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991.
C S Nielsen P H Hansen A Lihme P M Heegaard 《Journal of biochemical and biophysical methods》1989,20(1):69-79
Monitoring of acylation reactions during solid phase peptide synthesis is important to ensure high coupling yields in all steps of the synthesis. We describe in this paper a simple and reliable method for monitoring the time course of the acylation steps as well as the washing and deprotection steps during computer-controlled solid phase peptide synthesis. The method is based on the continuous measurement of electrical conductivity in the reaction vessel. It is shown that there is a close correspondence between the degree of acylation (as determined from the amount of 9-fluorenylmethoxycarbonyl- (Fmoc) groups released during deprotection) and the conductivity profile obtained during coupling of the amino acids to the growing peptide chain. The measurements are fed back to the computer providing data for software control of the duration of the acylation, deprotection and washing steps. The method is demonstrated with pentafluorophenol esters, but is equally applicable to dihydroxybenzotriazole esters and symmetric anhydrides using the Fmoc-polyamide strategy in a continuous flow set-up with dimethylformamide (DMF) as the general solvent. 相似文献
992.
Sensory and pulmonary irritation of butylamine was investigated in CF-1 and NMRI mice according to the American standard test method (ASTM E981-84). The method is based on the reflexively induced reduction of the respiratory rate of mice, when exposed to chemical irritants. Sensory irritation was investigated in normal mice, yielding RD50 values (concentration which reduces the respiratory rate by 50%) of 121 and 246 ppm for CF-1 and NMRI mice, respectively. The concentration-effect curves were parallel, but had significantly different elevations, indicating a lower sensitivity of NMRI mice. Pulmonary irritation was investigated in mice, inhaling through a tracheal cannula, yielding RD50 values of 300 and 362 ppm for CF-1 and NMRI mice, respectively. No statistically significant difference between either the slopes or the elevations of the concentration-effect curves was found, indicating the same level of sensitivity of CF-1 and NMRI mice regarding pulmonary irritation. It can be concluded that the 2 mice stocks gave qualitatively comparable responses, but regarding sensory irritation they responded differently quantitatively. Thus for sensory irritation investigations the RD50 values obtained with NMRI mice should be multiplied by 0.49 to obtain comparable values to those, expected in the recommended stock given by E981-84. 相似文献
993.
M H Perrin W H Fischer K S Kunitake A G Craig S C Koerber L A Cervini J E Rivier J C Groppe J Greenwald S M?ller Nielsen W W Vale 《The Journal of biological chemistry》2001,276(34):31528-31534
The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors. 相似文献
994.
Sucrose metabolism in cold-stored potato tubers with decreased expression of sucrose phosphate synthase 总被引:3,自引:0,他引:3
K.-P. Krause L. Hill R. Reimholz T. Hamborg Nielsen U. Sonnewald & M. Stitt 《Plant, cell & environment》1998,21(3):285-299
Transfer of potato tubers to low temperature leads after 2–4 d to a stimulation of sucrose synthesis, a decline of hexose-phosphates and a change in the kinetic properties, and the appearance of a new form of sucrose phosphate synthase (SPS). Antisense and co-suppression transformants with a 70–80% reduction in SPS expression have been used to analyse the contribution of SPS to the control of cold sweetening. The rate of sucrose synthesis in cold-stored tubers was investigated by measuring the accumulation of sugars, by injecting labelled glucose of high specific activity into intact tubers, and by providing 50 mol m–3 labelled glucose to fresh tuber slices from cold-stored tubers. A 70–80% decrease of SPS expression resulted in a reproducible but non-proportional (10–40%) decrease of soluble sugars in cold-stored tubers, and a non-proportional (about 25%) inhibition of label incorporation into sucrose, increased labelling of respiratory intermediates and carbon dioxide, and increased labelling of glucans. The maximum activity of SPS is 50-fold higher than the net rate of sugar accumulation in wild-type tubers, and decreased expression of SPS in the transformants was partly compensated for increased levels of hexose-phosphates. It is concluded that SPS expression per se does not control sugar synthesis. Rather, a comparison of the in vitro properties of SPS with the estimated in vivo concentrations of effectors shows that SPS is strongly substrate limited in vivo . Alterations in the kinetic properties of SPS, such as occur in response to low temperature, will provide a more effective way to stimulate sucrose synthesis than changes of SPS expression. 相似文献
995.
Structural similarity between legumin and vicilin storage proteins from legumes. 总被引:13,自引:1,他引:12 下载免费PDF全文
The primary structures for several members of both the vicilin and legumin families of storage proteins were examined using a computer routine based on amino acid physical characteristics. The comparison algorithm revealed that sequences from the two families could be aligned and share a number of predicted secondary structural features. The COOH-terminal half of the subunits in both families displayed a highly conserved core region that was largely hydrophobic and in which a high proportion of the residues were predicted to be in beta-sheet conformations. The central region of the molecules which contained mixed areas of predicted helical and sheet conformations showed more variability in residue selection than the COOH-terminal regions. The NH2-terminal segments of subunits from the two different families could not be aligned though they characteristically had a high proportion of residues predicted to be in helical conformations. The feature which most clearly distinguished subunits between the two families was an inserted span in the legumin group with a high proportion of acidic amino acids located between the central and COOH-terminal domains. Residues in this insertion were predicted to exist mainly in helical conformation. Since considerable size variation occurs in this area amongst the legumin subunits, alterations in this region may have a minimal detrimental effect on the structure of the proteins. 相似文献
996.
I. Matsuba Å. Lernmark B. Michelsen J. H. Nielsen J. Scholler H. Vissing B. Welinder N. Tommerup M. Mikkelsen H. Ishikawa Y. Ikeda T. Tanese 《In vitro cellular & developmental biology. Plant》1988,24(11):1071-1076
Summary Cross-culture contamination of cell lines propagated in continuous culture is a frequent event and particularly difficult
to resolve in cells expressing similar phenotypes. We demonstrate that DNA-DNA hybridization to blotted endonuclease-digested
cell DNA effectively detects cross-culture contamination to monitor inter-species as well as intra-species cross contamination.
An insulin-producing cell-line, Clone-16, originally cloned from a human fetal endocrine pancreatic cell line did not produce
human c-peptide as anticipated. DNA from these cells showed no hybridization to the human ALU sequence probe, BLUR, and lacked
restriction fragment length polymorphism typical for the human HLA-DQ β-chain gene. Although a human insulin gene probe showed
a weak, nonhuman hybridization pattern, a cDNA probe for the Syrian hamster insulin gene hybridized strongly consistent with
a single copy hamster insulin gene. Karyotyping confirmed the absence of human chromosomes in the Clone-16 cells while sizes,
centromere indices, and banding patterns were identical to Syrian hamster fibroblasts. We conclude that the insulin-producing
Clone-16 cells are of Syrian hamster origin and demonstrate the effective use of gene probes to control the origin of cell
cultures.
This paper is dedicated to the late Lis Lyngsie in much appreciation of her contributions to this study.
This work was supported in part by the National Institutes of Health, Bethesda, MD (grants DK 26190 and 33873). I. Matsuba
and B. Michelsen were supported by research fellowships from the Juvenile Diabetes Foundation International, and J. Scholler
from the Danish Medical Research Council (J.no. 12-5758). 相似文献
997.
Sequence comparison of the rDNA introns from six different species of Tetrahymena. 总被引:5,自引:4,他引:1 下载免费PDF全文
We have studied the sequence variation of the rDNA intron among six species of Tetrahymena. From these data, the intron appears to be relatively well conserved in evolution. We have evaluated the sequence variations among the most distant of these species in relation to the secondary structure model for the intron RNA of Cech et al. (Proc. Natl. Acad. Sci. U.S.A. 80, 3903 (83)). Most of the sequence variation in the four new sequences reported here is found in single stranded loops in the model. However, in four cases we found nucleotide substitutions in duplex stem regions, two of them involving compensating base pair changes. Interestingly, one of these is found in a region that is known to be dispensable in the in vitro splicing reaction suggesting differences between the in vivo and in vitro reactions. One of the single nucleotide deletions is found in the so-called "internal guide sequence" which has been implicated in the alignment process during splicing. In conclusion, none of the observed natural sequence variations are in disfavor of the proposed secondary structure model. 相似文献
998.
Summary The chemotactic responsiveness of blood monocytes was tested in 16 patients with nonseminomatous testicular carcinoma before, during, and after chemotherapy. All the patients initially had monocyte chemotaxis within the normal range. No correlation with the histology of the tumor, the clinical stage, or the presence in serum of -fetoprotein and human chorionic gonadotropin was observed. Plasma from the patients did not inhibit the chemotaxis of normal monocytes, and serum from the patients contained no chemotactic factor inhibitor. During intensive chemotherapy with cis-platinum, bleomycin, and vinblastine a reversible defect in chemotaxis occurred without correlation to the development of fever. Two months after the completion of chemotherapy the chemotactic responsiveness was unchanged compared with pretreatment values. In conclusion, this study shows normal monocyte chemotaxis in patients with testicular carcinoma, which is in contrast to reports on a variety of other solid tumors. 相似文献
999.
Recurrent mutation pressure does not explain the prevalence of the marker (X) syndrome 总被引:1,自引:1,他引:0
F. Vogel J. Krüger K. Brøndum Nielsen J. P. Fryns D. Schindler A. Schinzel A. Schmidt E. Schwinger 《Human genetics》1985,71(1):1-6
Summary In order to test the hypothesis that the high prevalence of the mar(X) syndrome is caused by a high mutation rate in male germ cells only, the fraction of new mutants among mothers of probands in 112 informative families has been examined by segregation analysis among their brothers and sisters. The estimated fraction of new mutants among these mothers is much lower than expected if a stable equilibrium existed between an unusually high mutation rate and a selective disadvantage of mentally retarded, male and female mar(X) carriers. Hence, the above-mentioned hypothesis could not be confirmed. 相似文献
1000.
Bahareh Jouleh Marta Erdal Tomas Mikal Eagan Per Bakke Amund Gulsvik Rune Nielsen 《BMC pulmonary medicine》2018,18(1):195