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61.
Mitsuhashi J 《In vitro cellular & developmental biology. Animal》2001,37(6):330-337
Summary A highly nutritive culture medium (MGM-464) was developed for insect cell primary culture. The new medium consists of 6 inorganic
salts, 4 organic acids, 21 amino acids, 3 sugars, 10 vitamins, and 8 other chemicals, including natural substances. The complete
medium was generated by adding 20 ml fetal bovine serum to 100 ml MGM-464. The detail of the composition of the medium is
given in a table, and the protocol to prepare the medium is described in the text. Among the 15 kinds of cultures made with
MGM-464, embryonic cells from a walking stick and ovarian cells from the common white were subcultured more than 70 times,
and embryonic cells of a chrysomelid beetle were subcultured more than 15 times. Other cultures could not be subcultured.
However, embryonic cells from the commercial silkworm and a cockroach, ovarial cells from the commercial silkworm and a sphingid
moth, nervous cells from the commercial silkworm and two sphingid moths, and cells from the dorsal vessel plus surrounding
tissue of the commercial silkworm survived for several mo. The cells from the honeybee embryos, aphid embryos, and planthopper
embryos were rather short-lived, and deteriorated after about 1 mo. 相似文献
62.
Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block 总被引:6,自引:0,他引:6
The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section. The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a denned cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses. 相似文献
63.
AIMS: The influence of the spore preparation on subsequent fungal growth of Penicillium chrysogenum was assessed. METHODS AND RESULTS: The influence of four factors [the nature of the diluting solution (physiological water and physiological water added with Tween-80), the age of the sporulating culture (4, 8 and 12 days), the strain (737, 738 and 740) and the inoculum size (102, 103, 104 and 105 spores ml(-1)] on two responses (i.e. the radial growth rate, mu, and the lag time, lambda) was studied using an experimental screening methodology. CONCLUSIONS: The main conclusion was the strong effect of the inoculum size on lambda. In contrast, the diluting solution had no effect on both the experimental responses. In order to obtain the highest growth rates, it is recommended to use 4-day-old sporulating cultures with an inoculum size of 102 spores ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for standardizing spore preparation in predictive mycology. The screening methodology is a powerful tool to determine the influence of qualitative and quantitative factors on various biological responses and can be applied widely in microbiology. 相似文献
64.
Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers 总被引:2,自引:0,他引:2
Summary. Methods for cryogenic fixation, freeze substitution, and embedding were developed to preserve the cellular structure and
protein localization of secondary-wall-stage cotton (Gossypium hirsutum L.) fibers accurately for the first time. Perturbation by specimen handling was minimized by freezing fibers still attached
to a seed fragment within 2 min after removal of seeds from a boll still attached to the plant. These methods revealed native
ultrastructure, including numerous active Golgi bodies, multivesicular bodies, and proplastids. Immunolocalization in the
context of accurate structure was accomplished after freeze substitution in acetone only. Quantitation of immunolabeling identified
sucrose synthase both near the cortical microtubules and plasma membrane and in a proximal exoplasmic zone about 0.2 μm thick.
Immunolabeling also showed that callose (β-1,3-glucan) was codistributed with sucrose synthase within this exoplasmic zone.
Similar results were obtained from cultured cotton fibers. The distribution of sucrose synthase is consistent with its having
a dual role in cellulose and callose synthesis in secondary-wall-stage cotton fibers.
Received August 19, 2002; accepted November 12, 2002; published online June 13, 2003
RID="*"
ID="*" Correspondence and reprints: Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, U.S.A.
E-mail: candace.haigler@ttu.edu 相似文献
65.
Several genetic and transgenic mouse models are currently being used for studying the regulation of myocardial contractility under normal conditions and in disease states. Little information has been provided, however, about myocardial energy metabolism in mouse hearts. We measured glycolysis, glucose oxidation and palmitate oxidation (using 3H-glucose, 14C-glucose and 3H-palmitate) in isolated working mouse hearts during normoxic conditions (control group) and following a 15 min global no-flow ischemic period (reperfusion group). Fifty min following reperfusion (10 min Langendorff perfusion + 40 min working heart perfusion) aortic flow, coronary flow, cardiac output, peak systolic pressure and heart rate were 44 ± 4, 88 ± 4, 57 ± 4, 94 ± 2 and 81 ± 4% of pre-ischemic values. Rates of glycolysis and glucose oxidation in the reperfusion group (13.6 ± 0.8 and 2.8 ± 0.2 mol/min/g dry wt) were not different from the control group (12.3 ± 0.6 and 2.5 ± 0.2 mol/min/g dry wt). Palmitate oxidation, however, was markedly elevated in the reperfusion group as compared to the control group (576 ± 37 vs. 357 ± 21 nmol/min/g dry wt, p < 0.05). This change in myocardial substrate utilization was accompanied by a marked fall in cardiac efficiency measured as cardiac output/oxidative ATP production (136 ± 10 vs. 54 ± 5 ml/mol ATP, p < 0.05, control and reperfusion group, respectively). We conclude that ischemia-reperfusion in isolated working mouse hearts is associated with a shift in myocardial substrate utilization in favour of fatty acids, in line with previous observations in rat. 相似文献
66.
大熊猫气味标记DNA的制备和序列分析 总被引:2,自引:0,他引:2
大熊猫气味标记在其个体间的通讯中具有重要意义。用不同方法收集了7只大熊猫个体的9个气味标记样品,运用Instagene Kit制备出了DNA。采用PCR扩增线粒体D-环区和细胞色素b基因、Thr-tRNA基因片段并作序列分析。结果提示,不同收集方式所得气味标记样品均有DNA,但用干棉花收集样品的方法最佳。该方法为大熊猫的遗传多样性研究提供了新的简捷有效的DNA来源。 相似文献
67.
Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale. 相似文献
68.
69.
Mark R. Condina Johan O. R. Gustafsson Manuela Klingler‐Hoffmann Christopher J. Bagley Shaun R. McColl Peter Hoffmann 《Proteomics》2010,10(13):2516-2530
The quality of MALDI‐TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix‐deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix‐deposition strategy for LC‐MALDI‐TOF/TOF MS using an automated instrument that produces a nebulised matrix “mist” under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5‐DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix‐deposition strategy with LC‐MALDI‐TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC‐ESI‐IT‐MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor‐stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC‐MALDI‐TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis. 相似文献
70.
《Anthropology & education quarterly》2006,37(2):110-127
Through an analysis of a contemporary rite of passage-the final stage of teacher preparation-I develop a new theory of liminality that both builds on and extends Victor Turner's enduring insights. The analysis focuses on how preservice teachers in an undergraduate education program engage in a process of identity formation within an asynchronous, nondimensional, liminal space made possible and shaped by e-mail and with the support of experienced mentor teachers. 相似文献