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821.
822.
LR James CH Griffiths J Garthwaite TC Bellamy 《British journal of pharmacology》2009,158(6):1454-1464
Background and purpose:
Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3′-5′ cyclic guanosine monophosphate (cGMP). Ca2+-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC50 values of 11, 80 and 180 µM respectively. Here we sought to elucidate the underlying mechanism(s).Experimental approach:
We used cerebellar cell suspensions to determine the influence of CaM antagonists on all steps of the NO-cGMP pathway. Homogenized tissue and purified enzyme were used to test effects of calmidazolium on sGC activity.Key results:
Inhibition of cGMP accumulation in the cells did not depend on changes in intracellular Ca2+ concentration. Degradation of cGMP and inactivation of NO were both inhibited by the CaM antagonists, ruling out increased loss of cGMP or NO as explanations. Instead, calmidazolium directly inhibited purified sGC (IC50= 10 µM). The inhibition was not in competition with NO, nor did it arise from displacement of the haem moiety from sGC. Calmidazolium decreased enzyme Vmax and Km, indicating that it acts in an uncompetitive manner.Conclusions and implications:
The disruption of every stage of NO signal transduction by common CaM antagonists, unrelated to CaM antagonism, cautions against their utility as pharmacological tools. More positively, the compounds exemplify a novel class of sGC inhibitors that, with improved selectivity, may be therapeutically valuable. 相似文献823.
Surgical resection remains the primary component of cancer therapy. The precision required to successfully separate cancer tissue from normal tissue relies heavily on the surgeon's ability to delineate the tumor margins. Despite recent advances in surgical guidance and monitoring systems, intra-operative identification of these margins remains imprecise and directly influences patient prognosis. If the surgeon had improved tools to distinguish these margins, tumor progression and unacceptable morbidity could be avoided. In this article, we review the history of chlorotoxin and its tumor specificity and discuss the research currently being generated to target optical imaging agents to cancer tissue. 相似文献
824.
825.
Chronic matrix metalloproteinase inhibition following myocardial infarction in mice: differential effects on short and long-term survival 总被引:2,自引:0,他引:2
Spinale FG Escobar GP Hendrick JW Clark LL Camens SS Mingoia JP Squires CG Stroud RE Ikonomidis JS 《The Journal of pharmacology and experimental therapeutics》2006,318(3):966-973
Left ventricular (LV) remodeling occurs after myocardial infarction (MI), and the matrix metalloproteinases (MMPs) contribute to adverse LV remodeling after MI. Short-term pharmacological MMP inhibition (MMPi; days to weeks) in animal models of MI have demonstrated a reduction in adverse LV remodeling. However, the long-term effects (months) of MMPi on survival and LV remodeling after MI have not been examined. MI was induced in adult mice (n = 131) and, at 3 days post-MI, assigned to MMPi [MI-MMPi: (s)-2-(4-bromo-biphenyl-4-sulfonylamino)-3-methyl-butyric acid (PD200126), 7.5 mg/day/p.o., n = 64] or untreated (MI-only, n = 67). Unoperated mice (n = 16) served as controls. The median survival in the MI-only group was 5 days, whereas median survival was significantly greater in the MI-MMPi group at 38 days (p < 0.05). However, with prolonged MMPi (>120 days), a significant divergence in the survival curves occurred in which significantly greater mortality was observed with prolonged MMPi (p < 0.05). LV echocardiography at 6 months revealed LV dilation in the MI-only and MI-MMPi groups (154 +/- 14 and 219 +/- 24 microl) compared with control (67 +/- 4 microl, p < 0.05), with a greater degree of dilation in the MI-MMPi group (p < 0.05). MMPi conferred a beneficial effect on survival early post-MI, but prolonged MMPi (>3 months) was associated with higher mortality and adverse LV remodeling. These unique results suggest that an optimal temporal window exists with respect to pharmacological interruption of MMP activity in the post-MI period. 相似文献
826.
827.
Hemant Kumar Vladimir Kasho Irina Smirnova Janet S. Finer-Moore H. Ronald Kaback Robert M. Stroud 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(5):1784-1788
Here we describe the X-ray crystal structure of a double-Trp mutant (Gly46→Trp/Gly262→Trp) of the lactose permease of Escherichia coli (LacY) with a bound, high-affinity lactose analog. Although thought to be arrested in an open-outward conformation, the structure is almost occluded and is partially open to the periplasmic side; the cytoplasmic side is tightly sealed. Surprisingly, the opening on the periplasmic side is sufficiently narrow that sugar cannot get in or out of the binding site. Clearly defined density for a bound sugar is observed at the apex of the almost occluded cavity in the middle of the protein, and the side chains shown to ligate the galactopyranoside strongly confirm more than two decades of biochemical and spectroscopic findings. Comparison of the current structure with a previous structure of LacY with a covalently bound inactivator suggests that the galactopyranoside must be fully ligated to induce an occluded conformation. We conclude that protonated LacY binds d-galactopyranosides specifically, inducing an occluded state that can open to either side of the membrane.The lactose permease of Escherichia coli (LacY), a paradigm for the major facilitator superfamily (MFS), binds and catalyzes transport of d-galactose and d-galactopyranosides specifically with an H+ (1, 2). In contrast, LacY does not recognize d-glucose or d-glucopyranosides, which differ only in the orientation of the C4-OH of the pyranosyl ring. By using the free energy released from the energetically downhill movement of H+ in response to the electrochemical H+ gradient (), LacY catalyzes the uphill (active) transport of galactosides against a concentration gradient. Because coupling between sugar and H+ translocation is obligatory, in the absence of , LacY also can transduce the energy released from the downhill transport of sugar to drive uphill H+ transport with the generation of , the polarity of which depends upon the direction of the sugar gradient.It also has been shown that LacY binds sugar with a pKa of ∼10.5 and that sugar binding does not induce a change in ambient pH; both findings indicate that the protein is protonated over the physiological range of pH (3–5). These observations and many others (1, 2) provide evidence for an ordered kinetic mechanism in which protonation precedes galactoside binding on one side of the membrane and follows sugar dissociation on the other side. Recent considerations (6) suggest that a similar ordered mechanism may be common to other members of the MFS.Because equilibrium exchange and counterflow are unaffected by imposition of , it is apparent that the alternating accessibility of sugar- and H+-binding sites to either side of the membrane is the result of sugar binding and dissociation and not of (reviewed in refs. 1 and 2). Moreover, downhill lactose/H+ symport from a high to a low lactose concentration exhibits a primary deuterium isotope effect that is not observed for -driven lactose/H+ symport, equilibrium exchange, or counterflow (7, 8). Thus, it is likely that the rate-limiting step for downhill symport is deprotonation (9, 10), whereas in the presence of either dissociation of sugar or a conformational change leading to deprotonation is rate-limiting.X-ray crystal structures of WT LacY (11), the conformationally restricted mutant C154G (12, 13), and a single-Cys mutant with covalently bound methanethiosulfonyl-galactopyranoside (MTS-Gal) (14) have been determined in an inward-facing conformation. The structures consist of two six-helix bundles that are related by a quasi twofold symmetry axis in the membrane plane, linked by a long cytoplasmic loop between helices VI and VII. Furthermore, in each six-helix bundle, there are two three-helix bundles with inverted symmetry (6, 15). The two six-helix bundles surround a deep hydrophilic cavity tightly sealed on the periplasmic face and open only to the cytoplasmic side (an inward-open conformation). Although crystal structures reflect only a single lowest-energy conformation under conditions of crystallization, the entire backbone appears to be accessible to water (16–18), and an abundance of biochemical and spectroscopic data (19–27) demonstrate that sugar binding causes the molecule to open alternatively to either side of the membrane, thereby providing strong evidence for an alternating-access model (reviewed in refs. 28 and 29).The initial X-ray structure of conformationally restricted C154G LacY was obtained with density at the apex of the central cavity, but because of limited resolution, the identity of the bound sugar at this site and/or side-chain interactions are difficult to specify with certainty. However, biochemical and spectroscopic studies show that LacY contains a single galactoside-binding site and that the residues involved in sugar binding are located at or near the apex of the central cavity. Although the specificity of LacY is strongly directed toward the C4-OH of the galactopyranosyl ring, other OH groups also are important in the following order: C4-OH >> C6-OH > C3-OH > C2-OH (30, 31). Cys-scanning mutagenesis, site-directed alkylation, and direct binding assays show that Glu126 (helix IV) and Arg144 (helix V) are irreplaceable for substrate binding and probably are charge-paired (4, 32–35). Trp151 (helix V), two turns removed from Arg144, stacks aromatically with the galactopyranosyl ring (36, 37). Glu269 (helix VIII), another irreplaceable residue (38–40), also is essential for sugar recognition and binding and cannot be replaced even with Asp without markedly decreasing affinity (4, 41). It has been shown recently (42) that Asn272 (helix VIII) also is essential for binding and transport. In contrast, Cys148 (helix V), which is protected from alkylation by substrate, and Ala122 (helix IV), where bulky replacements make LacY specific for galactose, are close to the binding site but probably do not contact the sugar directly.Among the conserved residues in LacY and other MFS members are two Gly–Gly pairs between the N- and C-terminal six-helix bundles on the periplasmic side of LacY at the ends of helices II and XI (Gly46 and Gly370, respectively) and helices V and VIII (Gly159 and Gly262, respectively) (43). When Gly46 (helix II) and Gly262 (helix VIII) are replaced with bulky Trp residues (Fig. 1), transport activity is abrogated with little or no effect on galactoside binding (44). Moreover, site-directed alkylation and stopped-flow binding kinetics indicate that the G46W/G262W mutant is open on the periplasmic side (open outward). In addition, the detergent-solubilized mutant exhibits much greater thermal stability than WT LacY (44).Open in a separate windowFig. 1.Side view of LacY G46W/G262W molecules A (Center) and B (Left) in an almost occluded, outward-facing conformation shown in green and gray ribbons, respectively. The two molecules in the asymmetric unit are shown adjacent to one another from the perspective of the membrane plane. The two molecules have a similar conformation with bound TDG, and the Trp replacements at positions 46 and 262 are shown. TDG and the Trp replacements are represented as spheres, with carbon atoms in magenta (for Trp) or orange (for TDG), oxygen atoms in red, nitrogen atoms in blue, and sulfur in yellow. Dashed lines depict the quasi twofold axes relating the N- and C-terminal helix bundles. (Right) The change in structure between LacY G46W/G262W (green; helices numbered) versus the apo WT structure (PDB ID code 2V8N, blue). The orientation matches chain A, and the alignment of the two structures is based on alignment of the N-terminal six-helix bundle of the apo structure onto the G46W/G262W.The G46W/G262W mutant now has been crystallized in the presence of a high-affinity lactose analog. Remarkably, the structure presented here depicts an almost occluded, outward-open conformation with a reliably defined model of the bound sugar molecule. 相似文献
828.
829.