全文获取类型
收费全文 | 169943篇 |
免费 | 13813篇 |
国内免费 | 13468篇 |
出版年
2024年 | 270篇 |
2023年 | 1913篇 |
2022年 | 4775篇 |
2021年 | 8037篇 |
2020年 | 5497篇 |
2019年 | 6796篇 |
2018年 | 6557篇 |
2017年 | 5019篇 |
2016年 | 6951篇 |
2015年 | 10118篇 |
2014年 | 12120篇 |
2013年 | 12737篇 |
2012年 | 15405篇 |
2011年 | 13948篇 |
2010年 | 8714篇 |
2009年 | 7760篇 |
2008年 | 9068篇 |
2007年 | 8138篇 |
2006年 | 7147篇 |
2005年 | 5865篇 |
2004年 | 4955篇 |
2003年 | 4401篇 |
2002年 | 3730篇 |
2001年 | 3207篇 |
2000年 | 2988篇 |
1999年 | 2860篇 |
1998年 | 1671篇 |
1997年 | 1694篇 |
1996年 | 1590篇 |
1995年 | 1417篇 |
1994年 | 1410篇 |
1993年 | 1030篇 |
1992年 | 1471篇 |
1991年 | 1195篇 |
1990年 | 925篇 |
1989年 | 848篇 |
1988年 | 723篇 |
1987年 | 609篇 |
1986年 | 578篇 |
1985年 | 557篇 |
1984年 | 332篇 |
1983年 | 319篇 |
1982年 | 228篇 |
1981年 | 163篇 |
1980年 | 158篇 |
1979年 | 177篇 |
1978年 | 112篇 |
1976年 | 100篇 |
1974年 | 126篇 |
1972年 | 112篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
991.
Peshwa MV Page LA Qian L Yang D van Schooten WC 《Biotechnology and bioengineering》1996,50(5):529-540
CD8(+) cytotoxic T-lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide-specific CD8(+) T-lymphocytes from the peripheral blood of healthy, human trophic leukemia virus-1 (HTLV-1) seronegative human leucocyte antigen (HLA)-A*0201 individuals. A combination of density-based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen-presenting cells (APCs), and T-lymphocytes. The DCs were pulsed with HLA-A*0201 binding peptides and cultured with autologous T-lymphocytes to generate peptide-specific CTLs. The CTLs were generated against a nine-amino-acid peptide from the Tax protein of HTLV-1. The CTLs were expanded according to a restimulation schedule employing peptide-pulsed autologous monocytes and low-dose interleukin-2 (IL-2) to numbers in excess of 100 x 10(6) cells following 5 weeks of culture. Expanded cells contained primarily CD3(+) T-cells, of which CD8(+) T-lymphocytes constituted greater than two-thirds of the cell population. Obtained CTLs exhibited potent antigen-specific lysis of peptide-pulsed target cells in a dose-dependent fashion in in vitro (51)Cr release cytotoxicity assay. This antigen-specific killing was shown to be HLA class I restricted and mediated by CD8(+) T-lymphocytes. Since the T-lymphocytes were obtained from HTLV-1 seronegative donors, the generation of peptide-specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. (c) 1996 John Wiley & Sons, Inc. 相似文献
992.
The effects of the impeller configuration, aeration rate, and agitation speed on oxygen transfer coefficient K(L)a were studied in a newly designed centrifugal impeller bioreactor (5-L). The oxygen transfer rates in the novel bioreactor were also compared with those in a cell-lift bioreactor with comparable dimensions. The cell-lift impeller produced much higher surface oxygen transfer rates than the centrifugal one at an agitation speed over 200 rpm. This result was in good agreement with our observation that the cell-lift impeller produced much higher unfavorable turbulence. In addition, the experiments using granulated agar particles as pseudo plant cells indicated that the K(L)a value decreased steadily with an increase in agar particle concentration, and the centrifugal impeller still demonstrated a larger K(L)a than the cell lift up to a high pseudo cell concentration of 19.5 g dry weight (DW)/L (under 150 rpm and 0.20 vvm) or 22.3 g DW/L (under 200 rpm and 0.20 vvm). Furthermore, the correlation between power number and impeller Reynolds number for both the centrifugal and the cell-lift impellers was successfully obtained, which could be used for predicting the power input required by each impeller. From the results obtained, the centrifugal impeller bioreactor is expected to have great potential in its application to shear-sensitive biological systems. 相似文献
993.
A method was developed to provide a real-time measurement of intracellular adenosine 5'-triphosophate (ATP) concentrations in growing Escherichia coli. The bacteria to be monitored must first be modified by inserting the cDNA for firefly luciferase expressed from a constitutive promoter. Such a construct leads to constant specific activity of firefly luciferase during both the lag phase and exponential growth. When the luciferase substrate, D-luciferin, is added to the medium, ATP within the cells is utilized in the luciferase-catalyzed reaction that produces light. The light is carried from the bioreactor to a computer-based detector by an optical fiber. The detected per cell light emission varies during exponential growth. Analysis of cytoplasm extracts shows that this variance is related to changes in the ATP concentration, which ranges from 1 to 6 times the literature value for K(M). Experimental analyses demonstrated that inner filter effects are not a significant factor affecting the use of this system. The method was tested in a benchtop fermentor at cell densities above 13 g/L dry cell weight. A correction factor based on the accumulated light data is calculated and used in real time to account for consumption of luciferin from the culture broth by the light producing reaction. Dissolved oxygen concentrations must be kept above 15% of air saturation to ensure constant light output, but no detectable increase in oxygen demand is seen. The method does not significantly affect growth or production rates. (c) 1996 John Wiley & Sons, Inc. 相似文献
994.
Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network 总被引:1,自引:0,他引:1
A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH(2). A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH(2) accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (<27%) cultures. The TCA cycle provides 51-68% of the total ATP production. The calculated stoichiometric oxygen consumption differs among the batch and fed-batch cultures, depending on the glucose concentration. This result suggests that the relationship between the oxygen uptake rate (OUR) and cell growth may change with the culture conditions. However, the calculated respiratory quotient (RQ) is relatively constant in all cases.A linear relationship is obtained between the specific ATP production rate and the specific cell growth rate. The maximum ATP yield and the maintenance ATP requirement are determined based on this linear relationship. The biosynthetic ATP demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand. (c) 1996 John Wiley & Sons, Inc. 相似文献
995.
Pei-Wen Chiang SuQing Wang Paul Smithivas Woo-Joo Song Saravanan Ramamoorthy Joseph Hillman Sheryl Puett Margaret L. Van Keuren Eric Crombez Arun Kumar Thomas W. Glover Diane E. Miller Chun-Hui Tsai C.Clare Blackburn Xiao-Ning Chen Zhiguang Sun Jan-Fang Cheng Julie R. Korenberg David M. Kurnit 《Genomics》1996,34(3):328
996.
997.
998.
In the search for candidate genes for the tuberous sclerosis (TSC1) disease locus on chromosome 9q34, we have isolated an overlapping series of 22 plasmid and phage cDNA clones covering nearly 7 kb and with an open reading frame of 5070 bp encoding a protein of 1690 amino acids. The putative protein product is a member of the kinesin superfamily and is homologous to the mouse KIF1A and theCaenorhabditas elegansunc-104 genes. Both KIF1A and unc-104 function in the anterograde axonal transport of synaptic vesicles. The human homolog is therefore termed H-ATSV (axonal transporter of synaptic vesicles, HGMW-approved nomenclature ATSV) Screening of DNA from 107 tuberous sclerosis patients and 80 unaffected individuals with H-ATSV cDNA probes by pulsed-field gel electrophoresis/Southern blotting following digestion by rare-cutting methylation-sensitive restriction enzymes showed variant banding patterns in three patients with tuberous sclerosis. However, further analysis indicated that these variant fragments represent a rare polymorphism probably associated with methylation of clustered restriction sites. There is no evidence to support H-ATSV as a candidate gene for TSC1. 相似文献
999.
Approximate methods for estimating the pattern of nucleotide substitution and the variation of substitution rates among sites 总被引:13,自引:6,他引:7
We propose two approximate methods (one based on parsimony and one on
pairwise sequence comparison) for estimating the pattern of nucleotide
substitution and a parsimony-based method for estimating the gamma
parameter for variable substitution rates among sites. The matrix of
substitution rates that represents the substitution pattern can be
recovered through its relationship with the observable matrix of site
pattern frequences in pairwise sequence comparisons. In the parsimony
approach, the ancestral sequences reconstructed by the parsimony algorithm
were used, and the two sequences compared are those at the ends of a branch
in the phylogenetic tree. The method for estimating the gamma parameter was
based on a reinterpretation of the numbers of changes at sites inferred by
parsimony. Three data sets were analyzed to examine the utility of the
approximate methods compared with the more reliable likelihood methods. The
new methods for estimating the substitution pattern were found to produce
estimates quite similar to those obtained from the likelihood analyses. The
new method for estimating the gamma parameter was effective in reducing the
bias in conventional parsimony estimates, although it also overestimated
the parameter. The approximate methods are computationally very fast and
appear useful for analyzing large data sets, for which use of the
likelihood method requires excessive computation.
相似文献