全文获取类型
收费全文 | 12265篇 |
免费 | 886篇 |
国内免费 | 8篇 |
出版年
2021年 | 121篇 |
2020年 | 94篇 |
2019年 | 124篇 |
2018年 | 151篇 |
2017年 | 158篇 |
2016年 | 216篇 |
2015年 | 369篇 |
2014年 | 367篇 |
2013年 | 555篇 |
2012年 | 685篇 |
2011年 | 599篇 |
2010年 | 461篇 |
2009年 | 372篇 |
2008年 | 547篇 |
2007年 | 546篇 |
2006年 | 545篇 |
2005年 | 551篇 |
2004年 | 541篇 |
2003年 | 558篇 |
2002年 | 537篇 |
2001年 | 132篇 |
2000年 | 99篇 |
1999年 | 137篇 |
1998年 | 169篇 |
1997年 | 154篇 |
1996年 | 145篇 |
1995年 | 156篇 |
1994年 | 127篇 |
1993年 | 161篇 |
1992年 | 113篇 |
1991年 | 138篇 |
1990年 | 120篇 |
1989年 | 103篇 |
1988年 | 122篇 |
1987年 | 107篇 |
1986年 | 91篇 |
1985年 | 111篇 |
1984年 | 168篇 |
1983年 | 141篇 |
1982年 | 140篇 |
1981年 | 151篇 |
1980年 | 125篇 |
1979年 | 109篇 |
1978年 | 131篇 |
1977年 | 101篇 |
1976年 | 87篇 |
1975年 | 94篇 |
1974年 | 80篇 |
1973年 | 67篇 |
1970年 | 61篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
941.
A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences
were derived from highly conservative regions of the pmoA gene, encoding the α-subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments
with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers
were specific for members of the genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs
in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA
extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently
separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In
PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers.
By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong
similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils.
Received: 1 September 2000 / Accepted: 2 October 2000 相似文献
942.
Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells 总被引:23,自引:0,他引:23
Schneider Eric; Schmid-Kotsas Alexandra; Zhao Jinshun; Weidenbach Hans; Schmid Roland M.; Menke Andre; Adler Guido; Waltenberger Johannes; Grunert Adolf; Bachem Max G. 《American journal of physiology. Cell physiology》2001,281(2):C532
The aim of thisstudy was to identify fibrogenic mediators stimulatingactivation, proliferation, and/or matrix synthesis of rat pancreaticstellate cells (PSC). PSC were isolated from the pancreas of normalWistar rats and from rats with cerulein pancreatitis. Cell activationwas demonstrated by immunofluorescence microscopy of smooth muscle-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin,and transforming growth factor (TGF)-1. Proliferationwas measured by bromodeoxyuridine incorporation. Matrix synthesis wasdemonstrated on the protein and mRNA level. Within a few days inprimary culture, PSC changed their phenotype from fat-storing toSMA-positive myofibroblast-like cells expressing platelet-derivedgrowth factor (PDGF) - and PDGF -receptors. TGF-1and tumor necrosis factor (TNF)- accelerated the change in thecells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basicfibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 ± 0.49- and 2.96 ± 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 ± 1.13-fold), 5 ng/ml TGF-1 (2.46 ± 0.89-fold), 20 ng/ml PDGF (2.27 ± 0.68-fold), and 50 ng/ml TGF- (1.87 ± 0.19-fold). As shownby RT-PCR, PSC express predominantly the splice variant EIII-A offibronectin. Immunofluorescence microscopy and Northern blot confirmedthat in particular bFGF and TGF-1 stimulated thesynthesis of fibronectin and collagens type I and III. In conclusion,our data demonstrate that 1) TGF-1 andTNF- accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF,TGF-1, PDGF, and, to a lesser extent, TGF- stimulateextracellular matrix synthesis of cultured rat PSC. 相似文献
943.
Glycoproteins, as a class of biomolecules, exhibit much more heterogeneous structures than non-glycosylated proteins. They present a challenging area of research. Model glycoproteins with well-defined protein and carbohydrate structures are helpful in the search for high-resolution methods for the separation of glycoproteins. Neoglycoproteins, maltose-modified chymotrypsin and lactose-modified chymotrypsin, were synthesised by modifying chymotrypsin with maltose and lactose, respectively, using the reductive amination method. Boronate chromatography was applied to isolate the neoglycoproteins from non-glycosylated substances. The use of Tris–HCl as a shielding reagent during the boronate chromatography proved to be efficient in eliminating unwanted interactions between the boronate ligand and the peptide backbone of chymotrypsin. The retention time of neoglycoproteins on the boronate column was increased with increasing the degree of modification. 相似文献
944.
In this technical note we describe the setup and application of automated sample preparation and usage of flow-through NMR equipment for the characterization of ligand binding on proteins. In addition, we focus on the perspectives of automated analysis of 2D HSQC spectra to identify changes in patterns indicative for ligand binding or changes of sample conditions. In this context we discuss a combination of statistical and non-statistical data analysis. 相似文献
945.
Gronwald W Kirchhöfer R Görler A Kremer W Ganslmeier B Neidig KP Kalbitzer HR 《Journal of biomolecular NMR》2000,17(2):137-151
A computer program (RFAC) has been developed, which allows the automated estimation of residual indices (R-factors) for protein NMR structures and gives a reliable measure for the quality of the structures. The R-factor calculation is based on the comparison of experimental and simulated 1H NOESY NMR spectra. The approach comprises an automatic peak picking and a Bayesian analysis of the data, followed by an automated structure based assignment of the NOESY spectra and the calculation of the R-factor. The major difference to previously published R-factor definitions is that we take the non-assigned experimental peaks into account as well. The number and the intensities of the non-assigned signals are an important measure for the quality of an NMR structure. It turns out that for different problems optimally adapted R-factors should be used which are defined in the paper. The program allows to compute a global R-factor, different R-factors for the intra residual NOEs, the inter residual NOEs, sequential NOEs, medium range NOEs and long range NOEs. Furthermore, R-factors can be calculated for various user defined parts of the molecule or it is possible to obtain a residue-by-residue R-factor. Another possibility is to sort the R-factors according to their corresponding distances. The summary of all these different R-factors should allow the user to judge the structure in detail. The new program has been successfully tested on two medium sized proteins, the cold shock protein (TmCsp) from Termotoga maritima and the histidine containing protein (HPr) from Staphylococcus carnosus. A comparison with a previously published R-factor definition shows that our approach is more sensitive to errors in the calculated structure. 相似文献
946.
Phenology in central Europe – differences and trends of spring phenophases in urban and rural areas 总被引:5,自引:0,他引:5
Roetzer T Wittenzeller M Haeckel H Nekovar J 《International journal of biometeorology》2000,44(2):60-66
In order to examine the impacts of both large-scale and small-scale climate changes (urban climate effect) on the development
of plants, long-term observations of four spring phenophases from ten central European regions (Hamburg, Berlin, Cologne,
Frankfurt, Munich, Prague, Vienna, Zurich, Basle and Chur) were analysed. The objective of this study was to identify and
compare the differences in the starting dates of the pre-spring phenophases, the beginning of flowering of the snowdrop (Galanthus nivalis) and forsythia (Forsythia sp.), and of the full-spring phenophases, the beginning of flowering of the sweet cherry (Prunus avium) and apple (Malus domestica), in urban and rural areas. The results indicate that, despite regional differences, in nearly all cases the species studied
flower earlier in urbanised areas than in the corresponding rural areas. The forcing in urban areas was about 4 days for the
pre-spring phenophases and about 2 days for the full-spring phenophases. The analysis of trends for the period from 1951 to
1995 showed tendencies towards an earlier flowering in all regions, but only 22% were significant at the 5% level. The trends
for the period from 1980 to 1995 were much stronger for all regions and phases: the pre-spring phenophases on average became
earlier by 13.9 days/decade in the urban areas and 15.3 days/decade in the rural areas, while the full-spring phenophases
were 6.7 days earlier/decade in the urban areas and 9.1 days/decade earlier in the rural areas. Thus rural areas showed a
higher trend towards an earlier flowering than did urban areas for the period from 1980 to 1995. However, these trends, especially
for the pre-spring phenophases, turned out to be extremely variable.
Received: 21 October 1999 / Revised: 5 April 2000 / Accepted: 25 April 2000 相似文献
947.
948.
Human quadriceps mitochondria were isolated from ca. 80 mg tissue in ca. 45% yield. The preparation is described with respect to content of mitochondrial markers and nine different respiratory activities. The specific state 3 activities were high in comparison with literature data, indicating high integrity and purity of the preparation. Examples of state 3 rates, in µmol O min-1 g protein-1 (25°C): pyruvate + malate, 400; succinate, 514; malate + glutamate, 444. The notion of high integrity was also supported by the reproducibility of the preparation and the magnitude of the respiratory control ratios and the P/O ratios. The mitochondria most likely had lost ca. 30% of their cytochrome c upon isolation, but it was substantiated that this loss had not influenced the state 3 rates. Functional assays of single reactions or groups of reactions could be based on respiration experiments. The respiratory chain activity, for instance, was measured as respiration of NADH in freeze-permeabilized mitochondria (1263 mol O min-1 g protein-1). Comparison of uncoupled rates of respiration and state 3 rates indicated that the ATP synthesis exerted major flux control over respiration of succinate + glutamate, malate + glutamate and pyruvate + malate. These reactions, showing very similar rates of ATP synthesis, could be used as a functional assay of ATP synthesis (1200 mol ATP min-1 g protein-1). Respiration of succinate, palmitoyl-carnitine + malate, or glutamate could not support the maximal rate of ATP synthesis and the upstream reactions probably exerted major flux control in these cases. The specific activities appeared very constant in this group of young men, only the respiratory activity with glutamate might show biological variation. 相似文献
949.
Noninvasive and/or nondestructive techniques are capable of providing more macro- or microstructural information about bone than standard bone densitometry. Although the latter provides important information about osteoporotic fracture risk, numerous studies indicate that bone strength is only partially explained by bone mineral density. Quantitative assessment of macro- and microstructural features may improve our ability to estimate bone strength. The methods available for quantitatively assessing macrostructure include (besides conventional radiographs) quantitative computed tomography (QCT) and volumetric quantitative computed tomography (vQCT). Methods for assessing microstructure of trabecular bone noninvasively and/or nondestructively include high-resolution computed tomography (hrCT), micro-computed tomography (muCT), high-resolution magnetic resonance (hrMR), and micromagnetic resonance (muMR). vQCT, hrCT and hrMR are generally applicable in vivo; muCT and muMR are principally applicable in vitro. Although considerable progress has been made in the noninvasive and/or nondestructive imaging of the macro- and microstructure of bone, considerable challenges and dilemmas remain. From a technical perspective, the balance between spatial resolution versus sampling size, or between signal-to-noise versus radiation dose or acquisition time, needs further consideration, as do the trade-offs between the complexity and expense of equipment and the availability and accessibility of the methods. The relative merits of in vitro imaging and its ultrahigh resolution but invasiveness versus those of in vivo imaging and its modest resolution but noninvasiveness also deserve careful attention. From a clinical perspective, the challenges for bone imaging include balancing the relative advantages of simple bone densitometry against the more complex architectural features of bone or, similarly, the deeper research requirements against the broader clinical needs. The considerable potential biological differences between the peripheral appendicular skeleton and the central axial skeleton have to be addressed further. Finally, the relative merits of these sophisticated imaging techniques have to be weighed with respect to their applications as diagnostic procedures requiring high accuracy or reliability on one hand and their monitoring applications requiring high precision or reproducibility on the other. 相似文献
950.
Detection of apoptosis in tissue sections 总被引:17,自引:0,他引:17
During the last few years, detection of apoptosis has evolved from a predominantly morphological basis to the use of ever more specific techniques. The methods widely used to visualize DNA fragmentation in tissue sections are now supplemented by a variety of specific antisera against components of the cell death pathways. Essential requirements for apoptosis detection techniques include high sensitivity for apoptotic cells, the ability to differentiate between apoptotic and necrotic cell death and other forms of DNA damage, and the distinction between different stages of the cell death process. In this overview, we will focus on recent technical advances in apoptosis detection covering improvements of in situ DNA fragmentation techniques, as well as pointing out some of the new tools available for the detection of apoptotic cells in tissue. 相似文献