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H Nunoi

Publications and source records attributed to H Nunoi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Drug-selected complete restoration of superoxide generation in Epstein-Barr virus-transformed B cells from p47phox-deficient chronic granulomatous disease patients by using a bicistronic retrovirus vector encoding a human multi-drug resistance gene (MDR1) and the p47phox gene.

Chronic granulomatous disease (CGD) is a group of disorders characterized by the failure of phagocytes to produce superoxide. One-third of the cases of CGD in the USA and Europe results from defects in the gene encoding p47phox, a cytoplasmic component of NADPH oxidase for superoxide generation. In this study, we constructed the bicistronic retrovirus vector Ha-MDR-IRES-p47, which carries cDNAs for a human multi-drug-resistance gene (MDR1) and p47phox. The amphotropic retroviral producer cells were generated, and the supernatant of the producer cells was used to transduce Epstein-Barr virus-transformed B (EBV-B) cells, established from B cells of p47phox-deficient CGD patients, as an in vitro model of gene therapy for p47phox-deficient CGD. The transduced cells expressed both P-glycoprotein and p47phox protein, and the expression levels were increased after appropriate vincristine selection. The levels of superoxide production in the vincristine-selected cells were increased to a level similar to normal EBV-B cells. This result suggests that it is possible to achieve 100% correction of the CGD defect in p47phox-deficient EBV-B cells by using the bicistronic vector. This strategy could be employed not only in vitro, but also in vivo, in the gene therapy of a number of inherited diseases.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Study on the superoxide-producing enzyme of eosinophils and neutrophils--comparison of the NADPH oxidase components.

It has been known that eosinophils produce more superoxide anion (O2-) than neutrophils. To elucidate the mechanism involved in the difference in the superoxide-producing activities, we compared the NADPH oxidase components and the translocation of the cytosolic components between eosinophils and neutrophils. Membrane-bound cytochrome b558, cytosolic p47-phox, p67-phox, and p40-phox were present in both neutrophils and eosinophils, but the amounts of these components were 1.5- to 3.3-fold greater in eosinophils than neutrophils. Upon activation, p47-phox, p67-phox, and p40-phox were translocated to the membranes in both leukocytes, but larger amounts were translocated in eosinophils than in neutrophils. Furthermore, the cross-mixing experiments using membrane and cytosol of eosinophils and neutrophils revealed that more cytosolic components were translocated, and more superoxide-producing activities were obtained using eosinophil fractions. Interestingly, Km values of activated oxidase for NADPH were almost the same in any combination of membrane and cytosol from both leukocytes, indicating that oxidase components are likely similar in both eosinophils and neutrophils. These observations suggest that NADPH oxidase components are more abundant in eosinophils than neutrophils, and, upon activation, larger amounts of NADPH oxidase complex are formed in eosinophils than in neutrophils.

Amino Acid Sequence↗
PubMed1997

Synthetic peptides corresponding to various hydrophilic regions of the large subunit of cytochrome b558 inhibit superoxide generation in a cell-free system from neutrophils.

Cytochrome b558 is a component of the superoxide-generating system in neutrophils and plays key roles in both the assembly of a functional complex with cytosolic proteins and shuttling an electron from NADPH to molecular oxygen. To determine the role of predicted hydrophilic domains of gp91-phox, a glycosylated subunit of the cytochrome, we synthesized peptides corresponding to the regions and tested whether they affected superoxide generation in the cell-free system obtained from human neutrophils. Among twelve peptides tested, six peptides, four of which correspond to previously unreported regions, inhibited superoxide generation in the cell-free system. All of the active peptides were effective when added to the system before activation with sodium dodecyl sulfate. Four peptides, including two peptides corresponding to two newly identified regions, inhibited the translocation of the cytosolic components, p47-phox and p67-phox. The extent of inhibition on translocation of these components varied depending on the peptide used.

Amino Acid Sequence↗
PubMed1997

Mutations of the CD40 ligand gene in 13 Japanese patients with X-linked hyper-IgM syndrome.

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency caused by a defective CD40 ligand. We identified mutations of the CD40 ligand gene in 13 unrelated Japanese XHIM patients. Of the four patients with missense mutations, one had a mutation within the transmembrane domain, and the three others had mutations affecting the TNF homology region of the extracellular domain. Two of the missense mutations resulted in the substitution of amino acids that are highly conserved in TNF family proteins. Three patients had nonsense mutations, all of which resulted in the truncation of the TNF homology domain of the CD40 ligand. Three patients had genomic DNA deletions of 2, 3 or 4 nucleotides, respectively. All of the deletions were flanked by direct repeat sequences, suggesting that these deletions were caused by slipped mispairing. Three patients had mutations within introns resulting in altered splicing, and multiple splicing products were found in one patient. Thus, each of the 13 Japanese patients had different mutations, 9 of them being novel mutations. These results indicate that mutations in XHIM are highly heterogeneous, although codon 140 seems to be a hot spot of the CD40 ligand gene since two additional point mutations were located at Trp 140, bringing the total numbers of mutations affecting codon 140 to six. In one XHIM family with a missense mutation, prenatal diagnosis was performed by single-strand conformation polymorphism analysis of genomic DNA of a male fetus.

Alternative Splicing↗
PubMed1996

Translocation of guinea pig p40-phox during activation of NADPH oxidase.

The superoxide-producing NADPH oxidase consists of membrane-associated cytochrome b558 and cytosolic components, p47-phox and p67-phox. Recently, we have found a novel cytosolic component, p40-phox, which is tightly associated with p67-phox. In this study, we examined the translocation of p40-phox during activation of NADPH oxidase in a cell-free system using the membrane and the purified p47-phox/p67-phox/p40-phox complex. p40-phox was translocated to the membrane by arachidonic acid in a dose-dependent manner. The translocation pattern of p40-phox was similar to those of p47-phox and p67-phox. However, immunoprecipitation assay revealed that p40-phox was dissociated from p47-phox and p67-phox during activation. The translocation of three cytosolic components was not affected by the deletion of GTP-gamma-s from the reaction mixture. Interestingly, a synthetic peptide corresponding to carboxyl-terminus of p40-phox inhibited the activation of NADPH oxidase and translocation of p40-phox, p47-phox, and p67-phox, suggesting that p40-phox might play a role in the activation of NADPH oxidase. These observations suggest that p40-phox is dissociated from p67-phox during activation, and translocates to the membrane by GTP-gamma-s-independent mechanism.

Amino Acid Sequence↗
PubMed1996

Modulation of degranulation and superoxide generation in human neutrophils by unsaturated fatty acids of odd carbon numbers.

Unsaturated fatty acids of odd carbons, 13:1(12), 17:1(10trans), 19:1(7) and 19:1(10) inhibited release of myeloperoxidase (MPO) from fMet-Leu-Phe-cytochalasin B-treated neutrophils. The inhibitory effect was smaller than that of aseanostatins which have been isolated as microbial-derived free fatty acids with a methyl blanch (i-14:0 and ai-15:0) (Journal of Antibiotics (1991) 44, 524-532). These unsaturated fatty acids also inhibited lactoferrin release by the same treatment. On the other hand, 13:1(12), 15:1(10) and 19:1(10) inhibited fMet-Leu-Phe-stimulated superoxide generation of neutrophils, and the fatty acids 15:1(10), 17:(10) and 19:2(10,13) induced superoxide generation in both unstimulated cells and the cell-free system. However, none of unsaturated fatty acids of odd carbons tested inhibited beta-glucuronidase release, whereas 15:1(10), 17:1(10), 19:1(10) and 19:2(10,13) rather enhanced an increase in beta-glucuronidase activity liberated from cells at high concentrations over 35 microM, indicating cellular damages by these fatty acids. These observations suggest that unsaturated free fatty acids having odd carbons such as 13, 15, 17 and 19 may act as modulators of neutrophil functions including degranulation and superoxide generation.

Adult↗
PubMed1996

Two-exon skipping due to a point mutation in p67-phox--deficient chronic granulomatous disease.

The cytosolic 67-kD protein in phagocytes (p67-phox) and B lymphocytes is one of essential components of the superoxide-generating system in these cells, and its defect causes an autosomal recessive type of chronic granulomatous disease (CGD). We performed mutation analysis of p67-phox mRNA from a CGD patient who lacks the protein and found an in-frame deletion from nucleotide 694 to 879, which corresponds to the entire sequence of exons 8 and 9. This sequence encodes one of two Src homology 3 domains and a part of proline-rich domain in p67-phox and lack of these domains seem to have influenced stability of this protein. To know causative reason for the deletion, we analyzed genomic DNA for p67-phox using two sets of primers that covered exons 8 and 9 with adjacent introns. The DNA fragments from the patient were shown to be same in length as those from control. However, the single-strand conformation-polymorphism analysis of the fragments showed that a patient's specimen that included the splice junction of exon 9 exhibited different mobility from the control. By sequencing of the fragment, a homozygous G to A replacement at position +1 of intron 9 was found to be a sole mutation, which reduced the matching score of the splicing sequence to the consensus calculated according to the formula proposed by Shapiro and Senapathy (Nucleic Acids Res 15:7155, 1987). The reduced matching score at the splice doner site (5' splice site) of intron 9 and the original low matching score at the acceptor site (3' splice site) of intron 7 may explain the skipping of exon 8 and 9, and another predicted mechanism is discussed on the basis of Shapiro and Senapathy's hypothesis.

Adult↗
PubMed1996

Suppression of superoxide anion production by interleukin-10 is accompanied by a downregulation of the genes for subunit proteins of NADPH oxidase.

Interleukin-10 (IL-10) inhibited the production of superoxide anion (02-) by both unactivated and interferon-gamma (IFN-gamma)-activated human monocytes. Simultaneous addition of IL-10 with IFN-gamma at the start of incubation was necessary for an optimal inhibitory effect. The degree of inhibition was substantially comparable to that of IL-4, and the combination of suboptimal concentrations of IL-10 and IL-4 produced an additive effect. A similar effect was also obtained when viral IL-10 (vIL-10) was used instead of IL-10. The inhibitory effect of IL-10 was accompanied by the reduced accumulation of transcripts for heavy chain subunit of cytochrome b558 (gp9l-phox) and 47-kD cytosolic factor (p47-phox), components of the O2--generating NADPH oxidase system. Reduction of the mRNAs was distinct within 24 hours. On the other hand, the induced O2- production by human monocytic leukemia cell lines (THP-1 and HL60) was not inhibited by IL-10. The amount of gp9l-phox and p47-phox mRNAs remained unchanged even in the presence of excess amount of IL-1O. Taken together, these results suggest that IL-10 inhibits 02- production by downregulation of the gp9l-phox and p47-phox genes in human monocytes.

Depression, Chemical↗
PubMed1995

AG dinucleotide insertion in a patient with chronic granulomatous disease lacking cytosolic 67-kD protein.

The 67-kD cytosolic protein (p67-phox) is an essential component of the superoxide-generating system in phagocytes, and its defect is known to cause chronic granulomatous disease (CGD). We sequenced p67-phox cDNA from one of seven patients found in Japan and his parents. In the patient's cDNA, homozygous AG dinucleotide insertion at position 399 (or 401) was found together with three other homozygous substitutions in a coding region (A-542 to G, T-895 to C and A-983 to G) compared with the sequence reported for HL-60 cells. In cDNA from his parents, the AG insertion was found to be heterozygous. In contrast, the other three substitutions were found homozygously in his father's specimen and the latter two in his mother's specimen. The substitution of A-542 to G was heterozygous in his mother's cDNA. The AG insertion would induce a frame shift and bring about a stop codon at the position of 433. However, the other three differences would give insignificant changes for the protein function, if any, because the substitutions of A-542 to G and A-983 to G result in the conservative amino acid transitions, ie, Lys-180 to Arg and Lys-327 to Arg, and that of T-895 to C no amino acid change. Neutrophils from the patients completely lacked superoxide generating activity whereas those from his parents generated substantial amounts of superoxide anion upon stimulation. Thus, it is concluded that the AG dinucleotide insertion is responsible for the disease in this patient.

Adult↗
PubMed1995

A new mutation in exon 12 of the gp91-phox gene leading to cytochrome b-positive X-linked chronic granulomatous disease.

We have previously reported a patient with cytochrome b-positive X-linked chronic granulomatous disease. Although the O2- production of neutrophils from the patient was completely defective, we presented data suggesting that the patient's cytochrome b was present at a normal level and possibly had normal spectroscopic features. Thus, to look for a mutation in the cytochrome b heavy chain (gp91-phox) gene, DNA analysis of gp91-phox cDNA derived from this patient was performed. As a result, we found that five nucleotides (1521 through 1525) within exon 12 were deleted, and a new sequence of eight nucleotides was inserted. This mutation converted Gln507-Lys508-Thr509 into His-Ile-Trp-Ala. Mismatched polymerase chain reaction showed that the mother has both wild and mutated alleles, confirming that this case was transmitted in an X-linked fashion. This mutation is different from those previously reported by others. The translocation of p47-phox and p67-phox to the membrane fraction occurred, indicating the complete formation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. We conclude that this case suggests that the structure encoded on exon 12 of gp91-phox is important for electron transfer.

Adult↗
PubMed1995

Structural organization of the gene for CD40 ligand: molecular analysis for diagnosis of X-linked hyper-IgM syndrome.

CD40 ligand (CD40L) on activated T cells plays a crucial role for Ig heavy-chain class switching and the mutation of the gene for this ligand in the X-chromosome causes immunodeficiency with hyper-IgM (X-HIM). We isolated and characterized the human genomic clone for CD40 L to obtain information about the transcriptional regulatory regions of the gene and to develop a molecular diagnostic method for X-HIM patients. The genomic DNA isolated was over 12 kb long containing 5 exons that cover full sequence of mRNA for the ligand. DNA motif analysis based on transcription factor database revealed the presence of a GATA 1 box at around -265 bp. The typical TATA box, CAT site or GC rich region was not found in the 5' flanking region. However, two possible TATA like sequences were found at around -27 and -136 bp. Using the oligonucleotide primers corresponding to the introns, we performed a PCR-SSCP analysis of each exon from a patient with X-HIM syndrome and detected abnormality in exon 5. When sequenced, dinucleotide deletion in this exon was found in the patient and in one X allele of his mother as the only different sequence from that of the control gene. This procedure is simple and could be used for diagnosis of the X-HIM syndrome.

Adolescent↗
PubMed1995

Characterization of the superoxide-generating system in human peripheral lymphocytes and lymphoid cell lines.

In B lymphocytes, but not T lymphocytes, isolated from human peripheral blood, we detected the four protein components essential for "the respiratory burst" by immunoblot analyses using peptide-directed antibodies. These are two membrane proteins, namely, 91- and 22-kDa subunits of cytochrome b558, and two cytosolic proteins with molecular masses of 47 and 65 kDa. Like in neutrophils, cytochrome b558 was expressed on the cell surface of peripheral B lymphocytes. Mean amounts (n = 8) of the 91-, 22-, 47-, and 65-kDa proteins, respectively, in peripheral B lymphocytes calculated from intensity of the blots were 0.011 +/- 0.003, 0.026 +/- 0.006, 0.179 +/- 0.022, and 0.039 +/- 0.013 relative to those in neutrophils on the basis of cell number. Epstein-Barr virus (EBV)-transformed cell lines derived from normal B lymphocytes and some B cell lines also possessed cytochrome b558 and two cytosolic proteins. Isolated human peripheral B lymphocytes generated the superoxide anion upon cross-linking of surface antigens such as IgM, IgD, IgG, HLA-DR, and CD19. EBV-transformants derived from normal peripheral B lymphocytes and B lymphoid cell lines also generated the superoxide anion when stimulated with various antibodies against surface antigens. These results indicate that peripheral B lymphocytes have substantial amounts of a superoxide-generating system identical to that in phagocytes and that the system is stimulated to generate the superoxide anion by the cross-linking of clonally expressed surface immunoglobulins or of certain surface antigens.

Antigens, Surface↗
PubMed1995

Induction of CD40 in promyelocytic HL60 cells cultured with retinoic acid and/or various cytokines.

The antigenic protein CD40 on the surface of B lymphocytes plays an important role in their proliferation, immunoglobulin class switching, and rescue from apoptosis in the germinal center through interaction with T lymphocytes expressing CD40 ligand. The protein is also found on the cell surface of other antigen-presenting cells such as monocytes, dendritic cells, and thymic epithelium cells, but its presence in other myeloid cells has not been reported. We show here that CD40 protein is induced in promyelocytic HL60 cells, when cultured with retinoic acid, a vitamin that converts them to granulocyte-like cells. The cultured cells also expressed CD15, a marker for granulocytes, and cytochrome b(558), an essential component of the superoxide-generating system in phagocytes, on their surface. No detectable amount of mRNA for CD40 was found in naive HL60 cells, whereas a large amount of the message was induced in the cells cultured with the vitamin. Although CD40 expression was enhanced when the cells were further cultured with GM-CSF or IFN-gamma, expression of CD14, a marker for monocytes, was also enhanced. HL60 cells, therefore, express CD40 protein during differentiation not only toward monocytes but also toward granulocytes, at least transiently.

Antigens, Surface↗
PubMed1995

Characterization of antineutrophil antibodies in patients with neutropenia associated with nutritional copper deficiency.

Six antineutrophil antibody (ANA)-positive patients with copper deficiency were classified into two groups; those with (group A, n = 3) and those without (group B, n = 3) neutropenia. The percent binding of ANA for normal peripheral neutrophils was similar in both groups (83.5 +/- 7.2 vs. 79.1 +/- 10.5%). The percent binding of sera to cultured promyelocytic leukemia cells (HL-60) was increased in group A (from 3.7 +/- 3.2 to 12.2 +/- 2.3%) but not in group B (from 65.3 +/- 21.7 to 40.7 +/- 6.3%) after stimulation of HL-60 with DMSO. The stimulated HL-60 cells expressed CD 16 and CD 11b antigens. In the presence of monoclonal antibody for CD 16, the titer of ANA was nil in group A and unchanged in group B. Thus, ANA of patients with neutropenia may recognize mainly the CD 16 antigen, the Fc gamma receptor III of neutrophils.

Adolescent↗
PubMed1995

Signaling through CD40 rescues IgE but not IgG or IgA secretion in X-linked immunodeficiency with hyper-IgM.

The ligand for CD40 (CD40L) is a membrane protein on activated T cells that induces B cell proliferation and differentiation. Several mutations of the CD40L gene were reported responsible for defective class switching of B cells in an X-linked immunodeficiency with hyper IgM (X-HIM). We studied four affected males from three families and found three independent mutations including new mutations of CD40L gene. In every X-HIM patient tested, however, anti-CD40 plus IL-10 did not induce class switching from IgM to IgG or IgA, even in the presence of Staphylococcus aureus Cowan I strain (SAC). CD4+ T cell clones, expressing CD40L on their surface, also did not rescue IgG or IgA induction by X-HIM peripheral blood B cells in vitro. But signaling through CD40 induced both B cell proliferation and IgE secretion when IL-4 was added to the culture. Taken together, these results show that in vitro signaling through CD40 rescues IgE but not IgG or IgA secretion by peripheral blood X-HIM B cells and suggest that in vivo CD40 and CD40L interaction might be necessary for IgG and IgA differentiation in X-HIM.

Amino Acid Sequence↗