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51.
Mechanistic Insights into Cyclic Peptide Generation by DnaE Split-Inteins through Quantitative and Structural Investigation
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Leonhard M. Kick Dr. Sabrina Harteis Dr. Maximilian F. Koch Dr. Sabine Schneider 《Chembiochem : a European journal of chemical biology》2017,18(22):2242-2246
Inteins carry out protein-splicing reactions, which are used in protein chemistry, protein engineering and biotechnological applications. Rearrangement of the order of the domains in split-inteins results in head-to-tail cyclisation of the target sequence, which can be used for genetic encoding and expression of libraries of cyclic peptides (CPs). The efficiency of the splicing reaction depends on the target sequence. Here we used mass spectrometry to assess in vivo cyclic peptide formation from different hexameric target sequences by the DnaE split-inteins from Synechocystis sp. and Nostoc punctiforme, revealing a strong impact of the target sequence and of the intein on the intracellular peptide concentration. Furthermore, we determined the crystal structures of their pre-splicing complexes, which allowed us to identify F-block Asp17 as crucial for the DnaE-mediated splicing reaction. 相似文献
52.
Sabrina Reich Dr. Hans Wolfgang Hoeffken Dr. Bettina Rosche Dr. Bettina M. Nestl Prof. Dr. Bernhard Hauer 《Chembiochem : a European journal of chemical biology》2012,13(16):2400-2407
The crystal structure of the “ene” nicotinamide‐dependent cyclohexenone reductase (NCR) from Zymomonas mobilis (PDB ID: 4A3U) has been determined in complex with acetate ion, FMN, and nicotinamide, to a resolution of 1.95 Å. To study the activity and enantioselectivity of this enzyme in the bioreduction of activated α,β‐unsaturated alkenes, the rational design methods site‐ and loop‐directed mutagenesis were applied. Based on a multiple sequence alignment of various members of the Old Yellow Enzyme family, eight single‐residue variants were generated and investigated in asymmetric bioreduction. Furthermore, a structural alignment of various ene reductases predicted four surface loop regions that are located near the entrance of the active site. Four NCR loop variants, derived from loop‐swapping experiments with OYE1 from Saccharomyces pastorianus, were analysed for bioreduction. The three enzyme variants, P245Q, D337Y and F314Y, displayed increased activity compared to wild‐type NCR towards the set of substrates tested. The active‐site mutation Y177A demonstrated a clear influence on the enantioselectivity. The loop‐swapping variants retained reduction efficiency, but demonstrated decreased enzyme activity compared with the wild‐type NCR ene reductase enzyme. 相似文献
53.
In recent years, researchers have begun to study inductive databases, a new generation of databases for leveraging decision support applications. In this context, the user interacts with the
DBMS using advanced, constraint-based languages for data mining where constraints have been specifically introduced to increase
the relevance of the results and, at the same time, to reduce its volume. In this paper we study the problem of mining frequent
itemsets using an inductive database. We propose a technique for query answering which consists in rewriting the query in
terms of union and intersection of the result sets of other queries, previously executed and materialized. Unfortunately,
the exploitation of past queries is not always applicable. We then present sufficient conditions for the optimization to apply
and show that these conditions are strictly connected with the presence of functional dependencies between the attributes
involved in the queries. We show some experiments on an initial prototype of an optimizer which demonstrates that this approach
to query answering is viable and in many practical cases it drastically reduces the query execution time. 相似文献
54.
Butler Stephen F.; Newman Frederick L.; Cacciola John S.; Frank Arlene; Budman Simon H.; McLellan A. Thomas; Ford Sabrina; Blaine Jack; Gastfriend David; Moras Karla; Salloum Ihsan M.; Barber Jacques P. 《Canadian Metallurgical Quarterly》1998,10(4):399
The Addiction Severity Index (ASI) is a reliable and valid measure of problem severity among addicted patients. Concerns have been raised about the reliability of the Interviewer Severity Rating (ISR), a summary score for each of 7 domains. As part of an effort to build a computer-administered ASI, regression equations were developed to predict the ISR. Repeated resampling of a large dataset, consisting of 1,124 ASIs conducted by trained interviewers, permitted derivation of stable regression equations predicting the ISR for each ASI domain from patients' answers to preselected interview items. The resulting 7 Predicted Severity Ratings (PSRs) were tested on 8, standardized vignettes, with "gold standard," expert-generated ISRs. Reliabilities compared well with those of intensively trained interviewers. The PSRs could provide an alternative to potentially unreliable interviewer ratings, enhancing the ASI's role in treatment planning and treatment matching and make possible a computer-administered version of the ASI. (PsycINFO Database Record (c) 2010 APA, all rights reserved) 相似文献
55.
An acoustic-emission characterization of the failure modes in polymer-composite materials 总被引:3,自引:0,他引:3
M Giordano A Calabro C Esposito A D'Amore L Nicolais 《Composites Science and Technology》1998,58(12):1923-1928
A new methodology for the analysis of failure modes in composite materials by means of acoustic emission techniques has been developed. A single-carbon-fiber composite based on a polyester matrix, has been used as a simple model. The occurrence of fiber-breakage during tensile loading tests has been observed by a polarized light microscope and concurrently detected by a resonant acoustic probe. The resonant probe has been used as a trigger for the reading of fiber failure events. Single acoustic emission events from a wide-band probe has been recorded for FFT Analysis. The single-fiber specimen, having a unique failure mode, has advantages for the standardization of AE techniques for the quantitative analysis of failures in polymer-composite materials.
The same procedure can be exploited to investigate other failure modes namely, fiber matrix solidus debonding and matrix cracking. 相似文献
56.
S Antinori L Galimberti GL Tadini AL Ridolfo C Parravicini R Esposito M Moroni 《Canadian Metallurgical Quarterly》1995,14(10):911-914
Two patients with AIDS and disseminated tuberculosis characterized by cutaneous involvement are reported. They developed a maculopapular skin eruption, from which a multidrug-resistant Mycobacterium tuberculosis strain was isolated. In both cases the clinical course was rapidly fatal. Tuberculosis cutis miliaris disseminata should be differentiated from the skin lesions frequently seen in HIV-infected patients, especially from folliculitis. In patients with tuberculosis, the appearance of cutaneous lesions may be due to the haematogenous dissemination of mycobacteria. Therefore, early identification of the causative organism by use of optimal microbiological methods is fundamental. 相似文献
57.
Frecer V Kabelác M De Nardi P Pricl S Miertus S 《Journal of molecular graphics & modelling》2004,22(3):209-220
We have designed small focused combinatorial library of hexapeptide inhibitors of NS3 serine protease of the hepatitis C virus (HCV) by structure-based molecular design complemented by combinatorial optimisation of the individual residues. Rational residue substitutions were guided by the structure and properties of the binding pockets of the enzyme's active site. The inhibitors were derived from peptides known to inhibit the NS3 serine protease by using unusual amino acids and alpha-ketocysteine or difluoroaminobutyric acid, which are known to bind to the S1 pocket of the catalytic site. Inhibition constants (Ki) of the designed library of inhibitors were predicted from a QSAR model that correlated experimental Ki of known peptidic inhibitors of NS3 with the enthalpies of enzyme-inhibitor interaction computed via molecular mechanics and the solvent effect contribution to the binding affinity derived from the continuum model of solvation. The library of the optimised inhibitors contains promising drug candidates-water-soluble anionic hexapeptides with predicted Ki* in the picomolar range. 相似文献
58.
59.
Falcieri E Burattini S Bortul R Luchetti F Tabellini G Tazzari PL Cappellini A Cocco L Martelli AM 《Microscopy research and technique》2003,62(3):192-200
Two distinct types of cell death have been described: apoptosis and necrosis. However, it is becoming increasingly clear that the differences between these two types are far less numerous than initially thought. Morphological analyses might provide important information to distinguish apoptotic from necrotic samples. We recently reported that in necrotic, but not apoptotic, HL-60 human myeloid leukaemia cells, the nuclear protein topoisomerase IIalpha concentrated in nucleoli. In order to ascertain whether or not this phenomenon was restricted to a peculiar cell type or could be detected also in cells of lymphoid lineage, we performed an investigation aimed at defining the localization of topoisomerase IIalpha in apoptotic and necrotic Jurkat human T lymphoblastoid cells. Immunofluorescence staining demonstrated that topoisomerase IIalpha was excluded from the condensed chromatin of apoptotic cells, whereas in necrotic cells it was localized in discrete nuclear dots. Immuno-electron microscopy analysis showed that topoisomerase IIalpha was undetectable in nucleoli of normal and apoptotic cells, whereas it was present in the nucleolus of necrotic cells irrespectively of the type of inducer used (ethanol, H(2)O(2), HgCl(2)). Taken together, our findings identify topoisomerase IIalpha as a potential morphological marker useful to discriminate between apoptotic and necrotic cells. 相似文献
60.
Suska F Källtorp M Esposito M Gretzer C Tengvall P Thomsen P 《Journal of materials science. Materials in medicine》2001,12(10-12):939-944
Machined, commercially pure titanium (Ti) disks were coated with approximately 400 nm copper (Cu) by physical vapor deposition or left uncoated. The kinetics of inflammatory cell recruitment, distribution and viability was evaluated around Ti, Cu, and in sham sites after 1, 3, 12, 18, 24, and 48 h in a rat subcutaneous (s.c.) model. Further analysis of the cells on implant surfaces was performed by ex vivo incubation of the disks. Ti and Cu stimulated an increased recruitment of inflammatory cells in comparison with sham sites. A markedly higher amount of cells, predominantly polymorpho-nuclear granulocytes (PMN), was detected around Cu after 18 h and onwards. More cells were found at the implant surfaces than in the surrounding exudates after 18 h. The total amount of lactate dehydrogenase (LDH), an indicator of plasma membrane injury, was higher in Cu exudates after 18 h in comparison with Ti and sham. In contrast, no differences in the proportion of dead cells (trypan blue dye uptake) were detected in the exudates. Further, LDH levels were higher around Ti than Cu during the initial 18 h of ex vivo incubation. The results of this study indicate that the early inflammatory process associated with a cytotoxic material in soft tissues is largely attributed to the induction of a markedly strong and prolonged chemotactic response. In contrast, this process is characterized by a higher amount of inflammatory cells around a biocompatible material than in sham sites, but with a transient course and total LDH similar to sham sites. 相似文献