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61.
62.
Cossu F Malvezzi F Canevari G Mastrangelo E Lecis D Delia D Seneci P Scolastico C Bolognesi M Milani M 《Protein science : a publication of the Protein Society》2010,19(12):2418-2429
Inhibitor of apoptosis proteins (IAPs) are negative regulators of apoptosis. As IAPs are overexpressed in many tumors, where they confer chemoresistance, small molecules inactivating IAPs have been proposed as anticancer agents. Accordingly, a number of IAP-binding pro-apoptotic compounds that mimic the sequence corresponding to the N-terminal tetrapeptide of Smac/DIABLO, the natural endogenous IAPs inhibitor, have been developed. Here, we report the crystal structures of the BIR3 domain of cIAP1 in complex with Smac037, a Smac-mimetic known to bind potently to the XIAP-BIR3 domain and to induce degradation of cIAP1, and in complex with the novel Smac-mimetic compound Smac066. Thermal stability and fluorescence polarization assays show the stabilizing effect and the high affinity of both Smac037 and Smac066 for cIAP1- and cIAP2-BIR3 domains. 相似文献
63.
Gourlay LJ Sommaruga S Nardini M Sperandeo P Dehò G Polissi A Bolognesi M 《Protein science : a publication of the Protein Society》2010,19(12):2430-2439
Lipopolysaccharide (LPS) biosynthesis represents an underexploited target pathway for novel antimicrobial development to combat the emergence of multidrug‐resistant bacteria. A key player in LPS synthesis is the enzyme D ‐arabinose‐5‐phosphate isomerase (API), which catalyzes the reversible isomerization of D ‐ribulose‐5‐phosphate to D ‐arabinose‐5‐phosphate, a precursor of 3‐deoxy‐D ‐manno‐octulosonate that is an essential residue of the LPS inner core. API is composed of two main domains: an N‐terminal sugar isomerase domain (SIS) and a pair of cystathionine‐β‐synthase domains of unknown function. As the three‐dimensional structure of an enzyme is a prerequisite for the rational development of novel inhibitors, we present here the crystal structure of the SIS domain of a catalytic mutant (K59A) of E. coli D ‐arabinose‐5‐phosphate isomerase at 2.6‐Å resolution. Our structural analyses and comparisons made with other SIS domains highlight several potentially important active site residues. In particular, the crystal structure allowed us to identify a previously unpredicted His residue (H88) located at the mouth of the active site cavity as a possible catalytic residue. On the basis of such structural data, subsequently supported by biochemical and mutational experiments, we confirm the catalytic role of H88, which appears to be a generally conserved residue among two‐domain isomerases. 相似文献
64.
Follicular cumulus-enclosed goat oocytes were matured in vitro in the presence of granulosa cells, follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol-17beta. While 86% of the oocytes from follicles 2 to 6 mm in diameter achieved meiotic maturation, only 24% of the oocytes from follicles 1 to 2 mm in diameter progressed to Metaphase II. Exposure of follicle-enclosed cumulus-oocyte complexes to 20 degrees C prior to culture resulted in 11.5% of the oocytes exhibiting abnormal meiotic spindle. This indicated that immature goat oocytes are particularly sensitive to temperature. Ejaculated spermatozoa were capacitated according to the technique previously proposed for ram sperm (1). The fertilization rates of ovulated and mechanically denuded in vitro-matured oocytes were 85 and 82.8%, respectively; 59.7% of ovulated and 57.1% of in vitro-matured oocytes were normally fertilized, as shown by the presence of both the female and the male pronucleus as well as by the remnants of the sperm tail in the ooplasm, 17 hours after insemination. Polyspermy was the main abnormality detected, and it affected almost 20% of the inseminated oocytes. The cleavage rate (two to fourcell stage) 41 hours after insemination of in vitro-matured and fertilized oocytes was 58%. 相似文献
65.
Silvia De Francia Antonio D’Avolio Francesca De Martino Elisa Pirro Lorena Baietto Marco Siccardi Marco Simiele Silvia Racca Giuseppe Saglio Francesco Di Carlo Giovanni Di Perri 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2009,877(18-19):1721-1726
A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 μl of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min. Mean intra-day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 78.1 ng/ml for imatinib and at 62.5 ng/ml for dasatinib and nilotinib. This novel developed methodology allows a specific, sensitive and reliable simultaneous determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in plasma of patients affected by chronic myeloid leukemia. 相似文献
66.
67.
Molecular insights into cell toxicity of a novel familial amyloidogenic variant of β2‐microglobulin 下载免费PDF全文
Manuela Leri Francesco Bemporad Reinier Oropesa‐Nuñez Claudio Canale Martino Calamai Daniele Nosi Matteo Ramazzotti Sofia Giorgetti Francesco S. Pavone Vittorio Bellotti Massimo Stefani Monica Bucciantini 《Journal of cellular and molecular medicine》2016,20(8):1443-1456
The first genetic variant of β2‐microglobulin (b2M) associated with a familial form of systemic amyloidosis has been recently described. The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild‐type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity (with possible functional unbalance and cytotoxicity) in GM1‐enriched domains only, thus establishing a link between aggregate‐membrane contact and cell damage. 相似文献
68.
Dr. C. De Martino M. L. Marcante A. Floridi G. Citro M. Bellocci A. Cantafora P. G. Natali 《Cell and tissue research》1977,176(1):69-90
Summary A cell line obtained from isolated seminiferous tubules of adult rat testis has been studied in vitro over a period of 35 days.Light and electron microscopic studies performed from hour 2 to the end of culture have shown the presence of a monomorphic cell population. After 5–6 days of culture the cells formed a monolayer. The cytoplasm of the cells contained numerous lipid bodies and produced numerous projections. The nucleus showed several indentations and one or more nucleoli. From the 9th to the 15th day of culture the cells developed a large amount of endoplasmic reticulum, Golgi apparatus and aggregates of electron dense granules. From the 20th to 40th day the cell cultures progressively degenerated.Immunochemical analysis of the culture medium revealed the presence of estradiol-17, which reached its maximum production rate from the 8th day to the 18th day of culture. Corresponding to cell involution estradiol concentration underwent a rapid decrease.On the basis of morphological and biochemical data the cells could be considered Sertoli cells.This work was supported by Grants n. 74.00155.04 and n. 75.01224.04 from the Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy, and by Istituto di Ricerca F. Angelini, Rome, ItalyPart of this work was presented at the 10th Italian Congress of Electron Microscopy. Ostuni 1–4 October 1975The excellent technical assistance of Miss Laura Vassallo, Daniela Venturini and Mr. Massimo Rosati and Mario Termine is deeply appreciated 相似文献
69.
Biochemical and morphological modifications in rabbit Achilles tendon during maturation and ageing. 下载免费PDF全文
G Cetta R Tenni G Zanaboni G De Luca E Ippolito C De Martino A A Castellani 《The Biochemical journal》1982,204(1):61-67
1. The plasma clearance of intravenously injected 125I-labelled mitochondrial malate dehydrogenase (half-life 7 min) was not influenced by previous injection of suramin and/or leupeptin (inhibitors of intralysosomal proteolysis). 2. Pretreatment with both inhibitors considerably delayed degradation of endocytosed enzyme in liver, spleen, bone marrow and kidneys. 3. The tissue distribution of radioactivity was determined at 30 min after injection, when only 3% of the dose was left in plasma. All injected radioactivity was still present in the carcass. The major part of the injected dose was found in liver (49%), spleen (5%), kidneys (13%) and bone, including marrow (11%). 4. Liver cells were isolated 15 min after injection of labelled enzyme. We found that Kupffer cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 9530 and 156 ml of plasma/day per g of cell protein respectively. Endothelial cells do not significantly contribute to uptake of the enzyme. 5. Uptake by Kupffer cells was saturable, whereas uptake by parenchymal cells was not. This suggests that these cell types endocytose the enzyme via different receptors. 6. Previous injection of carbon particles greatly decreased uptake of the enzyme by liver, spleen and bone marrow. 相似文献
70.
Alessandra Pesce Liesbet Thijs Marco Nardini Lorenza Sisinni Louise Gourlay Massimiliano Coletta Xuehua Wan Paolo Ascenzi Martino Bolognesi 《Journal of molecular biology》2009,386(1):246-35534
Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS162) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS162 as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS162 is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS162 is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS162 is a penta-coordinated species, where the proximal HisF8-Fe bond is severed. 相似文献