全文获取类型
收费全文 | 10299篇 |
免费 | 866篇 |
国内免费 | 5篇 |
专业分类
11170篇 |
出版年
2023年 | 44篇 |
2022年 | 50篇 |
2021年 | 111篇 |
2020年 | 80篇 |
2019年 | 88篇 |
2018年 | 221篇 |
2017年 | 163篇 |
2016年 | 252篇 |
2015年 | 490篇 |
2014年 | 533篇 |
2013年 | 624篇 |
2012年 | 815篇 |
2011年 | 761篇 |
2010年 | 499篇 |
2009年 | 396篇 |
2008年 | 624篇 |
2007年 | 640篇 |
2006年 | 609篇 |
2005年 | 575篇 |
2004年 | 492篇 |
2003年 | 525篇 |
2002年 | 460篇 |
2001年 | 122篇 |
2000年 | 104篇 |
1999年 | 97篇 |
1998年 | 78篇 |
1997年 | 68篇 |
1996年 | 59篇 |
1995年 | 47篇 |
1994年 | 40篇 |
1993年 | 41篇 |
1992年 | 42篇 |
1991年 | 63篇 |
1990年 | 80篇 |
1989年 | 66篇 |
1988年 | 80篇 |
1987年 | 63篇 |
1986年 | 50篇 |
1985年 | 46篇 |
1984年 | 49篇 |
1983年 | 41篇 |
1982年 | 40篇 |
1981年 | 40篇 |
1980年 | 31篇 |
1979年 | 36篇 |
1978年 | 43篇 |
1977年 | 35篇 |
1975年 | 30篇 |
1974年 | 40篇 |
1973年 | 29篇 |
排序方式: 共有10000条查询结果,搜索用时 0 毫秒
131.
Robin Teufel Johannes W. Kung Daniel Kockelkorn Birgit E. Alber Georg Fuchs 《Journal of bacteriology》2009,191(14):4572-4581
A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO2 fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.In the thermoacidophilic autotrophic crenarchaeum Metallosphaera sedula, CO2 fixation proceeds via a 3-hydroxypropionate/4-hydroxybutyrate cycle (8, 23, 24, 28) (Fig. (Fig.1).1). A similar cycle may operate in other autotrophic members of the Sulfolobales and in mesophilic Crenarchaea (Cenarchaeum sp. and Nitrosopumilus sp.) of marine group I. The cycle uses elements of the 3-hydroxypropionate cycle that was originally discovered in the phototrophic bacterium Chloroflexus aurantiacus (11, 16, 17, 19, 20, 32, 33). It involves the carboxylation of acetyl-coenzyme A (CoA) to malonyl-CoA by the biotin-dependent acetyl-CoA carboxylase. Malonyl-CoA is reduced via malonate semialdehyde to 3-hydroxypropionate (1), which is further reductively converted to propionyl-CoA (3). Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by a propionyl-CoA carboxylase that is similar or identical to acetyl-CoA carboxylase. In fact, only one copy of the genes for the acetyl-CoA/propionyl-CoA carboxylase subunits is present in most Archaea, suggesting that this is a promiscuous enzyme that acts on both acetyl-CoA and propionyl-CoA (24). (S)-Methylmalonyl-CoA is epimerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA by coenzyme B12-dependent methylmalonyl-CoA mutase.Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other members of the Sulfolobales. Enzymes are the following: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionyl-CoA synthetase (3-hydroxypropionate-CoA ligase, AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinate semialdehyde reductase (NADPH); 12, 4-hydroxybutyryl-CoA synthetase (4-hydroxybutyrate-CoA ligase, AMP-forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14, crotonyl-CoA hydratase; 15, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD+); 16, acetoacetyl-CoA β-ketothiolase. The two steps of interest are highlighted.In Chloroflexus succinyl-CoA is converted to (S)-malyl-CoA, which is cleaved by (S)-malyl-CoA lyase to acetyl-CoA (thus regenerating the CO2 acceptor molecule) and glyoxylate (16). Glyoxylate is assimilated into cell material by a yet not completely resolved pathway (37). In Metallosphaera succinyl-CoA is converted via 4-hydroxybutyrate to two molecules of acetyl-CoA (8), thus regenerating the starting CO2 acceptor molecule and releasing another acetyl-CoA for biosynthesis. Hence, the 3-hydroxypropionate/4-hydroxybutyrate cycle (Fig. (Fig.1)1) can be divided into two parts. The first part transforms one acetyl-CoA and two bicarbonates into succinyl-CoA, and the second part converts succinyl-CoA to two acetyl-CoA molecules.The reductive conversion of 3-hydroxypropionate to propionyl-CoA requires three enzymatic steps: activation of 3-hydroxypropionate to its CoA ester, dehydration of 3-hydroxypropionyl-CoA to acryloyl-CoA, and reduction of acryloyl-CoA to propionyl-CoA. In C. aurantiacus these three steps are catalyzed by a single large trifunctional enzyme, propionyl-CoA synthase (2). This 200-kDa fusion protein consists of a CoA ligase, a dehydratase, and a reductase domain. Attempts to isolate a similar enzyme from M. sedula failed. Rather, a 3-hydroxypropionyl-CoA synthetase was found (3), suggesting that the other two reactions may also be catalyzed by individual enzymes.Here, we purified the missing enzymes 3-hydroxypropionyl-CoA dehydratase and acryloyl-CoA reductase from M. sedula, identified the coding genes in the genome of M. sedula and other members of the Sulfolobales, produced recombinant enzymes as proof of function, and studied the enzymes in some detail. A comparison with the respective domains of propionyl-CoA synthase from C. aurantiacus indicates that the conversion of 3-hydroxypropionate to propionyl-CoA via the 3-hydroxypropionate route has evolved independently in these two phyla. 相似文献
132.
The absolute configuration of three 4‐aryl‐3,4‐dihydro‐2(1H)‐pyrimidones (Biginelli compounds, DHPMs) was established by comparison of the typical circular dichroism (CD) spectra of individual enantiomers with reference samples of known absolute configuration. The enantiomers were obtained by semipreparative separation of racemic mixtures on a Chiralcel OD‐H chiral stationary phase. The method was used to establish the enantiopreference of various lipases in biocatalytic kinetic resolution experiments employing activated DHPM esters. Chirality 11:659–662, 1999. © 1999 Wiley‐Liss, Inc. 相似文献
133.
Christian Beyer Georg Schett Steffen Gay Oliver Distler Jörg HW Distler 《Arthritis research & therapy》2009,11(2):220-9
Autoimmunity, microangiopathy and tissue fibrosis are hallmarks of systemic sclerosis (SSc). Vascular alterations and reduced
capillary density decrease blood flow and impair tissue oxygenation in SSc. Oxygen supply is further reduced by accumulation
of extracellular matrix (ECM), which increases diffusion distances from blood vessels to cells. Therefore, severe hypoxia
is a characteristic feature of SSc and might contribute directly to the progression of the disease. Hypoxia stimulates the
production of ECM proteins by SSc fibroblasts in a transforming growth factor-β-dependent manner. The induction of ECM proteins
by hypoxia is mediated via hypoxia-inducible factor-1α-dependent and -independent pathways. Hypoxia may also aggravate vascular
disease in SSc by perturbing vascular endothelial growth factor (VEGF) receptor signalling. Hypoxia is a potent inducer of
VEGF and may cause chronic VEGF over-expression in SSc. Uncontrolled over-expression of VEGF has been shown to have deleterious
effects on angiogenesis because it leads to the formation of chaotic vessels with decreased blood flow. Altogether, hypoxia
might play a central role in pathogenesis of SSc by augmenting vascular disease and tissue fibrosis. 相似文献
134.
135.
Karine Frénal Jean-Baptiste Marq Damien Jacot Valérie Polonais Dominique Soldati-Favre 《PLoS pathogens》2014,10(11)
The glideosome is an actomyosin-based machinery that powers motility in Apicomplexa and participates in host cell invasion and egress from infected cells. The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45. In Toxoplasma gondii, GAP45 also contributes to the cohesion of the pellicle, composed of the inner membrane complex (IMC) and the plasma membrane, during motor traction. GAP70 was previously identified as a paralog of GAP45 that is tailored to recruit MyoA at the apical cap in the coccidian subgroup of the Apicomplexa. A third member of this family, GAP80, is demonstrated here to assemble a new glideosome, which recruits the class XIV myosin C (MyoC) at the basal polar ring. MyoC shares the same myosin light chains as MyoA and also interacts with the integral IMC proteins GAP50 and GAP40. Moreover, a central component of this complex, the IMC-associated protein 1 (IAP1), acts as the key determinant for the restricted localization of MyoC to the posterior pole. Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome. Conversely, removal of MyoA leads to the relocalization of MyoC along the pellicle and at the apical cap that accounts for residual invasion. The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival. 相似文献
136.
137.
138.
Bédouet L Marie A Berland S Marie B Auzoux-Bordenave S Marin F Milet C 《Marine biotechnology (New York, N.Y.)》2012,14(4):446-458
A successful strategy for the identification of shell proteins is based on proteomic analyses where soluble and insoluble fractions isolated from organic shell matrix are digested with trypsin with the aim of generating peptides, which are used to identify novel shell proteins contained in databases. However, using trypsin as a sole degradative agent is limited by the enzyme's cleavage specificity and is dependent upon the occurrence of lysine and arginine in the shell protein sequence. To bypass this limitation, we investigated the ability of trifluoroacetic acid (TFA), a low-specificity chemical degradative agent, to generate clusters of analyzable peptides from organic shell matrix, suitable for database annotation. Acetic acid-insoluble fractions from Haliotis tuberculata shell were processed by trypsin followed by TFA digestion. The hydrolysates were used to annotate an expressed sequence tag library constructed from the mantle tissue of Haliotis asinina, a tropical abalone species. The characterization of sequences with repeat motifs featured in some of the shell matrix proteins benefited from TFA-induced serial cutting, which can result in peptide ladder series. Using the degradative specificities of TFA and trypsin, we were able to identify five novel shell proteins. This pilot study indicates that a mild chemical digestion of organic shell matrix combined with trypsin generates peptides suitable for proteomic analysis for better characterization of mollusc shell matrix proteins. 相似文献
139.
Pardo A Stöcker M Kampmeier F Melmer G Fischer R Thepen T Barth S 《Cancer immunology, immunotherapy : CII》2012,61(10):1617-1626
Purpose
Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.Methods
We transfected A-431 tumour cells with the far red–emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A’ (ETA’). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.Results
The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA’ resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.Conclusions
We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA’. 相似文献140.
Monteiro SP do Brasil PE Cabello GM de Souza RV Brasil P Georg I Cabello PH De Castro L 《Memórias do Instituto Oswaldo Cruz》2012,107(2):224-230
Severe forms of dengue, such as dengue haemorrhagic fever (DHF) and dengue shock syndrome, are examples of a complex pathogenic mechanism in which the virus, environment and host immune response interact. The influence of the host's genetic predisposition to susceptibility or resistance to infectious diseases has been evidenced in several studies. The association of the human leukocyte antigen gene (HLA) class I alleles with DHF susceptibility or resistance has been reported in ethnically and geographically distinct populations. Due to these ethnic and viral strain differences, associations occur in each population, independently with a specific allele, which most likely explains the associations of several alleles with DHF. As the potential role of HLA alleles in the progression of DHF in Brazilian patients remains unknown, we then identified HLA-A alleles in 67 patients with dengue fever and 42 with DHF from Rio de Janeiro, Brazil, selected from 2002-2008 by the sequence-based typing technique. Statistical analysis revealed an association between the HLA-A*01 allele and DHF [odds ratio (OR) = 2.7, p = 0.01], while analysis of the HLA-A*31 allele (OR = 0.5, p = 0.11) suggested a potential protective role in DHF that should be further investigated. This study provides evidence that HLA class I alleles might be important risk factors for DHF in Brazilian patients. 相似文献