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21.
Giuseppe Stabile Antoine Lepillier Ermenegildo De Ruvo Marco Scaglione Matteo Anselmino Frederic Sebag Domenico Pecora Mark Gallagher Mariano Rillo Graziana Viola Luca Rossi Valerio De Santis Maurizio Landolina Antonello Castro Massimo Grimaldi Nicolas Badenco Maurizio Del Greco Antonio De Simone Ennio Pisan Salim Abbey Filippo Lamberti Antonio Pani Giulio Zucchelli Giuseppe Sgarito Daniela Dugo Emanuele Bertaglia Teresa Strisciuglio Francesco Solimene 《Journal of cardiovascular electrophysiology》2020,31(7):1694-1701
22.
Filippo Torroni Erminia Romeo Francesca Rea Paola De Angelis Francesca Foschia Simona Faraci Giovanni Federici di Abriola Anna Chiara Contini Tamara Caldaro Luigi Dall’Oglio 《World journal of gastrointestinal endoscopy》2014,6(7):318-323
AIM: To assess the usefulness of the balloon assisted enteroscopy in preventing surgical intervention in pa-tients with Peutz-Jeghers syndrome (PJS) having a small bowel large polyps. METHODS: Seven consecutive asymptomatic pts(age 15-38 years) with PJS have been collected; six under-went polypectomy using single balloon enteroscopy(Olympus SIF Q180) with antegrade approach using push and pull technique. SBE system consists of the SIF-Q180 enteroscope, an overtube balloon control unit(OBCU Olympus Balloon Control Unit) and a dispos-able silicone splinting tube with balloon(ST-SB1). All procedures were performed under general anesthesia. Previously all pts received wireless capsule endos-copy(WCE). Prophylactic polypectomy was reservedmainly in pts who had polyps 15 mm in diameter. The balloon is inflated and deflated by a balloon control unit with a safety pressure setting range from-6.0 kPa to +5.4 kPa. Informed consent has been obtained from pts or parents for each procedure.RESULTS: Six pts underwent polypectomy of small bowel polyps; in 5 pts a large polyp 15 mm(range 20-50 mm in diameter) was resected; in 1 patient with WCE negative, SBE was performed for previous surgi-cal resection of gastrointestinal stromal tumors. In 2 pts endoscopic clips were placed due to a polypectomy. No surgical complication have been reported. SBE with resection of small bowel large polyps in PJS pts was useful to avoid gastrointestinal bleeding and emergency laparotomy due to intestinal intussuscep-tions. No gastrointestinal tumors were found in sub-sequent enteroscopic surveillance in all seven pts. In order surveillance, all pts received WCE, upper en-doscopy, ileocolonoscopy every 2 years. No pts had extraintestinal malignant lesions. SBE was performed when WCE was positive for significant polyps( 15 mm).CONCLUSION: The effective of prophylactic polyp-ectomy of small bowel large polyps( 15 mm) could be the first line treatment for conservative approach in management of PJS patients. 相似文献
23.
Michael R. Sawaya Duilio Cascio Mari Gingery Jose Rodriguez Lukasz Goldschmidt Jacques-Philippe Colletier Marc M. Messerschmidt Sébastien Boutet Jason E. Koglin Garth J. Williams Aaron S. Brewster Karol Nass Johan Hattne Sabine Botha R. Bruce Doak Robert L. Shoeman Daniel P. DePonte Hyun-Woo Park Brian A. Federici Nicholas K. Sauter Ilme Schlichting David S. Eisenberg 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(35):12769-12774
It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.The advent of X-ray free-electron lasers (XFELs) has made it possible to obtain atomic resolution macromolecular structures from crystals with sizes approximating only 1/60th of the volume of a single red blood cell. Brief, intense pulses of coherent X-rays, focused on a spot of 3-μm diameter, have produced 1.9-Å-resolution diffraction data from a stream of lysozyme crystals, each crystal no bigger than 3 μm3 (1). A stream of crystals, not just one crystal, is required to collect the many tens of thousands of diffraction patterns that compose a complete data set. No single crystal can contribute more than one diffraction pattern because the XFEL beam is so intense and the crystals so small that the crystals are typically vaporized after a single pulse. Impressively, a photosystem I crystal no bigger than 10 unit cells (300 nm) on an edge produced observable subsidiary diffraction peaks between Bragg reflections, details which would be unobservable from conventionally sized crystals (2). With this new ability to collect diffraction patterns from crystals of unprecedentedly small dimensions, it is conceivable that high-resolution diffraction data could be collected from crystals in vivo. The structure obtained in this manner would be unaltered from that occurring naturally in a living cell, free from distortion that might otherwise potentially arise from nonphysiological conditions imposed by recrystallization. A practical advantage would also be gained by eliminating the need for a protein purification step, whether the in vivo grown crystals were naturally, or heterologously expressed (3).The nascent field of serial femtosecond crystallography (SFX) has published results on nine different macromolecular systems since its inception in 2009 (3, 9). The crystals for this study were not grown in artificial crystallization chambers as has been the protocol of conventional macromolecular crystallography since the 1950s. Instead, crystals were grown in cells. Specifically, they were grown in Sf9 insect cells, heterologously expressing Trypanosoma brucei cathepsin B. These in vivo-grown crystals were used for the XFEL diffraction experiment. To this end, the cells were lysed and the crystals were extracted before injecting them in the XFEL beam for data collection. This last purification step seems to be the only major departure from our goal of obtaining high-resolution structural information from crystal inclusions in vivo, without requiring the crystal to be extracted from the cell that assembled it. Here we attempt to go one step further than previous studies—to record diffraction from crystals within living cells.
Open in a separate window*The available XFEL energy was limited to 2 keV (6.2 Å wavelength) when these experiments were conducted.Our target for in vivo crystal structure determination is the insecticidal Cry3A toxin from Bacillus thuringiensis (Bt). The bacterium naturally produces crystals of toxin during sporulation (16). Presumably, the capacity for in vivo crystallization evolved in Bt as a mechanism to store the toxin in a concentrated, space-efficient manner. Since the 1920s, farmers have used the crystalline insecticidal proteins to control insect pests; its production as a natural pesticide is now a commercial enterprise. Attempts to structurally characterize the toxins date back to more than 40 y ago with the first report of diffraction from isolated crystals that were packed together in powder form to obtain a measurable signal; X-ray sources available at the time were relatively weak (17). More than 20 y later, the structure was determined at 2.5-Å resolution by single crystal diffraction using a synchrotron X-ray source (18). However, to achieve this result, the authors dissolved the naturally occurring microcrystals and recrystallized the toxin using the hanging drop vapor diffusion method. To date, more than a dozen Bt toxin structures have been reported from various strains [Protein Data Bank (PDB) ID codes 1cby, 1ciy, 1i5p, 1ji6, 1w99, 2d42, 2c9k, 2rci, 3eb7, 2ztb, 3ron, 4d8m, 4ato, 4ary, and 4arx], but none using naturally occurring crystals, and all of the crystals had lost their native context.In pursuit of in vivo diffraction, we took advantage of the Bt subsp. israelensis strain 4Q7/pPFT3As to produce the largest in vivo crystals achievable. This strain contains the plasmid pPFT3As, which increases expression of Cry3A by 12.7-fold over wild type by using strong promoters and an mRNA stabilizing sequence (19). The level of Cry3A production is such that the cell essentially distorts to take on the shape of the enclosed crystal. The calculated average crystal volume is 0.7 µm3 (19), almost accounting for the volume of the cell. To explore the possibilities for in situ data collection of in vivo microcrystals, we injected both the crystals in cells and crystals that we isolated from cells in the XFEL beam and collected SFX diffraction data. Our experiments revealed that the cell wall and other cellular components are not an obstacle to achieving 2.9-Å-resolution diffraction, and analogous studies in other systems might be similarly successful. 相似文献
Table 1.
SFX publications from XFEL sources to datePublication date | System | Product | Resolution (Å) | Title of publication | Authors | Reference |
Feb 2011* | Photosystem I | Structure | 8.7 | Femtosecond X-ray protein nanocrystallography | Chapman et al. | 2 |
Dec 2011* | Lysozyme | Structure | 8.7 | Radiation damage in protein serial femtosecond crystallography using an X-ray free-electron laser | Lomb et al. | 4 |
Jan 2012* | Photosystem I-Ferredoxin | Data | 11 | Time-resolved protein nanocrystallography using an X-ray free-electron laser | Aquila et al. | 5 |
Jan 2012* | Cathepsin B | Data | 7.5 | In vivo protein crystallization opens new routes in structural biology | Koopman et al. | 3 |
Jan 2012* | Photosynthetic Reaction Center | Structure | 7.4 | Lipidic phase membrane protein serial femtosecond crystallography | Johansson et al. | 6 |
Jun 2012 | Photosystem II | Structure | 6.6 | Room temperature femtosecond X-ray diffraction of photosystem II microcrystals | Kern et al. | 7 |
Jul 2012 | Lysozyme | Structure | 1.9 | High-resolution protein structure determination by serial femtosecond crystallography | Boutet et al. | 1 |
Nov 2012 | Thermolysin | Data | 4.0 | Nanoflow electrospinning serial femtosecond crystallography | Sierra et al. | 8 |
Jan 2013 | Cathepsin B | Structure | 2.1 | Natively inhibited Trypsanosoma brucei cathepsin B structure determined by using an X-ray laser | Redecke et al. | 9 |
Apr 2013 | Photosystem II | Structure | 5.7 | Simultaneous femtosecond X-ray spectroscopy and diffraction of photosystem II at room temperature | Kern et al. | 10 |
May 2013 | Lysozyme | Structure | 3.2 | Anomalous signal from S atoms in protein crystallographic data from an X-ray free-electron laser | Barends et al. | 11 |
Sept 2013 | Ribosome | Data | <6 | Serial femtosecond X-ray diffraction of 30S ribosomal subunit microcrystals in liquid suspension at ambient temperature using an X-ray free-electron laser | Demirci et al. | 12 |
Dec 2013 | Photosynthetic Reaction Center | Structure | 3.5 | Structure of a photosynthetic reaction center determined by serial femtosecond crystallography | Johansson et al. | 13 |
Dec 2013 | Serotonin receptor | Structure | 2.8 | Serial femtosecond crystallography of G protein-coupled receptors | Liu et al. | 14 |
Jan 2014 | Lysozyme + Gd | Structure | 2.1 | De novo protein crystal structure determination from XFEL data | Barends et al. | 15 |
This study | Cry3A toxin, isolated crystals and whole cells | Structure | 2.8, 2.9 | 2.9 Å-Resolution protein crystal structure obtained from injecting bacterial cells into an X-ray free-electron laser beam | Sawaya et al. | This study |
24.
Jingling Liao Chun-Xiang Wu Christopher Burlak Sheng Zhang Heather Sahm Mu Wang Zhong-Yin Zhang Kurt W. Vogel Mark Federici Steve M. Riddle R. Jeremy Nichols Dali Liu Mark R. Cookson Todd A. Stone Quyen Q. Hoang 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(11):4055-4060
Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (RocR1441H) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD) (1–5). LRRK2 is a large (2,527-aa) multidomain protein consisting of seven putative domains (2), including a Ras-like GTPase domain called Ras of complex proteins (Roc), followed by a domain called C-terminal of Roc (COR), which is then followed by a kinase domain (Kin). It remains unclear how perturbations of these activities result in disease; however, the most common mutation in LRRK2-associated PD, G2019S in the kinase domain, shows higher kinase activity than wild type; therefore, its overactivation might be associated with disease pathogenesis (6).The tandem Roc-COR-Kin arrangement suggests that their activities might be coupled such that the GTPase activity of Roc might modulate the kinase activity. Indeed, several studies have shown that GTP binding to the Roc domain regulates the activity of the Kin domain (7, 8). Moreover, a PD-associated mutation in the Roc domain (R1441C) has been shown to have higher kinase activity (9), thus suggesting that mutations in the Roc domain, also up-regulate kinase activity.Understanding the function of Roc and its mechanism of action is important for understanding the mechanism of PD pathogenesis and therapeutic development. However, because of the lack sufficient quantity of protein samples amendable for detailed investigations, the biochemical properties and enzymatic activities of the Roc domain of LRRK2 are poorly understood.Here, we describe a stably folded construct of human Roc domain that enabled us to investigate quantitatively its biochemical and enzymatic properties. The results revealed that a PD-causing mutation R1441H in the Roc domain renders it less active at hydrolyzing GTP, as well as having higher affinity for GTP, than its wild-type counterpart, thereby increasing the residence time of its GTP-bound “active state,” which is associated with PD pathogenesis (8). 相似文献
25.
26.
27.
Peyvandi F Klamroth R Carcao M Federici AB DI Minno G Jiménez-Yuste V Rodriguez Merchán EC 《Haemophilia》2012,18(Z2):24-36
Development of FVIII inhibitors is currently the most severe and challenging complication of haemophilia A treatment and represents a very large economic burden for a chronic disease. As a result, clinical research is making major efforts to optimize the therapeutic approaches for this condition. In this section we will review some important aspects of the management of haemophilia in adults, including an overview of bleeding in women with von Willebrand disease, an analysis of FVIII consumption in patients with severe haemophilia A, an update of the ongoing RES.I.ST study, long-term prophylaxis and experience from the Pro.Will study, current evidence relating to economic aspects of the treatment of haemophilic patients with inhibitors (based on the PROFIT study), and an overview of musculoskeletal complications in adults with severe bleeding disorders. 相似文献
28.
Mekinian A Néel A Sibilia J Cohen P Connault J Lambert M Federici L Berthier S Fiessinger JN Godeau B Marie I Guillevin L Hamidou M Fain O;Club Rhumatismes et Inflammation French Vasculitis Study Group Société Nationale Française de Médecine Interne 《Rheumatology (Oxford, England)》2012,51(5):882-886
29.
OBJECTIVE: the aim of this study is to determine the clinical characteristics of drug-induced agranulocytosis in rheumatology. PATIENTS AND METHOD: it is a retrospective monocentric study, including all cases of rheumatologic drug-induced agranulocytosis followed between January 1985 and December 2005. RESULTS: eight female and 4 men, mean age 62.5 years (range: 17-79), were included in the present study. The causative drugs were: anti-inflammatory agents (n=5, 41.6%), methotrexate (n=3, 25%), sulfasalazine (n=2, 16.6%), noramidopyrine (n=1) and dapsone (n=1). Main clinical features included infectious disorders in 9 cases (75%) with 4 cases of septicemia (33.3%) and 2 cases of septic shock (16.6%). The mean neutrophils count was 0.132 x 10(9)/L (range, 0-0.4). Outcome was favorable in 11 patients (91.6%). One patient died of septic complications. CONCLUSION: in rheumatology, drug-induced agranulocytosis is a rare but life-threatening disorder. 相似文献
30.
Intraspinal neural stem cell transplantation in amyotrophic lateral sclerosis: Phase 1 trial outcomes
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