| Project/Area Number |
05454189
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Bacteriology (including Mycology)
|
|---|
| Research Institution | The University of Tokyo |
|---|
Principal Investigator |
KANEGASAKI Shiro Inst.of Med.Sci., Univ.of Tokyo Professor, 医科学研究所, 教授 (10012767)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
ONO Yasuo Inst.of Med.Sci., Univ.of Tokyo Reserach Associate, 医科学研究所, 教務職員 (10177272)
KURIBAYASHI Futoshi Inst.of Med.Sci., Univ.of Tokyo Joshu, 医科学研究所, 助手 (60251443)
KOBAYASHI Sonoko Inst.of Med.Sci., Univ.of Tokyo Reserach Associate, 医科学研究所, 教務職員 (00013764)
NUNOI Hiroyuki Inst.of Med.Sci., Univ.of Tokyo Joshu, 医科学研究所, 助手 (50218260)
IMAJOH-OHMI Shinobu Inst.of Med.Sci., Univ.of Tokyo Associate Professor, 医科学研究所, 助教授 (20160046)
|
|---|
| Project Period (FY) |
1993 – 1994
|
| Project Status |
Completed (Fiscal Year 1994)
|
| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1994: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1993: ¥4,000,000 (Direct Cost: ¥4,000,000)
|
|---|
| Keywords | Super oxide / Cytochrome / Neutrophils / Synthetic peptides / NADPH / Phagocytes / スーパーオキシド / b型シトクロム / 47kDa蛋白質 / 電気穿孔法 |
|---|
| Research Abstract |
1)Cytochrome b558 in phagocytes is a transmembrane protein composed of large and small subunits, and considered to play a key role in O_2^--generation during the respiratory burst. We showed previously that two synthetic peptides corresponding to the COOH-terminal region of each subunit inhibit NADPH-dependent oxygen uptake induced by sodium dodecylsulfate (SDS) in a cell free system consisting of plasma membrane and cytosol. Using a cross-linking reagent containing disulfide bound cleavable under reducing conditions (DTBP), we showed that the cytosolic 47-kDa protein, an essential component of the O_2^--generating system, interacted with the synthetic peptides during activation. We now extend the observations to intact neutorphils. The synthetic peptide corresponding to the COOH-terminal region of the large subunit was introduced into neutrophils by electropolation and the cells were stimulated with chemotactic peptide f-Met-Leu-Phe, phorbolmyristate acetate or SDS.The synthetic pepti
… More
de but not unrelated peptide was found to inhibit O_2^--generation induced by the simuli. The synthetic peptide conjugaated with a photoreactive cross-linking agent (Sulfo-SADP) was introduced into the cells by the same procedure but under dark conditions. After stimulaltion with the stimuli, the cells were exposed to UV light. Photoaffinity labeled proteins in the cells were analyzed by SDS-PAGE and subsequently by immunoblotting method using antibody against the synthetic peptide. The cytosolic 47-kDa protein was found to be cross-linked with the synthetic peptide. The results cleanly indicate that the cytosolic COOH-terminal region of cytochrome b_<558> subunit is the binding site for the cytosolic 47-kDa protein, and that the binding takes place during activation of the system.
2)We focused on a hydrophilic region bearing the beta-turn structure near the predicted heme-binding site in the small subunit of the cytochrome (i.e.His-94) and synthesized peptides corresponding to this region. The peptides inhibited superoxide generation in a cell-free system. The shortest sequence that gave a half inhibitory concentration (IC_<50>) lower than 50 muM was TRNYYVRAVL.Substitution of alanine for any of two tyrosine or central valine residues markedly increased IC_<50>, there by 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 b_<558> and cytosolic components.
3)We evaluate activities of neutrophils from immunocompromized hosts by measuring chemiluminescence. Previously, we have shown that the assay is a very sensitive and reliable way for determining the opsonic activity of serum. By using the method, we compared the serum opsonic activity of patients infected with human HIV and that of healthy persons. Sera from the patients exhibited lower opsonic activity than pooled normal serum. The low opsonic activity was found in both asymtomatic HIV-seropositive individuals and patients with acquired immmunodeficiency syndrome. Less
|
