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941.
Søren B. Nielsen Kristina Wilhelm Jürgen Schleucher Daniel Otzen 《Journal of molecular biology》2010,398(2):351-11509
The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to α-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of α-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appear to be central to its biological action, similar to human α-lactalbumin made lethal to tumor cells. Here, we describe the interaction of ELOA with phospholipid membranes. Confocal scanning laser microscopy shows that ELOA, but not native EL, accumulates on the surface of giant unilamellar vesicles, without inducing significant membrane permeability. Quartz crystal microbalance with dissipation data indicated an essentially non-disruptive binding of ELOA to supported lipid bilayers, leading to formation of highly dissipative and “soft” lipid membrane; at higher concentrations of ELOA, the lipid membrane desorbs from the surface probably as bilayer sheets of vesicles. This membrane rearrangement occurred to a similar extent when free oleic acid (OA) was added, but not when free OA was removed from ELOA by prior incubation with bovine serum albumin, emphasizing the role of OA in this process. NMR data indicated an equilibrium between free and bound OA, which shifts towards free OA as ELOA is progressively diluted, indicating that OA is relatively loosely bound. Activity measurements together with fluorescence spectroscopy and circular dichroism suggested a conversion of ELOA towards a more native-like state on interaction with lipid membranes, although complete refolding was not observed. Altogether, these results suggest that ELOA may act as an OA carrier and facilitate OA transfer to the membrane. ELOA's properties illustrate that protein folding variants may possess specific functional properties distinct from the native protein. 相似文献
942.
Ralstonia eutropha (formerly Alcaligenes eutrophus) is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and molecular engineering of this organism. We report here a versatile gene expression method for the genetic engineering of R. eutropha. This method, based on a simplified electroporation protocol, uses a recombinant plasmid, pBS29-P2, containing a Pseudomonas syringae promoter (P2) and two antibiotic-resistance markers (i.e., genes coding for kanamycin (Km)- and tetracycline (Tc)-resistance). Using this method, we successfully achieved transformation of wild-type R. eutropha and its poly(hydroxyalkanoate)-negative mutant, R. eutropha PHB−4, with various pBS29-P2-based recombinants. A transformation frequency as high as 4 × 103 Km-resistance colonies/μg DNA was obtained per electroporation experiment. We further demonstrated the successful expression of a heterologous gene coding for green-fluorescent-protein by fluorescence measurement. In addition, our results indicated the expression of a truncated but active Streptomyces coelicolor α-galactosidase in R. eutropha. 相似文献
943.
A new population of the invasive American Eastern grey squirrel (Sciurus carolinensis) has recently settled in central Italy from an accidental release in Perugia, Umbria in the early 2000s. The grey squirrel is known to compete with and exclude native red squirrels (S. vulgaris) in the British Isles and Northern Italy, so it represents a potentially important new conservation threat to the red squirrel subspecies of south and central Italy, S. vulgaris italicus and S. v. meridionalis, which are endemic to peninsular Italy. The grey squirrel population range in Perugia is currently expanding at a rate of about 0.29 km/year (SD 0.19), slower than grey squirrel invasions elsewhere in Europe. Nuclear DNA analysed at 12 different microsatellite loci revealed that the grey squirrels in Perugia have extremely low genetic diversity, consistent with a small founder size. Genetic assignment tests indicate that the Perugia population was founded by translocations from an established population in Piedmont, Italy. No genetic substructure is evident yet in the Perugia population. These results together have serious consequences for the management of the grey squirrel invasion in Perugia and the conservation of the red squirrel subspecies: the Perugia grey squirrel population should be eradicated if politically feasible; otherwise new releases of grey squirrels, especially from sources other than the Piedmont population, should be prevented because such introductions could increase genetic diversity, thereby potentially increasing population range expansion rate to the much higher levels seen for more diverse grey squirrel populations elsewhere in Europe. 相似文献
944.
Harald Biessmann Evi Andronopoulou Max R. Biessmann Vassilis Douris Spiros D. Dimitratos Elias Eliopoulos Patrick M. Guerin Kostas Iatrou Robin W. Justice Thomas Kr?ber Osvaldo Marinotti Panagiota Tsitoura Daniel F. Woods Marika F. Walter 《PloS one》2010,5(3)
Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP–ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects. 相似文献
945.
Bruce A. C. Cree John D. Rioux Jacob L. McCauley Pierre-Antoine F. D. Gourraud Philippe Goyette Joseph McElroy Philip De Jager Adam Santaniello Timothy J. Vyse Peter K. Gregersen Daniel Mirel David A. Hafler Jonathan L. Haines Margaret A. Pericak-Vance Alastair Compston Stephen J. Sawcer Jorge R. Oksenberg Stephen L. Hauser IMAGEN IMSGC 《PloS one》2010,5(6)
Background
In Northern European descended populations, genetic susceptibility for multiple sclerosis (MS) is associated with alleles of the human leukocyte antigen (HLA) Class II gene DRB1. Whether other major histocompatibility complex (MHC) genes contribute to MS susceptibility is controversial.Methodology/Principal Findings
A case control analysis was performed using 958 single nucleotide polymorphisms (SNPs) spanning the MHC assayed in two independent datasets. The discovery dataset consisted of 1,018 cases and 1,795 controls and the replication dataset was composed of 1,343 cases and 1,379 controls. The most significantly MS-associated SNP in the discovery dataset was rs3135391, a Class II SNP known to tag the HLA-DRB1*15:01 allele, the primary MS susceptibility allele in the MHC (O.R. = 3.04, p<1×10−78). To control for the effects of the HLA-DRB1*15:01 haplotype, case control analysis was performed adjusting for this HLA-DRB1*15:01 tagging SNP. After correction for multiple comparisons (false discovery rate = .05) 52 SNPs in the Class I, II and III regions were significantly associated with MS susceptibility in both datasets using the Cochran Armitage trend test. The discovery and replication datasets were merged and subjects carrying the HLA-DRB1*15:01 tagging SNP were excluded. Association tests showed that 48 of the 52 replicated SNPs retained significant associations with MS susceptibility independently of the HLA-DRB1*15:01 as defined by the tagging SNP. 20 Class I SNPs were associated with MS susceptibility with p-values ≤1×10−8. The most significantly associated SNP was rs4959039, a SNP in the downstream un-translated region of the non-classical HLA-G gene (Odds ratio 1.59, 95% CI 1.40, 1.81, p = 8.45×10−13) and is in linkage disequilibrium with several nearby SNPs. Logistic regression modeling showed that this SNP''s contribution to MS susceptibility was independent of the Class II and Class III SNPs identified in this screen.Conclusions
A MHC Class I locus contributes to MS susceptibility independently of the HLA-DRB1*15:01 haplotype. 相似文献946.
Daniel Ambort Florence Brellier Christoph Becker-Pauly Walter Stöcker Snezana Andrejevic-Blant Matthias Chiquet Erwin E. Sterchi 《Matrix biology》2010,29(1):31-42
The metalloprotease meprin has been implicated in tissue remodelling due to its capability to degrade extracellular matrix components. Here, we investigated the susceptibility of tenascin-C to cleavage by meprinβ and the functional properties of its proteolytic fragments. A set of monoclonal antibodies against chicken and human tenascin-C allowed the mapping of proteolytic fragments generated by meprinβ. In chicken tenascin-C, meprinβ processed all three major splicing variants by removal of 10 kDa N-terminal and 38 kDa C-terminal peptides, leaving a large central part of subunits intact. A similar cleavage pattern was found for large human tenascin-C variant where two N-terminal peptides (10 or 15 kDa) and two C-terminal fragments (40 and 55 kDa) were removed from the intact subunit. N-terminal sequencing revealed the exact amino acid positions of cleavage sites. In both chicken and human tenascin-C N-terminal cleavages occurred just before and/or after the heptad repeats involved in subunit oligomerization. In the human protein, an additional cleavage site was identified in the alternative fibronectin type III repeat D. Whereas all these sites are known to be attacked by several other proteases, a unique cleavage by meprinβ was located to the 7th constant fibronectin type III repeat in both chicken and human tenascin-C, thereby removing the C-terminal domain involved in its anti-adhesive activity. In cell adhesion assays meprinβ-digested human tenascin-C was not able to interfere with fibronectin-mediated cell spreading, confirming cleavage in the anti-adhesive domain. Whereas the expression of meprinβ and tenascin-C does not overlap in normal colon tissue, inflamed lesions of the mucosa from patients with Crohn's disease exhibited many meprinβ-positive leukocytes in regions where tenascin-C was strongly induced. Our data indicate that, at least under pathological conditions, meprinβ might attack specific functional sites in tenascin-C that are important for its oligomerization and anti-adhesive activity. 相似文献
947.
Nicole M Gerardo Boran Altincicek Caroline Anselme Hagop Atamian Seth M Barribeau de Martin Vos Elizabeth J Duncan Jay D Evans Toni Gabaldón Murad Ghanim Adelaziz Heddi Isgouhi Kaloshian Amparo Latorre Andres Moya Atsushi Nakabachi Benjamin J Parker Vincente Pérez-Brocal Miguel Pignatelli Yvan Rahbé John S Ramsey Chelsea J Spragg Javier Tamames Daniel Tamarit Cecilia Tamborindeguy Caroline Vincent-Monegat Andreas Vilcinskas 《Genome biology》2010,11(2):1-17
948.
Kentaro Suzuki Yasuha Adachi Tomokazu Numata Shoko Nakada Motoko Yanagita Naomi Nakagata Sylvia M. Evans Daniel Graf Aris Economides Ryuma Haraguchi Anne M. Moon Gen Yamada 《PloS one》2012,7(9)
Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4flox/flox) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4flox/flox conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia. 相似文献
949.
Julie Della-Maria Muralidhar L. Hegde Daniel R. McNeill Yoshihiro Matsumoto Miaw-Sheue Tsai Tom Ellenberger David M. Wilson III Sankar Mitra Alan E. Tomkinson 《The Journal of biological chemistry》2012,287(46):39233-39244
XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair. 相似文献
950.
AL-12182, a novel 11-oxa prostaglandin analog with topical ocular hypotensive activity in the monkey
Selliah RD Hellberg MR Sharif NA McLaughlin MA Williams GW Scott DA Earnest D Haggard KS Dean WD Delgado P Gaines MS Conrow RE Klimko PG 《Bioorganic & medicinal chemistry letters》2004,14(17):4525-4528
A series of 11-oxa prostaglandin analogs was evaluated for FP receptor binding and activation. Several compounds having aryloxy-terminated lower chains were found to be potent agonists. Topical ocular dosing of AL-12182, the isopropyl ester prodrug of the potent agonist 13, lowered intraocular pressure in the monkey by 40% accompanied by minimal conjunctival hyperemia in the rabbit. AL-12182 was synthesized on multigram scale starting with D-sorbitol. 相似文献