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Biomedical subjects

A Irie

Publications and source records attributed to A Irie.

At least 37 records · Page 2Linked to original sources
PubMed1999

Alpha 3 beta 1 adhesion to laminin-5 and invasin: critical and differential role of integrin residues clustered at the boundary between alpha 3 N-terminal repeats 2 and 3.

Integrin/ligand interaction is a therapeutic target for many diseases. We previously reported that residues critical for ligand binding are clustered in N-terminal repeat 3 (in the predicted 2-3 loop) of alpha 4, alpha 5 and alpha IIb. Here we have localized residues critical for ligand binding in the alpha 3 subunit of integrin alpha 3 beta 1 with distinct ligand specificity (laminin-5). We identified an alpha 3 epitope common to several function-blocking anti-alpha 3 antibodies at the boundary between repeats 1 and 2 (residues 75-80). We found that swapping the predicted 4-1 loop (residues 153-165) at the boundary between repeats 2 and 3 with the corresponding alpha 4 sequence and mutating Thr-162 and Gly-163 residues in this predicted loop block laminin-5 binding. Thr-162 and Gly-163 and the antibody epitope are separated in the primary structure; however, they are close to each other in the proposed beta-propeller model. Mutating residues recently reported to block (Tyr-186 and Trp-188) or enhance (Asp-122) laminin-5 binding to alpha 3 beta 1 [Krukonis, E. S., Dersch, P., Eble, J. A., and Isberg, R. R.(1998) J. Biol. Chem. 273, 31837-31843] did not affect laminin-5 binding under the assay conditions used. Thr-162 and Gly-163 are not critical for adhesion to invasin, indicating that laminin-5 and invasin may use different recognition mechanisms, and that mutation of Thr-162 and Gly-163 does not drastically affect the integrity of alpha 3 beta 1. These results suggest that residues critical for ligand binding may be similarly (but not identically) located in repeat 3 of the alpha subunit regardless of ligand specificity.

Adhesins, Bacterial↗
PubMed1999

BRCA1 gene mutation and loss of heterozygosity on chromosome 17q21 in primary prostate cancer.

The tumor suppressor gene BRCA1 on chromosome 17q21 has been characterized and shown to be mutated in patients with familial breast and ovarian cancer. Several studies examined the relatives of women with breast cancer and noted an association with ovarian and prostate cancer. This study investigated 24 human prostate cancer specimens for BRCA1 gene mutations and loss of heterozygosity (LOH) on chromosome 17q21 assessed by the polymerase chain reaction. LOH was identified using 7 highly polymorphic tandem repeat markers on chromosome 17q21, in addition to an analysis of the whole coding region of the BRCA1 gene. Four of the 24 prostate cancer specimens showed LOH at one or more loci, all of which were histologically poorly differentiated (4 of 11) and stage D (4 of 15). One of the 24 cases showed a germ-line mutation of the BRCA1 gene, and a sister of this patient died of ovarian cancer. It appears that the BRCA1 gene is not frequently involved in the development of primary prostate cancer.

BRCA1 Protein↗
PubMed1999

Augmentation of immune response by altered peptide ligands of the antigenic peptide in a human CD4+ T-cell clone reacting to TEL/AML1 fusion protein.

The 12;21 chromosomal translocation occurs in leukemic cells from 20(30% of patients with B-lineage childhood acute lymphoblastic leukemia, the result being the TEL/AML1 fusion gene carrying a sequence different from TEL or AML1. Because the protein newly formed by TEL/ AML1 fusion is probably not tolerated by human immune system, the fusion region is a good candidate for tumor antigen expressed only in TEL/ AML1-positive leukemic cells. We established two human CD4+ alphabeta T-cell clones (T31.1 and Y41.2) reacting to the TEL/AML1 fusion region, from two unrelated healthy donors. In order to do this, we stimulated peripheral blood mononuclear cells with synthetic peptides corresponding to the TEL/ AML1 fusion region. Both T31.1 and Y41.2 proliferated in response to TEL/ AML1 fusion protein as well as to a peptide IGRIAECILGMNPSR, in the context of HLA-DP5 and DP17, respectively, and killed B lymphoblastoid cells pulsed with the peptide. Furthermore, these T-cell clones proliferated in response to several altered peptide ligands carrying a single residue substitution in the TEL/AML1 peptide, and some induced augmentation of proliferation and production of Th1-type cytokines. These superagonistic altered peptide ligands can be given consideration for anti-leukemic immunotherapy.

Adjuvants, Immunologic↗
PubMed1999

Therapeutic efficacy of an adenovirus-mediated anti-H-ras ribozyme in experimental bladder cancer.

Ras oncogenes are thought to play a critical role in cellular proliferation and tumorigenesis. Reversal of the malignant phenotype, inhibition of tumor growth, and decreased tumorgenicity have been demonstrated with the use of anti-H-ras ribozymes. In this study, the therapeutic efficacy of a hammerhead ribozyme targeting the mutated H-ras oncogene was investigated in an experimental bladder cancer model using a recombinant adenovirus as delivery vehicle. Tumors were established in nude mice by subcutaneous injection of EJ human bladder carcinoma cells harboring a point mutation of the H-ras gene. The tumors were treated with intralesional injections of an adenovirus expressing an anti-H-ras ribozyme (rAd-Hras Rz) by different schedules at serial titers, and the tumor inhibition efficacy was analyzed. The viral infection efficacy and kinetics of ribozyme expression were also evaluated. Intralesional injection of rAd-Hras Rz resulted in significant antineoplastic effects in a dose-dependent fashion. Complete regression of the tumor was achieved by rAd-Hras Rz in several cases without recurrence during the 50-day observation period. Although there was moderate vector-associated cytotoxicity in this cell line, complete regressions were not observed in the cases treated with control adenovirus vectors or vectors expressing an inactive anti-H-ras ribozyme or anti-H-ras antisense oligonucleotides. These results suggest the efficacy of a ribozyme-encoding adenovirus in the experimental gene therapy of human bladder cancer.

Adenoviridae↗
PubMed1999

[Elevation of serum and urine tumor necrosis factor levels after transurethral resection of the prostate].

BACKGROUND: Recent studies have suggested that inflammatory cytokines are major mediator of the acute phase protein response after surgery. The aim of the present study is to investigate the relationship between the degree of surgical trauma and the change of serum and urine cytokine levels after transurethral resection of the prostate (TUR-P). METHOD: Serum and urine concentrations of tumor necrosis factor-alpha (TNF), interleukin-6 (IL 6), and interleukin-1 (IL 1) were evaluated in 55 patients who underwent TUR-P and in 23 patients who underwent abdominal surgery. The samples were collected periodically before and after an intervention, and the concentrations of cytokines were measured by enzyme-linked immunosorbent assay. RESULTS: The concentration of serum TNF was significantly increased 6 hours after TUR-P. Since serum TNF level was not increased after abdominal surgery, serum TNF level was significantly higher after TUR-P than after abdominal surgery. Serum IL 6 and IL 1 levels were not increased after TUR-P. Urine levels of TNF, IL 6 and IL 1 were significantly increased after TUR-P, meanwhile no significant elevation of urine cytokine levels was recognized in the patients who underwent abdominal surgery. The elevation of urine cytokine levels was thought to be caused by the increased production of cytokines at the surgically resected sites. The urine TNF level after TUR-P was increased related to the resected tissue volume and irrigation fluid volume. The preoperative urinary tract infection caused excessive elevation of the urine TNF level after TUR-P. The urine TNF level after TUR-P also tended to be increased depending on the degree of postoperative pyrexia. CONCLUSION: These results indicate the unique response of TNF to TUR-P. Measurement of serum and urine TNF levels after TUR-P can be a useful index for evaluating the perioperative condition of the patients undergoing TUR-P.

Adult↗
PubMed1998

CMP-N-Acetylneuraminic acid hydroxylase is exclusively inactive in humans.

We cloned cDNAs for mouse and human CMP-N-acetylneuraminic acid (CMP-NeuAc) hydroxylases and showed that the human CMP-NeuAc hydroxylase protein is inactive because of a partial deletion in the hydroxylase gene. We report here that no other active CMP-NeuAc hydroxylases are present in humans. Southern blot analysis showed that the human homologue of the mouse CMP-NeuAc hydroxylase is one gene in the human genome and no other homologues of the mouse hydroxylase exist in human genome. The mouse and the human CMP-NeuAc hydroxylases were mapped to chromosome 13A3 and chromosome 6p22, respectively, by fluorescence in situ hybridization. The chromosomal location of the human hydroxylase is syntenic to that of the mouse hydroxylase. These results demonstrate that the human CMP-NeuAc hydroxylase is the only homologue of the mouse hydroxylase, and CMP-NeuAc hydroxylase is exclusively inactive in humans.

Animals↗
PubMed1998

The molecular basis for the absence of N-glycolylneuraminic acid in humans.

N-Glycolylneuraminic acid (NeuGc) is abundantly expressed in most mammals, but it is not detectable in humans. The expression of NeuGc is controlled by cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase activity. We previously cloned a cDNA for mouse CMP-NeuAc hydroxylase and found that the human genome contains a homologue. We report here the molecular basis for the absence of NeuGc in humans. We cloned a cDNA for human CMP-NeuAc hydroxylase from a HeLa cell cDNA library. The cDNA encodes a 486-amino acid protein, and its deduced amino acid sequence lacks a domain corresponding to the N-terminal 104 amino acids of the mouse CMP-NeuAc hydroxylase protein, although the human protein is highly identical (93%) to the rest of the mouse hydroxylase protein. The N-terminal truncation of the human hydroxylase is caused by deletion of a 92-base pair-long exon in human genomic DNA. The human hydroxylase expressed in COS-7 cells exhibited no enzymatic activity, and a mouse hydroxylase mutant, which lacks the N-terminal domain, was also inactive. A chimera composed of the human hydroxylase and the N-terminal domain of the mouse hydroxylase displayed the enzyme activity. These results indicate that the human homologue of CMP-NeuAc hydroxylase is inactive because it lacks an N-terminal domain that is essential for enzyme activity. The absence of NeuGc in human glycoconjugates is due to a partial deletion in the gene that encodes CMP-NeuAc hydroxylase.

Amino Acid Sequence↗
PubMed1998

New antibacterial diterpenoids from the Sarcodon scabrosus fungus.

The structures of new antibacterial diterpenoids that had been isolated from Sarcodon scabrosus were established by chemical and spectral means to be sarcodonin L (2) and M (3), both having the cyathane skeleton. Other antibacterial compounds were identified to be allocyathin B2 (1), sarcodonin G (4) and sarcodonin A (5) by comparing their spectral data with those of authentic samples.

Anti-Bacterial Agents↗
PubMed1998

Comparison of bromodeoxyuridine labeling indices of smears and sections from mouse NR-S1 tumor.

Bromodeoxyuridine (BrdU) labeling indices (LIs) of smears and sections from mouse NR-S1 tumor were labeled in vivo or in vitro and compared to confirm the reliability of smears labeled in vitro. In vivo labeling was carried out by administering 40 mg/kg 5-bromo-2'-deoxyuridine while in vitro labeling was performed by incubating the tumor specimens in RPMI 1640 culture medium containing 200 microM 5-bromo-2'-deoxyuridine under 3 atmospheres. BrdU LIs of sections labeled in vitro (9.49 +/- 0.16) showed lower values than those labeled in vivo (14.95 +/- 0.36). BrdU LIs of smears labeled in vitro (9.87 +/- 0.23) also showed lower values than those labeled in vivo (14.83 +/- 0.49). However, the BrdU LIs of smears were similar to those of sections labeled in vivo and in vitro. In vitro BrdU immunochemistry in smears seems useful for evaluating the biological properties of tumor tissues.

Animals↗
PubMed1998

[Mutation of p53 gene and genomic instability in testicular tumors].

BACKGROUND: During the past decade, studies of human cancer have begun to yield molecular information on the identify of the multiple genetic changes in the development and progression of tumorigenesis. We investigated alterations of p53 and genomic instability in testicular tumors. MATERIALS AND METHODS: Polymerase chain reaction (PCR) single-strand conformation polymorphism was performed for analysis from exons 5 to 8 of p53 gene in 22 cases and PCR-microsatellite instability analysis using 8 microsatellite markers were conducted in 19 cases of testicular tumor. RESULTS: No mutations were noted for exons 5 to 8 of the p53 gene. Differences in unrelated microsatellites for tumor and corresponding normal DNA were detected in 5 of 19 (26.3%) cases examined. Alterations noted in more than 2 microsatellites were observed in 3 of 19 (15.8%) and categorized as replication error (RER) phenotype. Two of 7 (28.6%) seminomatous and 1 of 12 (8.3%) non-seminomatous testicular tumors patients showed RER. Two of 16 (12.5%) stage T1-3N0M0 and 1 of 3 (33.3%) stage T1-3N1-3M0-1 showed RER. CONCLUSIONS: Alterations in microsatellite instability may be involved in the development of testicular tumor.

DNA Replication↗
PubMed1997

Defining the topology of integrin alpha5beta1-fibronectin interactions using inhibitory anti-alpha5 and anti-beta1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the alpha5 subunit.

The high affinity interaction of integrin alpha5beta1 with the central cell binding domain (CCBD) of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III repeat) and a second site (in the adjacent 9th type III repeat) which synergizes with RGD. We have attempted to map the fibronectin binding interface on alpha5beta1 using monoclonal antibodies (mAbs) that inhibit ligand recognition. The binding of two anti-alpha5 mAbs (P1D6 and JBS5) to alpha5beta1 was strongly inhibited by a tryptic CCBD fragment of fibronectin (containing both synergy sequence and RGD) but not by GRGDS peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5 to alpha5beta1. In contrast, binding of the anti-beta1 mAb P4C10 to alpha5beta1 was inhibited to a similar extent by GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding. P1D6 inhibited the interaction of a wild type CCBD fragment with alpha5beta1 but had no effect on the binding of a mutant fragment that lacked the synergy region. The epitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the alpha5 subunit. Our results indicate that the synergy region is recognized primarily by the alpha5 subunit (in particular by its NH2-terminal repeats) but that the beta1 subunit plays the major role in binding of the RGD sequence. These findings provide new insights into the mechanisms, specificity, and topology of integrin-ligand interactions.

Animals↗
PubMed1997

Multiple loop structures critical for ligand binding of the integrin alpha4 subunit in the upper face of the beta-propeller mode 1.

A non-I-domain integrin, alpha4beta1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of alpha4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of alpha4 (which spans repeats 2-5 of the seven N-terminal repeats) with the corresponding regions of alpha5. Swapping residues 112-131 in repeat 2, or residues 237-247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40-52 in repeat 1, residues 151-164 in repeat 3, or residues 282-288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112-131, 151-164, and 186-191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published beta-propeller folding model of the integrin alpha4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65-72], in which seven four-stranded beta-sheets are arranged in a torus around a pseudosymmetric axis. The regions of alpha4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the beta-propeller model, although they are not adjacent in the primary structure.

Amino Acid Sequence↗
PubMed1997

Structural basis of integrin-mediated signal transduction.

Integrins are a family of alpha/beta heterodimers of cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions. Both alpha and beta subunits have a large extracellular domain and a short cytoplasmic domain. The alpha subunit has seven sequence repeats of 60-70 residues in its N-terminal region. The beta-propeller model, in which seven four-stranded beta-sheets are arranged in a torus around a pseudosymmetry axis, has been proposed as a structural model of these seven repeats. Several predicted loops critical for ligand binding have been identified in the upper face of the proposed beta-propeller model. Several alpha subunits (e.g., alpha 2, alpha L and alpha M) have I-domains of about 200 residues inserted between their second and third repeats. These I-domains adopt a Rossman-fold structure and have major ligand and cation binding sites (the MIDAS site) on their surfaces. The beta subunit has an I-domain-like structure in its N-terminal region. This structure includes multiple sequences/conserved oxygenated residues critical for ligand binding (e.g., Asp-119 in beta 3), and non-conserved residues critical for ligand specificities. Several "activation-dependent" epitopes have been identified in the Cys-rich (stalk) region of beta 1. It has yet to be determined how these multiple ligand binding sites in the alpha and beta subunits are involved in ligand binding, and how conformational changes on activation/ligand occupancy relate to signal transduction.

Animals↗
PubMed1996

Critical residues of integrin alphaIIb subunit for binding of alphaIIbbeta3 (glycoprotein IIb-IIIa) to fibrinogen and ligand-mimetic antibodies (PAC-1, OP-G2, and LJ-CP3).

Integrin alphaIIbbeta3 plays a critical role in platelet aggregation through its interaction with fibrinogen. Elucidation of the mechanisms of alphaIIbbeta3-fibrinogen interaction is critical to understanding hemostasis and thrombosis. Here we report that mutations of Gly-184, Tyr-189, Tyr-190, Phe-191, and Gly-193 within the predicted turn structure of the third amino-terminal repeat of alphaIIb significantly block binding of alphaIIbbeta3 to soluble fibrinogen. These mutations also block binding of alphaIIbbeta3 to ligand-mimetic monoclonal antibodies PAC-1, OP-G2, LJ-CP3, which have an RGD-related RYD sequence in their antigen-binding sites. These mutations do not significantly affect the expression of alphaIIbbeta3, in contrast to most of the natural alphaIIb mutations occurring in Glanzmann's thrombasthenic patients. The data suggest that these residues are critically involved in alphaIIbbeta3-ligand interactions.

Amino Acid Sequence↗