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Biomedical subjects

H Nunoi

Publications and source records attributed to H Nunoi.

At least 55 records · Page 3Linked to original sources
PubMed1995

[Neutropenia in patient with X-linked hyper-IgM syndrome].

The X-linked form of hyper-IgM syndrome (HIGM1) is a rare disorder characterized by the inability of B cells to undergo isotype switch by a deficiency of CD40 ligand (CD40L) on activated T lymphocytes. The patients suffer from recurrent infections not only due to a lack of B lymphocyte activation but also due to defect of T lymphocyte functions. In addition, neutropenia is frequently accompanied by these symptoms. A patient with HIGM1, we experienced, suffered from recurrent infections and neutropenia. But he had a normal number of hematopoietic stem cell by the in vitro colony forming assay. CD34+ myeloid stem cell has been known to express CD40. We speculated by these facts that myeloid cell numbers are regulated by CD40-CD40L interaction.

Adolescent↗
PubMed1994

Peptides corresponding to the region adjacent to His-94 in the small subunit of cytochrome b558 inhibit superoxide generation in a cell-free system from human neutrophils.

Synthetic peptides corresponding to the region adjacent to His-94 in the small subunit of cytochrome b558 inhibited superoxide generation in a cell-free system. The shortest sequence that gave a half inhibitory concentration (IC50) lower than 50 microM was TRNYYVRAVL. Substitution of alanine for any of two tyrosine or central valine residues markedly increased IC50, thereby indicating that these residues are critical for the activity. Since the inhibition was observed when the peptide was added to the system before but not after the stimulation with sodium dodecylsulfate, the peptides seem to interact irreversibly with the cytochrome molecule and hinder electron transport or alternatively to interfere with the association between cytochrome b558 and cytosolic components.

Amino Acid Sequence↗
PubMed1994

Analysis of the NADPH oxidase components during differentiation of HL-60 cells to eosinophilic lineage.

HL-60 cells were induced to differentiate into eosinophil-like cells with sodium butyrate after passage under mild alkaline condition. The differentiating cells gradually possessed the Luxol-fast-blue (LFB) staining-positive granules and the capacity to produce superoxide. The increase in the amounts of cytochrome b-558 paralleled the superoxide anion generating activity. Immunoblot analysis demonstrated that p47-phox cytosolic oxidase protein appeared 1 day after differentiation, and increased up to 7 days. On the other hand, p67-phox cytosolic oxidase protein appeared in 3 days, and increased gradually up to 7 days. The oxidase activity did not appear until p67-phox protein was expressed in the cytosol during eosinophilic differentiation, indicating that p67-phox protein is likely to be a key protein of cytosolic factors also in eosinophilic differentiating cells. The amounts of p47-phox and p67-phox translocated to the plasma membrane in response to phorbol myristate acetate (PMA) increased with increasing amounts of cytochrome b-558 in the membrane. Our data demonstrate that the appearance of NADPH oxidase activity during eosinophilic differentiation is dependent on the levels of p47-phox and p67-phox cytosolic proteins translocated to the plasma membrane and the amount of cytochrome b-558 in the membrane as observed with neutrophils and monocytes.

Cell Differentiation↗
PubMed1994

Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase.

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.

Amino Acid Sequence↗
PubMed1994

Homologous dinucleotide (GT or TG) deletion in Japanese patients with chronic granulomatous disease with p47-phox deficiency.

The cytosolic 47-kDa protein designated as p47-phox (phagocyte oxidase) is one of the essential components of the superoxide-generating system in phagocytes, and its defect is known to cause chronic granulomatous disease (CGD). Five unrelated CGD patients with p47-phox deficiency were found among 82 CGD patients in Japan. We sequenced the cDNAs and the genomic DNAs corresponding to p47-phox derived from these patients. In all cases examined, the defect was identified to be a GT (or TG) dinucleotide deletion at bases 75/76 (or 74/75, respectively) in the coding sequence for the protein. The same mutation was reported previously for a total of 9 alleles from 5 CGD patients in England and in the United States. It seems, therefore, that the dinucleotide GT deletion is the common mutation in 47-phox deficient CGD due to certain structural issues.

Adolescent↗
PubMed1994

A family of selective immunodeficiency with normal immunoglobulins: possible autosomal dominant inheritance.

We report here our findings in two Japanese siblings who experienced recurrent bacterial and viral infections since early infancy. Recent symptoms included diarrhoea, conjunctivitis, rashes, headache, sore throat, joint pain, vomiting and vertigo, all similar to those seen in toxic shock syndrome, except for shock. These symptoms improved following gammaglobulin treatment. Staphylococcus aureus with coagulase type IV was continuously isolated from nasal smears producing toxic shock syndrome toxin-1 (TSST-1). Serum antibodies did not or only poorly responded to TSST-1, diphtheria toxoid, varicella virus and rubella virus, whereas total and subclass levels of serum immunoglobulin and in vitro DNA synthesis of lymphocytes stimulated by TSST-1, Staph. aureus, varicella vaccine and mitogens were normal. In the family, ten other members in three generations (five males: five females) including the mother had similar clinical symptoms. Thus, the disease may be inherited in an autosomal dominant fashion.

Antibodies, Bacterial↗
PubMed1993

Combination of arachidonic acid and guanosine 5'-O-(3-thiotriphosphate) induce translocation of rac p21s to membrane and activation of NADPH oxidase in a cell-free system.

The superoxide-generating NADPH oxidase system in phagocytes consists of membrane-associated cytochrome b558 and three cytosolic components named p67-phox, p47-phox, and rac p21s. In a cell-free system consisting of membrane and cytosol, the oxidase can be activated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and an unsaturated fatty acid such as arachidonic acid (AA). Incubation of cytosol and membrane with AA alone caused clear translocation of p47-phox and p67-phox to the membrane, but only slight translocation of rac p21s. GTP gamma S alone did not significantly induce the translocation of rac p21s. However, GTP gamma S in combination with AA markedly augmented rac p21s translocation to the membrane. The translocation of rac p21s is not induced by GDP or GDP beta S. These results indicate that the GTP-bound active form of rac p21s is the entity that is translocated to the membrane by the action of AA.

Amino Acid Sequence↗
PubMed1993

Characterization of the GTP-dependent activation of the superoxide-producing NADPH oxidase in a cell-free system of pig neutrophils.

We characterized the cell-free activating system of the superoxide (O2-)-producing NADPH oxidase of pig neutrophils. Activation of the oxidase required both the membrane and cytosolic fractions in the presence of sodium dodecyl sulfate. Chromatography on 2',5'-ADP-Sepharose resulted in separation of the cytosolic fraction into two fractions, the flow-through and bound fractions, which synergistically supported the O2- production with the membrane fraction in the absence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), whereas only the bound fraction besides the membrane fraction was required for the activation in the presence of GTP gamma S. The effective factors in the bound fraction were further purified by gel filtration on Superdex G-200 and anion exchange chromatography on Mono Q and found to be p47-phox and p63-phox. The purified recombinant p47-phox and p65-phox replaced corresponding native factors for the activation. These results suggest that the membrane fraction from pig neutrophils contains the GTP-binding protein responsible for the activation. Furthermore, the presence of the GTP-binding protein for the activation in the flow-through fraction from 2',5'-ADP-Sepharose was also shown on the basis of the findings that extensive dialysis of the flow-through fraction resulted in complete loss of the ability to activate the oxidase with the recombinant factors and the washed membrane of human neutrophils which contained no GTP-binding protein for the activation and the lost ability was recovered by the addition of GTP gamma S. Thus, activation of the oxidase in the cell-free system of pig neutrophils absolutely requires the GTP-binding protein which localizes in the membrane fraction or in the cytosolic fraction.

Animals↗
PubMed1992

Post-translational processing of rac p21s is important both for their interaction with the GDP/GTP exchange proteins and for their activation of NADPH oxidase.

rac1 and rac2 p21s are ras p21-like small GTP-binding proteins which are implicated in the NADPH oxidase-catalyzed superoxide generation in phagocytes. rac1 and rac2 p21s have a Cys-A-A-Leu (A = aliphatic amino acid) structure in their C-terminal region which may undergo post-translational processing including prenylation, proteolysis, and carboxyl methylation. We studied the function of this post-translational processing of rac p21s in their interaction with the stimulatory and inhibitory GDP/GTP exchange proteins for rac p21s, named smg GDS and rho GDI, and in their NADPH oxidase activation. We produced human recombinant rac1 and rac2 p21s in insect cells and purified them from the membrane and soluble fractions as the post-translationally processed and unprocessed forms, respectively. Post-translationally processed rac1 and rac2 p21s were sensitive to both smg GDS and rho GDI, but post-translationally unprocessed rac1 and rac2 p21s were insensitive to them. The GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-bound form of post-translationally processed rac1 and rac2 p21s stimulated the NADPH oxidase activity, but post-translationally unprocessed rac1 and rac2 p21s were far less effective. These results indicate that both rac1 and rac2 p21s stimulate the NADPH oxidase activity and that their post-translational processing is important not only for their interaction with smg GDS and rho GDI but also for their NADPH oxidase activation.

Amino Acid Sequence↗
PubMed1992

Role of Mg2+ in activation of NADPH oxidase of human neutrophils: evidence that Mg2+ acts through G-protein.

The membrane fraction and three cytosolic proteins of neutrophils, p47-phox, p67-phox and a G-protein, are involved in the cell-free activation of the O2(-)-generating NADPH oxidase in the presence of SDS, though it has been controversial whether the G-protein is required or just enhancing the activity. We have used the three cytosolic factors, the solubilized membrane fraction, GTP gamma S and SDS, and found that both G-protein and GTP gamma S are essential for the activation of the NADPH oxidase. The effect of GTP gamma S is modified by Mg2+: the cations enhance the O2- generation at low concentrations of GTP gamma S, whereas they attenuate the activity at higher concentrations of GTP gamma S. In presence of 10 microM GTP gamma S, the maximal activity is observed at 0.1 microM Mg2+, which is several-fold higher than that at 1 mM Mg2+. The omission of Mg2+ followed by the chelation with EDTA results in loss of the activation, which is completely restored by the addition of Mg2+. Thus, Mg2+ seems to modulate the activation of the NADPH oxidase at the level of the G-protein.

Antibodies, Monoclonal↗
PubMed1992

Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins.

The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.

Amino Acid Sequence↗
PubMed1992

Leukocyte adhesion deficiency: identification of novel mutations in two Japanese patients with a severe form.

Leukocyte adhesion deficiency is a disorder with mutations of the gene for the beta subunit, a component common to three adhesion molecules; LFA-1, Mac-1 and p150,95. The molecular basis of the disorder was studied in two patients with its severe form. In the first patient, the mutant gene expressed an aberrant mRNA, 1.2 kb longer than usual, resulting from a G to A substitution at the splice donor site of a 1.2 kb intron. Several aberrantly spliced messages, arising from splicing at cryptic donor sites, were also identified. The beta subunit proteins deduced from the mRNA sequences lacked half the carboxyl terminal portion. In the second patient, the mutation was a G to A transition at nucleotide 454, which resulted in an Asp128 to Asn substitution of the beta subunit. The 128th Asp residue is located in a region crucial for the association with alpha subunits and strictly conserved among the integrin beta subunits so far analyzed.

Adult↗
PubMed1992

[Molecular bases of chronic granulomatous disease--analysis of the involvement of cytosol factors for NADPH oxidase].

During the past 5 years, the discovery of cell-free superoxide generation system (Bromberg Y, Pick E: Cell Immunol 88:213-221, 1984) has been revolutionized our understanding of phagocyte superoxide generation. Using cell-free system, it was clarified that NADPH oxidase for superoxide generation was comprised of components present in both the plasma membrane as well as in the cytosol. This oxidase could be kept inactive by keeping its components separated from each other within the cell and then quickly bringing them together in the plasma membrane upon activation. We analyzed cytosol components with column method, and clarified that 3 neutrophil cytosol factors (NCF-1/-2/-3) was necessary for reconstitute the cytosol activity which was missing in an autosomal recessive type of Chronic Granulomatous Disease (CGD) patients (Nunoi H, et al:Science 242:1298-1301, 1988). NCF-1/-2 were analyzed with B1 antibody and found their molecular weight as 47 and 67 kilodalton respectively. One of autosomal CGD patients was missing NCF-67k and the others were missing NCF-47k. NCF-47k and -67k were cloned with this antibody and sequenced and expressed as recombinant NCF-47k/-67k using baculovirus/insect cell system. Using these recombinants, we are trying to purify NCF-3 which is reported as small G protein in these days. Using monoclonal antibodies against these recombinants, we analyzed tissues with immunohistochemical methods and are trying to classify the type of CGD patients in Japan. In summary, at least five oxidase components now have been identified (alpha and beta chain of cytochrome b558 (gp91-phox, p22-phox) and NCF-47k/-67k/-3 (p47-phox/p67-phox/delta-1 or SOCI)).

Cytosol↗
PubMed1991

Characterization of neutrophil NADPH oxidase factors p47-phox and p67-phox from recombinant baculoviruses.

Superoxide production by phagocytic blood cells involves assembly of an active NADPH oxidase complex from components found both in membrane and cytosolic locations in resting cells. We recently cloned cDNAs encoding two cytosolic components (p47-phox and p67-phox) of the oxidase that are deficient in distinct forms of autosomal recessive chronic granulomatous disease. The precise roles of p47-phox and p67-phox were explored further using purified factors produced in large quantities using recombinant baculoviruses to infect cultured Sf9 insect cells. Neither p47-phox nor p67-phox are thought to represent the flavoprotein components of the oxidase, since neither of the purified recombinant factors contained or bound FAD. Recombinant p47-phox and p67-phox are capable of restoring the deficient cytosol from chronic granulomatous disease patient neutrophils to nearly normal levels in a cell-free reconstitution system. Both p47-phox and p67-phox, used together in the absence of neutrophil cytosol, are incapable of supporting cell free production of superoxide, confirming the involvement of other soluble factor(s) in the assembly of an active oxidase in vitro.

Baculoviridae↗
PubMed1990

Evidence for a functional cytoplasmic domain of phagocyte oxidase cytochrome b558.

Cytoplasmic domains of transmembrane proteins play a critical role in cellular processes involving interactions between membrane and cytosolic components. Activation of the phagocytic cell respiratory burst oxidase, the electron transport chain responsible for superoxide anion (O2-.) production, requires membrane components including cytochrome b558 and several cytosolic proteins; but the biochemical interactions of these components are poorly understood. Cytochrome b558 is an electron transport component of the oxidase. A role for cytochrome b558 in the organization or integration of other oxidase components has also been hypothesized. Antibodies binding the cytoplasmic carboxyl-terminal tail of the transmembrane 91-kDa subunit of cytochrome b558 specifically inhibited an amphiphile-activated cell-free O2-.-generating system that requires neutrophil membranes and cytosol. Synthetic peptides encompassing a 7-amino acid carboxyl-terminal sequence (RGVHFIF) within the same region of the 91-kDa subunit blocked activation of the oxidase by arachidonate, but did not affect activity of the assembled oxidase when added after arachidonate to the cell-free O2-.-generating system. The same peptides inhibited activation of the respiratory burst when allowed to diffuse into electrically permeabilized neutrophils before stimulation with formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate. These studies define a functional cytoplasmic domain of the transmembrane 91-kDa subunit of cytochrome b558 which may mediate interactions with other cellular proteins essential to activation of the phagocyte respiratory burst.

Amino Acid Sequence↗
PubMed1990

Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src.

Chronic granulomatous diseases (CGDs) are characterized by recurrent infections resulting from impaired superoxide production by a phagocytic cell, nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase. Complementary DNAs were cloned that encode the 67-kilodalton (kD) cytosolic oxidase factor (p67), which is deficient in 5% of CGD patients. Recombinant p67 (r-p67) partially restored NADPH oxidase activity to p67-deficient neutrophil cytosol from these patients. The p67 cDNA encodes a 526-amino acid protein with acidic middle and carboxyl-terminal domains that are similar to a sequence motif found in the noncatalytic domain of src-related tyrosine kinases. This motif was recently noted in phospholipase C-gamma, nonerythroid alpha-spectrin (fodrin), p21ras-guanosine triphophatase-activating protein (GAP), myosin-1 isoforms, yeast proteins cdc-25 and fus-1, and the 47-kD phagocyte oxidase factor (p47), which suggests the possibility of common regulatory features.

Amino Acid Sequence↗