simultaneous injection of F4168 and B16F10/L5 melanoma cells into mice did not enhance metastasis formation in lungs, whereas hTNF-alpha significantly promoted it. Enhancement of spontaneous lymph node metastasis of B16F10/L5 cells was also evident in TNF-alpha- but not in F4168-treated mice. In the spontaneous lymph node metastasis model of MethA fibrosarcoma, F4168 injection inhibited metastasis formation more effectively than hTNF-alpha. B16F10/L5 melanoma cells treated with hTNF-alpha enhanced not only their binding activity to laminin but also their invasive potential into Matrigel, whereas F4168 showed no such enhancement. These results suggest that F4168 is a low-toxicity mutein of hTNF-alpha.

Amino Acid Sequence↗
PubMed1995

Novel mutein of tumor necrosis factor alpha (F4614) with reduced hypotensive effect.

To eliminate systemic toxicity, including the hypotension associated with human tumor necrosis factor alpha (TNF-alpha), we constructed mutant proteins (muteins) by mean of genetic engineering. A novel mutein, F4614, containing mutations of 5Thr-->Gly and 6Pro-->Asp, which resulted in the introduction of cell-adhesive Arg-Gly-Asp and 29Arg-->Val, had remarkably reduced hypotensive effects and lower lethality. We present evidence that the Arg-->Val mutation at position 29 is largely responsible for the reduced hypotensive effect. This effect of F4614 was thought to be closely correlated with its low inducibility of nitric oxide and prostaglandin E2 in vivo. In addition, the therapeutically effective dose of F4614 to MethA fibrosarcoma-transplanted mice was increased compared with that of TNF-alpha, indicating a wide therapeutic index. These results indicated that F4614 has several advantages as a systemic therapeutic drug in the treatment of cancer.

Amino Acid Sequence↗