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921.
Inhibition of soluble guanylate cyclase by ODQ 总被引:6,自引:0,他引:6
Zhao Y Brandish PE Di Valentin M DiValentin M Schelvis JP Babcock GT Marletta MA 《Biochemistry》2000,39(35):10848-10854
The heme in soluble guanylate cyclases (sGC) as isolated is ferrous, high-spin, and 5-coordinate. [1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one] (ODQ) has been used extensively as a specific inhibitor for sGC and as a diagnostic tool for identifying a role for sGC in signal transduction events. Addition of ODQ to ferrous sGC leads to a Soret shift from 431 to 392 nm and a decrease in nitric oxide (NO)-stimulated sGC activity. This Soret shift is consistent with oxidation of the ferrous heme to ferric heme. The results reported here further define the molecular mechanism of inhibition of sGC by ODQ. Addition of ODQ to the isolated sGC heme domain [beta1(1-385)] gave the same spectral changes as when sGC was treated with ODQ. EPR and resonance Raman spectroscopy was used to show that the heme in ODQ-treated beta1(1-385) is indeed ferric. Inhibition of the NO-stimulated sGC activity by ODQ is due to oxidation of the sGC heme and not to perturbation of the catalytic site, since the ODQ-treated sGC has the same basal activity as untreated sGC (68 +/- 12 nmol min(-)(1) mg(-)(1)). In addition, ODQ-oxidized sGC can be re-reduced by dithionite, and this re-reduced sGC has identical NO-stimulated activity as the original ferrous sGC. Oxidation of the sGC heme by ODQ is fast with a second-order rate constant of 8.5 x 10(3) M(-)(1) s(-)(1). ODQ can also oxidize hemoglobin, indicating that the reaction is not specific for the heme in sGC versus that in other hemoproteins. 相似文献
922.
Mutation of W215 compromises thrombin cleavage of fibrinogen, but not of PAR-1 or protein C 总被引:3,自引:0,他引:3
W215 is a highly conserved residue that shapes the S3 and S4 specificity sites of thrombin and participates in an edge-to-face interaction with residue F8 of the fibrinogen Aalpha chain. Protein C and the platelet receptor PAR-1 carry an acidic residue at P3 and bind to the active site of thrombin without making contact with W215. This suggested that mutation of W215 could dissociate the cleavage of fibrinogen from that of protein C and PAR-1. Replacement of W215 with Phe produces modest effects on thrombin function, whereas the W215Y replacement compromises significantly the catalytic activity toward all chromogenic and natural substrates that are tested. Replacement of W215 with Ala almost obliterates Na(+) binding, reduces the level of fibrinogen cleavage 500-fold, but decreases the levels of protein C activation and PAR-1 cleavage only 3- and 25-fold, respectively. The W215A mutant cleaves PAR-1 with a specificity constant that is more than 13-fold higher than that of fibrinogen and protein C and is the first thrombin derivative to be described that functions as an almost exclusive activator of PAR-1. The environment of W215 influences differentially three physiologically important interactions of thrombin, which should assist in the study of each of these functions separately in vivo. 相似文献
923.
In the process of characterizing the Na(+)-binding properties of factor Xa, a specific inhibition of this enzyme by quaternary amines was identified, consistent with previous observations. The binding occurs with K(i) in the low millimolar range, with trimethylphenylammonium (TMPA) showing the highest specificity. Binding of TMPA inhibits substrate hydrolysis in a competitive manner, does not inhibit the binding of p-aminobenzamidine to the S1 pocket, and is positively linked to Na(+) binding. Inhibition by TMPA is also seen in thrombin and tissue plasminogen activator (tPA), though to a lesser extent compared to factor Xa. Computer modeling using the crystal structure of factor Xa suggests that TMPA binds to the S2/S3 specificity sites, with its hydrophobic moiety making van der Waals interactions with the side chains of Y99, F174, and W215, and the charged amine coupling electrostatically with the carboxylates of E97. Site-directed mutagenesis of factor Xa, thrombin, and tPA confirms the predictions drawn by docking calculations and reveal a dominant role for residue Y99. Binding of TMPA to factor Xa is drastically (25-fold) reduced by the Y99T replacement. Likewise, the Y99L substitution compromises binding of TMPA to tPA. On the other hand, the affinity of TMPA is enhanced 4-fold in thrombin with the substitution L99Y. The identification of a binding site for quaternary amines in factor Xa has a bearing on the rational design of selective inhibitors of this clotting enzyme. 相似文献
924.
The combined action of IL-15 and IL-12 gene transfer can induce tumor cell rejection without T and NK cell involvement 总被引:6,自引:0,他引:6
Di Carlo E Comes A Basso S De Ambrosis A Meazza R Musiani P Moelling K Albini A Ferrini S 《Journal of immunology (Baltimore, Md. : 1950)》2000,165(6):3111-3118
The cooperative antitumor effects of IL-12 and IL-15 gene transfer were studied in the N592 MHC class I-negative small cell lung cancer cell line xenotransplanted in nude mice. N592 cells engineered to secrete IL-15 displayed a significantly reduced tumor growth kinetics, and a slightly reduced tumor take rate, while N592 engineered with IL-12 displayed only minor changes in their growth in nude mice. However, N592 cells producing both cytokines were completely rejected, and produced a potent local bystander effect, inducing rejection of coinjected wild-type tumor cells. N592/IL-12/IL-15 cells were completely and promptly rejected also in NK-depleted nude mice, while in granulocyte-depleted animals a slight delay in the rejection process was observed. Immunohistochemical analyses of the N592/IL-12/IL-15 tumor area in intact nude mice revealed the presence of infiltrating macrophages, granulocytes, and NK cells, and expression of inducible NO synthase and of secondary cytokines such as IL-1beta, TNF-alpha, and IFN-gamma, and at higher levels GM-CSF, macrophage-inflammatory protein-2, and monocyte chemoattractant protein-1. In NK cell-depleted nude mice, numerous macrophages and granulocytes infiltrated the tumor, and a strong expression of macrophage-inflammatory protein-2 and inducible NO synthase was also observed. Finally, macrophages cocultured with N592/IL-12/IL-15 produced NO in vitro, and inhibited tumor cell growth, further suggesting their role as effector cells in this model. 相似文献
925.
Saviano M Isernia C Rossi F Di Blasio B Iacovino R Mazzeo M Pedone C Benedetti E 《Biopolymers》2000,53(2):189-199
A solid state analysis of the cyclic octapeptide c(-Pro(1)-Pro-Phe-Phe-Ac(6)c-Ile-D-Ala-Val(8)-) (C8-CLA), containing the Pro-Pro-Phe-Phe sequence, followed by the bulky helicogenic C(alpha,alpha)-dialkylated 1-aminocyclohexane-1-carboxylic acid (Ac(6)c) residue and a D-Ala residue in position 7, has been carried out by x-ray diffraction.The crystals, grown from a DMSO solution, are monoclinic, space group P2(1) with a = 13.458(3) A, b = 19. 404(5) A, c = 21.508(4) A, and beta = 90.83(6) degrees, with two independent cyclic molecules in the asymmetric unit, two DMSO molecules, and three water molecules. The structure has been solved using the half and bake procedure by Sheldrick, and refined to final R1 and wR2 indices of 0.0613 and 0.1534 for 9867 reflections with I > 2sigma(I).This cyclic peptide, a deletion analogue of the naturally occurring cyclic nonapeptide cyclolinopeptide A [c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), CLA] has been designed to study the influence of the ring size reduction on the conformational behavior of CLA and more in general to obtain structural information on asymmetric cyclic octapeptides.The compound exhibits, in the solid state, a "banana-twisted" conformation with a cis peptide bond located between the two proline residues. Five intramolecular H bonds stabilize the structure: one type VIa beta-turn, two consecutive type III/I beta-turns, one gamma-turn, and one C(16) bend.The structure has also been compared with either the solution structure previously reported by us and obtained by nmr and computational analysis, and with solid state structural data reported in the literature on cyclic octapeptides. 相似文献
926.
The effect of lysine residues on the deamidation reaction of the asparagine side chain has been studied on the peptide and on its lysine-acetylated derivative in a wide range of pH values. The amino acid sequence of these peptides is similar to the local sequence flanking the labile Asn-67 in RNAse A. The experimental data show that Lys influences both the deamidation rate and the relative yield of the two reaction products, i.e., the aspartic acid and beta-aspartic acid containing peptide. These effects are pH dependent and can be rationalized based on the mechanism previously proposed for the deamidation reaction via succinimide derivative. 相似文献
927.
Ollmann M Young LM Di Como CJ Karim F Belvin M Robertson S Whittaker K Demsky M Fisher WW Buchman A Duyk G Friedman L Prives C Kopczynski C 《Cell》2000,101(1):91-101
The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage. 相似文献
928.
An adult male mountain hare (Lepus timidus), shot during the hunting season on the slopes of Mt. Civetta (eastern Alps, Veneto, northeastern Italy), was eviscerated and diffuse nodular lesions of verminous pneumonia were observed. Adult helminths were collected and male specimens were identified as Protostrongylus pulmonalis. Histologically, a non-purulent interstitial pneumonia and a desquamative bronchiolitis and alveolitis, along with thickening of alveolar septa, were apparent in the nodules. In several enlarged alveoli, a large number of eggs and larvae of nematodes were found. This is the first recorded case of protostrongyliasis in mountain hares from Italy and from an alpine population. 相似文献
929.
930.
Fiore JR Laddago V Lepera A La Grasta L Di Stefano M Saracino A Lopalco P Pastore G Angarano G 《The new microbiologica》2000,23(1):85-92
The presence and antigen specificity of IgG and secretory-IgA (s-IgA) to HIV-1 were evaluated in cervicovaginal lavages (CVL) from 26 infected and 10 high-risk seronegative women. All the seropositive women had detectable IgG recognizing several viral antigens, while a smaller percentage of women demonstrated s-IgA to the virus. In addition, s-IgA were of limited specificity and provided weak reactivities on Immunoblot bands; an almost constant absence of s-IgA to gp120 was also observed. Neither the presence nor the specificity of either IgG or s-IgA to the virus in CVL prevented the shedding of HIV-1 in this body fluid; in fact, viral RNA was detected in all the women studied and the amounts of viral shedding was unrelated to the genital antibody response. On the other hand, none of the high-risk seronegative women had detectable antibodies to HIV-1 in CVL of either the IgG or s-IgA isotype. Our results a) confirm an impairment of mucosal antibody response during HIV-1 infection and suggest that mucosal immunity is not able to prevent viral shedding in the female genital tract and thus cannot modulate the infectivity of genital secretions; aa) do not provide evidence for a mucosal "memory/protective" antibody response in the genital tract of high-risk seronegative women. 相似文献