摘要:

The cofactor requirements of macrophage nitric oxide (NO·) synthase suggest involvement of an NADPH‐dependent flavoprotein. This prompted us to test the effect of the flavoprotein inhibitors diphenyleneiodonium (DPI), di‐2‐thienyliodonium (DTI), and iodoniumdiphenyl (ID) on the NO· synthases of macrophages and endothelium. DPI, DTI, and ID completely inhibited NO· synthesis by mouse macrophages, their lysates, and partially purified macrophage NO· synthase. Inhibition of NO· synthase by these agents was potent (IC50‘s 50–150 nM), irreversible, dependent on time and temperature, and independent of enzyme catalysis. The inhibition by DPI was blocked by NADPH, NADP+, or 2'5‘‐ADP, but not by NADH. Likewise, FAD or FMN, but not riboflavin or adenosine 5‐diphosphoribose, protected NO· synthase from inhibition by DPI. Neither NADPH nor FAD reacted with DPI. Once NO· synthase was inhibited by DPI, neither NADPH nor FAD could restore its activity. DPI also inhibited acetylcholine‐induced relaxation of norepinephrine‐preconstricted rabbit aortic rings (IC50300 nM). Inhibition of acetylcholine‐induced relaxation persisted for at least 2 h after DPI was washed out. In contrast, DPI had no effect on norepinephrine‐induced vasoconstriction itself nor on vasorelaxation induced by the NO·‐generating agent sodium nitroprusside. These results suggest that NO∗ synthesis in both macrophages and endothelial cells depends on an NADPH‐utilizing flavoprotein. As a new class of NO· synthase inhibitors, DPI and its analogs are likely to prove useful in analyzing the physiologic and pathophysiologic roles of NO·.—Stuehr, D. J.; Fasehun, O. A.; Kwon, N. S.; Gross, S. S.; Gonzalez, J. A.; Levi, R.; Nathan, C. F. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs.FASEB J.5: 98–103; 1991.

ISSN:0892-6638    

DOI:10.1096/fasebj.5.1.1703974

出版商:Wiley    

年代:1991

数据来源: WILEY

摘要:

BALB/3T3 cells were transfected with the glycoprotein D (gD) gene of herpes simplex virus (HSV) and a cell line expressing gD on the cell surface was isolated. In vitro,51Cr release tests showed that the transfected cells were destroyed by anti‐HSV antibody in the presence of complement. To investigate in vivo immune response, the gD‐transfected cells were transplanted into the footpads of syngeneic HSV‐immunized or unimmunized BALB/c mice. In unimmunized mice, transfected cells remained intact for 7 days or longer, and the site of injection showed only slight lymphocyte infiltration. In contrast, in immunized mice, transfected cells elicited massive lymphocyte infiltration and were mostly destroyed by day 4. Analysis of infiltrating cells revealed that they were mainly Thy1+and CD8+lymphoyctes along with small numbers of CD5+, CD4+, and B lymphocytes. These studies show that transfected murine cells expressing gD can be used to study the in vivo immune response to single viral proteins and they argue that the immune response contributes to the pathogenesis of HSV infection.—Nakayama, H.; Shibata, M.; Wohlenberg, C.; Rooney, J. F.; Notkins, A. L. Transplantation of syngeneic transfected cells to probe the in vivo immune response to viral proteins.FASEB J.5: 104–108; 1991.

ISSN:0892-6638    

DOI:10.1096/fasebj.5.1.1846831

出版商:Wiley    

年代:1991

数据来源: WILEY

摘要:

Band 3 is a ubiquitous membrane transport protein found in Golgi, mitochondrial, nuclear, and cell membranes. It is the most heavily used anion transport system in the body because it is responsible for CO2exchange in all tissues and organs and for acid‐base balance. The anion transport regions are mapped along the band 3 molecule using synthetic peptides (pep) from extracellular regions of band 3 and/or suspected anion transport regions. Assays include anion transport/inhibition and immunoblotting with anti‐idiotypic antibodies to a transport inhibitor. Results indicate that anion binding/transport regions of band 3 reside within residues 549–594, (588–594 being the most active) and 804–839 (822–839 being the most active), and 869–883. Pep‐COOH (residues 812–827), which is part of senescent cell antigen, is an anion binding site with most of the activity localized to residues 813–818 (the six amino acids on the amino side of pep‐COOH). The stilbene disulfonate inhibitors of transport bind to peptide 812–830, and possibly peptides 788–805 and 800–818, as determined with anti‐idiotypic antibodies. Residues 538–554, which have been reported to be a transport segment of band 3, do not bind sulfate. Band 3 external loops containing residues 539–553 and 812–830, and internal segments containing residues 588–594 and 869–883, are in close spacial proximity in the membrane. The contribution of lysine and/or arginine to anion transport is examined by synthesizing peptides in which glycines or arginines are substituted for lysines or arginines. Lysines can contribute to anion binding but are not required.—Kay, M. M. B. Molecular mapping of human band 3 anion transport regions using synthetic peptides.FASEB J.5: 109–115; 1991.

ISSN:0892-6638    

DOI:10.1096/fasebj.5.1.1991578

出版商:Wiley    

年代:1991

数据来源: WILEY

ISSN:0892-6638    

DOI:10.1096/fasebj.5.1.1991579

出版商:Wiley    

年代:1991

数据来源: WILEY