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991.
Influence of rhamnose substituents on the potency of SL0101, an inhibitor of the Ser/Thr kinase, RSK
Smith JA Maloney DJ Clark DE Xu Y Hecht SM Lannigan DA 《Bioorganic & medicinal chemistry》2006,14(17):6034-6042
We have previously reported the isolation of kaempferol 3-O-(3',4'-di-O-acetyl-alpha-l-rhamnopyranoside) from Forsteronia refracta [Xu, Y.-M.; Smith, J. A.; Lannigan, D. A.; Hecht, S. M. Biorg. Med. Chem.2006, 14, 3974-3977.]. This flavonoid glycoside, termed SL0101, is a specific inhibitor of p90 ribosomal S6 kinase (RSK) with a dissociation constant of 1 microM. In intact cells, however, the EC50 for inhibition of RSK activity is 50 microM, which suggests that the efficacy of SL0101 could be limited by cellular uptake. Therefore, we investigated the possibility of developing a more potent RSK inhibitor by synthesizing SL0101 analogs with increased hydrophobic character. The total syntheses of kaempferol 3-O-(3',4'-di-O-butyryl-alpha-L-rhamnopyranoside) (Bu-SL0101) and kaempferol 3-O-(2',3',4'-tri-O-acetyl-alpha-L-rhamnopyranoside) (3Ac-SL0101) were performed. The IC50 for inhibition of RSK activity in in vitro kinase assays for the analogs was similar to that obtained for SL0101. 3Ac-SL0101 demonstrated the same remarkable specificity for inhibiting RSK activity in intact cells as SL0101; however, Bu-SL0101 was not completely specific. 3Ac-SL0101 was approximately 2-fold more potent at inhibiting MCF-7 cell proliferation compared to SL0101 and preferentially decreased MCF-7 cell growth, as compared to the growth of the normal human breast line, MCF-10A. Thus the discovery of 3Ac-SL0101 as a more potent RSK-specific inhibitor than SL0101 should facilitate the development of RSK inhibitors as anti-cancer chemotherapeutic agents. 相似文献
992.
The ERV/ALR sulfhydryl oxidase domain is a versatile module adapted for catalysis of disulfide bond formation in various organelles and biological settings. Its four-helix bundle structure juxtaposes a Cys-X-X-Cys dithiol/disulfide motif with a bound flavin adenine dinucleotide (FAD) cofactor, enabling transfer of electrons from thiol substrates to non-thiol electron acceptors. ERV/ALR family members contain an additional di-cysteine motif outside the four-helix-bundle core. Although the location and context of this "shuttle" disulfide differs among family members, it is proposed to perform the same basic function of mediating electron transfer from substrate to the enzyme active site. We have determined by X-ray crystallography the structure of AtErv1, an ERV/ALR enzyme that contains a Cys-X4-Cys shuttle disulfide and oxidizes thioredoxin in vitro, and compared it to ScErv2, which has a Cys-X-Cys shuttle and does not oxidize thioredoxin at an appreciable rate. The AtErv1 shuttle disulfide is in a region of the structure that is disordered and thus apparently mobile and exposed. This feature may facilitate access of protein substrates to the shuttle disulfide. To test whether the shuttle disulfide region is modular and can confer on other enzymes oxidase activity toward new substrates, we generated chimeric enzyme variants combining shuttle disulfide and core elements from AtErv1 and ScErv2 and monitored oxidation of thioredoxin by the chimeras. We found that the AtErv1 shuttle disulfide region could indeed confer thioredoxin oxidase activity on the ScErv2 core. Remarkably, various chimeras containing the ScErv2 Cys-X-Cys shuttle disulfide were found to function efficiently as well. Since neither the ScErv2 core nor the Cys-X-Cys motif is therefore incapable of participating in oxidation of thioredoxin, we conclude that wild-type ScErv2 has evolved to repress activity on substrates of this type, perhaps in favor of a different, as yet unknown, substrate. 相似文献
993.
"UnPAKing" human immunodeficiency virus (HIV) replication: using small interfering RNA screening to identify novel cofactors and elucidate the role of group I PAKs in HIV infection
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In order to identify novel proviral host factors involved in human immunodeficiency virus (HIV) infection, we performed a screen of a small interfering RNA (siRNA) library targeting 5,000 genes with the highest potential for being targets for therapeutics. Many siRNAs in the library against known host factors, such as TSG101, furin, and CXCR4, were identified as inhibitors by the screen and thus served as internal validation. In addition, many novel factors whose knockdown inhibited infection were identified, including Pak3, a member of the serine/threonine group I PAK kinases. The HIV accessory factor Nef has been shown to associate with a PAK kinase, leading to enhanced viral production; however, the exact identity of the kinase has remained controversial. Prompted by the Pak3 screen hit, we further investigated the involvement of group I PAK kinases in HIV using siRNA. Contrary to the current literature, Pak1 depletion strongly inhibited HIV infection in multiple cell systems and decreased levels of integrated provirus, while Pak2 depletion showed no effect. Overexpression of a constitutively active Pak1 mutant also enhanced HIV infection, further supporting its role as the dominant PAK involved. 相似文献
994.
Feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine 总被引:1,自引:0,他引:1
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Mungall BA Middleton D Crameri G Bingham J Halpin K Russell G Green D McEachern J Pritchard LI Eaton BT Wang LF Bossart KN Broder CC 《Journal of virology》2006,80(24):12293-12302
Nipah virus (NiV) and Hendra virus (HeV) are paramyxoviruses capable of causing considerable morbidity and mortality in a number of mammalian species, including humans. Case reports from outbreaks and previous challenge experiments have suggested that cats were highly susceptible to NiV infection, responding with a severe respiratory disease and systemic infection. Here we have assessed the cat as a model of experimental NiV infection and use it in the evaluation of a subunit vaccine comprised of soluble G glycoprotein (sG). Two groups of two adult cats each were inoculated subcutaneously with either 500 or 5,000 50% tissue culture infective dose(s) (TCID(50)) of NiV. Animals were monitored closely for disease onset, and extensive analysis was conducted on samples and tissues taken during infection and at necropsy to determine viral load and tissue tropism. All animals developed clinical disease 6 to 9 days postinfection, a finding consistent with previous observations. In a subsequent experiment, two cats were immunized with HeV sG and two were immunized with NiV sG. Homologous serum neutralizing titers were greater than 1:20,000, and heterologous titers were greater than 1:20,000 to 16-fold lower. Immunized animals and two additional naive controls were then challenged subcutaneously with 500 TCID(50) of NiV. Naive animals developed clinical disease 6 to 13 days postinfection, whereas none of the immunized animals showed any sign of disease. TaqMan PCR analysis of samples from naive animals revealed considerable levels of NiV genome in a wide range of tissues, whereas the genome was evident in only two immunized cats in only four samples and well below the limit of accurate detection. These results indicate that the cat provides a consistent model for acute NiV infection and associated pathogenesis and an effective subunit vaccine strategy appears achievable. 相似文献
995.
996.
Allison SJ Baldock P Sainsbury A Enriquez R Lee NJ Lin EJ Klugmann M Klugman M During M Eisman JA Li M Pan LC Herzog H Gardiner EM 《The Journal of biological chemistry》2006,281(33):23436-23444
Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment. 相似文献
997.
Frank DJ Martin SR Gruender BN Lee YS Simonette RA Bayley PM Miller KG Beckingham KM 《The Journal of biological chemistry》2006,281(34):24728-24736
Myosin VI, a ubiquitously expressed unconventional myosin, has roles in a broad array of biological processes. Unusual for this motor family, myosin VI moves toward the minus (pointed) end of actin filaments. Myosin VI has two light chain binding sites that can both bind calmodulin (CaM). However unconventional myosins could use tissue-specific light chains to modify their activity. In the Drosophila testis, myosin VI is important for maintenance of moving actin structures, called actin cones, which mediate spermatid individualization. A CaM-related protein, Androcam (Acam), is abundantly expressed in the testis and like myosin VI, accumulates on these cones. We have investigated the possibility that Acam is a testis-specific light chain of Drosophila myosin VI. We find that Acam and myosin VI precisely colocalize at the leading edge of the actin cones and that myosin VI is necessary for this Acam localization. Further, myosin VI and Acam co-immunoprecipitate from the testis and interact in yeast two-hybrid assays. Finally Acam binds with high affinity to peptide versions of both myosin VI light chain binding sites. In contrast, although Drosophila CaM also shows high affinity interactions with these peptides, we cannot detect a CaM/myosin VI interaction in the testis. We conclude that Acam and not CaM acts as a myosin VI light chain in the Drosophila testis and hypothesize that it may alter the regulation of myosin VI in this tissue. 相似文献
998.
Khawaja XZ Smith DL Nawoschik SP Zhang J Dunlop J Dilks DW Olsen M Schechter LE 《Journal of neurochemistry》2006,98(1):134-145
We present evidence that the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor antagonist, N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY-100635), can induce receptor internalization in a human (h)5-HT(1A) receptor Chinese hamster ovary (CHO-K1) cell system. Exposure of h5-HT(1A) CHO cells to WAY-100635 decreased the cell-surface h5-HT(1A) receptor density in a way that was both time (24-72 h) and concentration (1-100 nm) dependent.[(3)H]WAY-100635 and [(3)H]8-hydroxy-dipropylaminotetralin ([(3)H]8-OH-DPAT) saturation analyses demonstrated a significant reduction (50-60%) in total h5-HT(1A) receptor number in the WAY-100635-treated (100 nm; 72 h) compared with control cells. In WAY-100635-treated cells, the 8-OH-DPAT-mediated inhibition of forskolin (FSK)-stimulated cAMP accumulation was right-shifted and the maximal inhibitory response of 8-OH-DPAT was impaired compared with control cells. Similar results were obtained for 8-OH-DPAT-mediated Ca(2+) mobilization after WAY-100635 treatment. h5-HT(1A) receptors labeled with [(3)H]WAY-100635, as well as [(3)H]4-(2'-Methoxy)-phenyl-1-[2'-(N-2'-pyridinyl)-p-fluorobenzamido]ethyl-piperazine (MPPF), exhibited a time-dependent rate of cellular internalization that was blocked by endocytotic suppressors and was pertussis-toxin insensitive. In contrast, quantitative autoradiographic studies demonstrated that chronic treatment of rats with WAY-100635 for two weeks produced a region-specific increase in the 5-HT(1A) receptor density. In conclusion, prolonged exposure of an h5-HT(1A) cell-based system to the 5-HT(1A) antagonist, WAY-100635, induced a paradoxical internalization of cell surface receptor resulting in depressed functional activity. This suggests that an antagonist can influence 5-HT(1A) receptor recycling in vitro differently to in vivo regulatory conditions. 相似文献
999.
Murine models of allergic asthma have been used to understand the mechanisms of development and pathology in this disease. In addition, knockout mice have contributed significantly to our understanding of the roles of specific molecules and cytokines in these models. However, results can vary significantly depending on the mouse strain used in the model, and in particularly in understanding the effect of specific knockouts. For example, it can be equivocal as to whether specific gene knockouts affect the susceptibility of the mice to developing the disease, or lead to resistance. Here we used a house dust mite model of allergic airway inflammation to examine the response of two strains of mice (C57BL/6 and BALB/c) which differ in their responses in allergic airway inflammation. We demonstrate an algorithm that can facilitate the understanding of the behavior of these models with regards to susceptibility (to allergic airway inflammation) (Saai) or resistance (Raai) in this model. We verify that both C57BL/6 and BALB/c develop disease, but BALB/c mice have higher Saai for development. We then use this approach to show that the absence of the Tec family kinase Itk, which regulates the production of Th2 cytokines, leads to Raai in the C57BL/6 background, but decreases Saai on the BALB/c background. We suggest that the use of such approaches could clarify the behavior of various knockout mice in modeling allergic asthma. 相似文献
1000.