全文获取类型
收费全文 | 55010篇 |
免费 | 4617篇 |
国内免费 | 36篇 |
出版年
2023年 | 269篇 |
2022年 | 580篇 |
2021年 | 1054篇 |
2020年 | 772篇 |
2019年 | 835篇 |
2018年 | 1207篇 |
2017年 | 1003篇 |
2016年 | 1638篇 |
2015年 | 2348篇 |
2014年 | 2308篇 |
2013年 | 2887篇 |
2012年 | 3427篇 |
2011年 | 3161篇 |
2010年 | 1932篇 |
2009年 | 1871篇 |
2008年 | 2290篇 |
2007年 | 2270篇 |
2006年 | 2066篇 |
2005年 | 2377篇 |
2004年 | 2358篇 |
2003年 | 1962篇 |
2002年 | 1592篇 |
2001年 | 1468篇 |
2000年 | 1395篇 |
1999年 | 1253篇 |
1998年 | 573篇 |
1997年 | 543篇 |
1996年 | 594篇 |
1995年 | 461篇 |
1994年 | 468篇 |
1993年 | 438篇 |
1992年 | 936篇 |
1991年 | 839篇 |
1990年 | 770篇 |
1989年 | 764篇 |
1988年 | 759篇 |
1987年 | 691篇 |
1986年 | 650篇 |
1985年 | 626篇 |
1984年 | 584篇 |
1983年 | 443篇 |
1982年 | 339篇 |
1981年 | 335篇 |
1980年 | 316篇 |
1979年 | 437篇 |
1978年 | 363篇 |
1977年 | 298篇 |
1975年 | 300篇 |
1974年 | 302篇 |
1973年 | 300篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
991.
V. Kumar J. L. de la Fuente A. L. Leitão P. Liras J. F. Martin 《Applied microbiology and biotechnology》1996,45(5):621-628
The bla gene of the cephamycin cluster of Nocardia lactamdurans has been subcloned in the shuttle plasmids pULVK2 and pULVK2A and amplified in N. lactamdurans LC411. The transformants showed two- to threefold higher β-lactamase activity. Formation of β-lactamase preceded the onset
of cephamycin biosynthesis. The β-lactamase of N. lactamdurans inactivated penicillins and, to a lesser extent, cephalosporin C but did not hydrolyse cephamycin C. This β-lactamase was
highly sensitive to clavulanic acid (50% inhibition was observed at 0.48 μg/ml clavulanic acid). The N. lactamdurans bla gene was disrupted in vivo by inertion of the kanamycin-resistance gene. Three bla-disrupted mutants, BD4, BD8 and BD12, were selected that lacked β-lactamase activity. Overexpresion of the bla gene resulted in N. lactamdurans transformants that were resistant to penicillin whereas mutants in which the bla gene was disrupted were supersensitive to this antibiotic. The three N. lactamdurans mutants with the bla gene disrupted showed a significant increase of cephamycin biosynthesis in solid medium, whereas transformants with the amplified
bla gene produced reduced levels of cephamycin. The cephamycin-overproducing Merck strain N. lactamdurans MA4213 showed no detectable levels of β-lactamase activity. The β-lactamase plays a negative role in cephamycin biosynthesis
in solid medium, but not in liquid medium.
Received: 26 July 1995/Received revision: 18 December 1995/Accepted: 8 January 1996 相似文献
992.
We describe the application of lamin immunocytochemistry (ICC) and single- or double-target fluorescence in situ hybridization (FISH) on 4 microm thick frozen tissue sections as a method to facilitate scoring of aberrant chromosome copy numbers in colonic tumors. Analysis of FISH signals in colon tissue sections is often hampered by overlap and truncation of epithelial nuclei, due to the density of the epithelial cells. Furthermore, on the basis of nuclear staining it is often difficult to determine whether or not nuclei are overlapping, or adjoining. Therefore, reliable evaluation of (F)ISH signals to screen for genomic changes was until now mainly restricted to isolated nuclei obtained from relatively thick tissue sections. In this study the applicability of lamin ICC, to stain the nuclear periphery and to distinguish individual nuclei, combined with the FISH procedure is explored to solve this problem for colon epithelium. For ICC we applied the alkaline phosphatase (APase)-Fast Red detection method, since the fluorescent precipitate of this reaction resists extensive proteolytic digestion as needed for efficient FISH on tissue sections. Chromosome copy numbers could easily be determined in 4 microm thick frozen tissue sections by combining lamin ICC and FISH. The ratio of the copy numbers of the chromosomes 7 and 17 could be determined in frozen tissue sections after combined lamin ICC and double-target FISH. It is concluded that the combination of lamin ICC and FISH improves chromosome copy number analysis and can be used to investigate genomic changes in different tumor compartments in thin frozen tissue sections. 相似文献
993.
D. de Beer V. O’Flaharty J. Thaveesri P. Lens W. Verstraete D. de Beer 《Applied microbiology and biotechnology》1996,46(2):197-201
Extracellular polysaccharides (EPS) were quantified in dense granules and loose flocs by chemical analysis of the uronic
acid content. Their distribution within the aggregates was determined by microscopic staining. Granules contained a higher
amount of EPS (1–1.6 mg/g volatile suspended solids, VSS) than flocs (0.3 mg/g VSS). In granules approximately 50% of the
total amount of EPS was present in a 40-μm-thick zone on the surface. The remainder was dispersed in the rest of the aggregate.
In flocs the highest concentration was present in the centre and the EPS layer on the surface was not found. Tests showed
that flocculent sludge was very sensitive to flotation, while the studied granules did not float. The lower susceptibility
to flotation of granules as compared to flocs was attributed to the presence of the hydrophilic EPS coating that prevents
attachment of gas bubbles.
Received: 21 November 1995/Received last revision: 15 April 1996/Accepted: 22 April 1996 相似文献
994.
Associations between different agonistic and affiliative behavioural patterns of female domestic cats (Felis silvestris catus) were studied. In three groups of intact cats living in confinement frequencies of fourteen agonistic and affiliative behavioural
patterns were recorded. The technique of factor analysis (Principal Components Analysis followed by varimax rotation on a
dyads X behavioural patterns matrix) was used to detect clusters in these behavioural patterns. Five factors (or types of
interindividual relationships) were extracted per group. They accounted collectively for at least 77% of the total variance
present in the data. Although differences existed between groups with respect to behavioural patterns included in each factor,
four clusters of behaviours could be discriminated: (I) social rubbing, lordosis and rolling in front of partner (sexual behaviour),
(II) allogrooming, social sniffing, nosing, sniffing rear and treading (inspection-affiliative behaviour), (III) offensive
behaviour and staring, and (IV) defensive behaviour and staring. The role of these clusters in group living is discussed. 相似文献
995.
Ana Maria Landeira-Fernandez Maricilia S. Costa Leopolde de Meis 《Bioscience reports》1996,16(6):439-451
Vesicles derived from maize roots retain a membrane bound H+-ATPase that is able to pump H+ at the expense of ATP hydrolysis. In this work it is shown that heparin, fucose-branched chondroitin sulfate and dextran sulfate 8000 promote a shift of the H+-ATPase optimum pH from 6.0 to 7.0. This shift is a result of a dual effect of the sulfated polysaccharides, inhibition at pH 6.0 and activation at pH 7.O. At pH 6.0 dextran 8000 promotes an increase of the apparent Km for ATP from 0.28 to 0.95 mM and a decrease of the Vmax from 14.5 to 7.1 mol Pi/mg · 30 min–1. At pH 7.0 dextran 8000 promotes an increase in Vmax from 6.7 to 11.7 mol Pi/mg · 30 min–1. In the presence of lysophosphatidylcholine the inhibitory effect of the sulfated polysaccharides observed at pH 6.0 was not altered but the activation of pH 7.0 decreased. It was found that in the presence of sulfated polysaccharides the ATPase became highly sensitive to K+ and Na+. Both the inhibition at pH 6.0 and the activation promoted by the polysaccharide were antagonized by monovalent cations (K+>Na+Li+).Abbreviations Mops
4-morpholinopropanesulfonic acid
- EDTA
ethylenediaminetetraacetic acid
- ACMA
9-amino-6-chloro-2-methoxyacridine
- FCCP
carbonyl cyanide p(trifluoromethoxy)-phenylhyrazone 相似文献
996.
J. Martin Sanchez F. Ruiz Martinez J. M. Salinas Martinez de Lecea C. Sanchez Rabasco C. Acedo Sanchez M. C. Sanchiz Marin V. Delgado Florencio F. Morillas Marquez 《Systematic parasitology》1996,33(3):177-182
This study presents the results of the characterisation of 17 strains ofLeishmania by isoenzyme electrophoresis from a focus of leishmaniasis in southern Spain: two from human visceral leishmaniasis, four from human cutaneous leishmaniasis and 11 from sandflies. The 17 strains are grouped in 6 zymodemes characterised by their variability as regards to the electrophoretic mobility of the enzymes MDH, G6PD, NP and ME. Thus, we confirm the high intraspecific variability ofLeishmania (L.) infantum in a focus of southern Spain, as already suggested by previous studies. Zymodemes GR-15 and GR-17 are also described for the first time in Spain, and they characteristically possess the same relative electrophoretic mobility in the enzyme ME (93). Sixteen zymodemes of theL. infantum complex found in southern Spain were numerically analysed on the basis of the enzymatic profiles of 122Leishmania strains characterised from this area. 相似文献
997.
Alexander Schouten Jan Roosien Fred A. van Engelen G. A. M. de Jong A. W. M. Borst-Vrenssen Jacoline F. Zilverentant Dirk Bosch Willem J. Stiekema Fred J. Gommers Arjen Schots Jaap Bakker 《Plant molecular biology》1996,30(4):781-793
The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed. 相似文献
998.
Three new polymorphic equine microsatellites: HLM2, HLM3, HLM5 总被引:1,自引:0,他引:1
999.
Inactivation of Btk by insertion of lacZ reveals defects in B cell development only past the pre-B cell stage. 总被引:9,自引:0,他引:9
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
R W Hendriks M F de Bruijn A Maas G M Dingjan A Karis F Grosveld 《The EMBO journal》1996,15(18):4862-4872
Bruton's tyrosine kinase (Btk) is a cytoplasmic protein kinase that is defective in X-linked agammaglobulinaemia in man and in X-linked immunodeficiency in the mouse. There is controversy regarding the stages of B cell development that are dependent on Btk function. To determine the point in B cell differentiation at which defects in Btk become apparent, we generated a mouse model by inactivating the Btk gene through an in-frame insertion of a lacZ reporter by homologous recombination in embryonic stem cells. The phenomenon of X-chromosome inactivation in Btk+/- heterozygous female mice enabled us to evaluate the competition between B cell progenitors expressing wild-type Btk and those expressing the Btk-/lacZ allele in each successive step of development. Although Btk was already expressed in pro-B cells, the first selective disadvantage only became apparent at the transition from small pre-B cells to immature B cells in the bone marrow. A second differentiation arrest was found during the maturation from IgD(low)IgM(high) to IgD(high)IgM(low) stages in the periphery. Our results show that Btk expression is essential at two distinct differentiation steps, both past the pre-B cell stage. 相似文献
1000.
Deep soil heterogeneity and fine root distribution in forests and pastures of eastern Amazonia 总被引:2,自引:1,他引:1
Little is known about deep soil heterogeneity, or its relationship with fine root distribution. Beneath a mature, closed-canopy forest of eastern Amazonia, and the pastures and secondary forests that are derived from this forest, soil soft spots and hollow chambers occur to at least 9 meters depth. We measured the vertical distribution of these soil patches, and compared chemical characteristics, mycorrhizal infection, and root density of soil soft spots with the surrounding matrix of more homogeneous soil. Soil soft spots and chambers varied little with depth, but occupied the greatest soil volume (0.8 to 1.2%) from 4 to 6 m depth in the mature forest. Soft spots had lower pH, P availability and arbuscular mycorrhizal infection, and higher K availability than surrounding soil. Root length density was 2 to 15 times higher in soft spots than in surrounding soil. In the pastures, roots were found only in soil soft spots at depths of >3 m. Pastures and secondary forest had more soil chambers in the upper meter of soil than mature forest, but were otherwise indistinguishable in their patterns of deep soil heterogeneity. Soil soft spots may be vestiges of cutter ant nest chambers, while hollow chambers are cutter ant chambers and root channels. Chambers may act as conduits for root penetration and water penetration to deep soil.Abbreviations AM
arbuscular mycorrhizae
- RLD
root length density (root length per unit of soil volume) 相似文献