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31.
Terry L. Riss Kenneth P. Karey B. Daniel Burleigh David Farker David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(11):1099-1106
Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant
insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on
poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's
modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin,
100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free
growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold
more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The
concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED50), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like
growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin,
ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED50, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth
factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures.
This work was supported by a contract from IMCERA Bioproducts, Inc. 相似文献
32.
Kenneth P. Karey Terry L. Riss B. Daniel Burleigh David Parker David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(11):1107-1113
Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized,
resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (125I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation
at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding
sites with K
d
=59.6 pM and an estimated 1.57×105 receptors/cell. Half-maximal displacement of bound125I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective
competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal
growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for125I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more
tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that125I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol.
This work was supported by a contract from IMCERA Bioproducts, Inc. 相似文献
33.
A. A. Galoyan B. Ya. Gurvits L. A. Shuvalova Michael T. Davis John E. Shively Terry D. Lee 《Neurochemical research》1992,17(8):773-777
A new class of stimulators of basal activity of a number of calmodulin-dependent enzymes have been previously isolated from bovine hypothalamus. One of these stimulators, denoted as C3, has been purified to homogeneity by reverse phase HPLC and tentatively identified as thymosin 4 (1–39) by mass spectrometry and Edman microsequence analysis. The stimulating effect of C3 on rabbit skeletal muscle MLCK basal activity was compared with that of thymosin 1 and thymosin 4 (16–38). Evidence is presented that all the indicated compounds are Ca2+-independent high-affinity MLCK stimulators. The potency of the stimulators in activating the enzyme was: C3>4>(CaM+Ca2+>1.This revised version was published online in June 2005 with corrections to the author name Gurvits. 相似文献
34.
Shyam K. Sharan Bernadette Holdener-Kenny David W. Threadgill Terry Magnuson 《Mammalian genome》1992,3(2):79-83
Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early post-implantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm. 相似文献
35.
Eugene M. Rinchik Terry Magnuson Bernadette Holdener-Kenny Gavin Kelsey Albert Bianchi Claudio J. Conti Fran?ois Chartier Kathryn A. Brown Stephen D. M. Brown Josephine Peters 《Mammalian genome》1992,3(Z1):S104-S120
Chair of Committee for Mouse Chromosome 7 相似文献
36.
François Baneyx Amanda Ayling Terry Palumbo Daniel Thomas George Georgiou 《Applied microbiology and biotechnology》1991,36(1):14-20
Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn2+ ions.
Offsprint requests to: G. Georgiou 相似文献
37.
Terry B. White Dianne K. Hammond Hernán Vásquez Henry W. Strobel 《Molecular and cellular biochemistry》1991,102(1):61-69
Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IAl or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P4501Al and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P4501A1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.Some of the data presented here were taken from a thesis submitted by D.K.H. in partial fulfillment of the requirements for the Ph.D. degree in the University of Texas Graduate School of Biomedical Sciences. 相似文献
38.
Cytogenetic Definition and Morphogenetic Analysis of Delta, a Gene Affecting Neurogenesis in Drosophila Melanogaster 总被引:4,自引:3,他引:1
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We have conducted a genetic analysis of a small interval of the third chromosome known to include Delta (Dl), a locus that affects the segregation of the ectoderm into neural and epidermal lineages during embryogenesis and the morphogenesis of some ectodermally derived structures, in Drosophila melanogaster. This analysis has led to the definition of seven independent complementation groups, one of which is Delta, within the interval extending from 91F6-13 to 92A2. Among the extant mutations in these seven loci, only mutations in Dl lead to the so-called neurogenic phenotype: hypertrophy of the nervous system and reduction of the epidermis. Combined cytogenetic and genetic analyses allow us to define absolute proximal (91F5-92A1) and distal (92A2) cytogenetic limits for the Dl locus. We have isolated hypomorphic and amorphic alleles of Dl and find that, for any given allele, there is an inverse correlation between neural hypertrophy and epidermal reduction in embryos and a direct correlation between the severity of embryonic phenotypes in mutant homozygotes and hemizygotes and the imaginal phenotype in heterozygous adults. 相似文献
39.
pH-dependent lysis of liposomes by adenovirus 总被引:16,自引:0,他引:16
Purified adenovirus induced a dose-dependent release of the water-soluble markers calcein and carboxyfluorescein from liposomes. Marker release was strongly dependent on pH, and at temperatures below 5 degrees C, the rate of release showed an optimum at a pH of about 6. This pH dependence parallels disruption of endocytic vesicles by adenovirus and the permeabilization that adenovirus induces on the cell surface. There did not seem to be a striking dependence on the lipid composition of the liposomes. Electron microscopy using a negative stain shows liposomes bound to adenovirus. In some cases, the liposomes were still intact, but many liposomes, which were attached to the vertices of the virus, appeared lysed. These data support the notion that adenovirus, which enters the host cell by receptor-mediated endocytosis, gains access to the cytoplasm by a subsequent pH-dependent disruption of the membrane of the endocytic vesicle. 相似文献
40.
Present models of turbellarian evolution depict the organism with a frontal organ — a complex of glands whose necks emerge at the anterior tip of the body — and therefore imply that this organ is homologous throughout the Turbellaria. However, comparisons of representatives of the Acoela and Macrostomida, two putatively primitive orders of the Turbellaria, show that frontal organs in these two are not similar in ultrastructure or histochemistry. The acoel Convoluta pulchra had a prominent cluster of frontal mucous glands whose necks emerged together in a frontal pore at the exact apical pole of the organism, and an array of smaller glands of at least five other types opened at the anterior end, separately from and ventral to this pore. The frontal organs (Stirndrüsen) of two species of Macrostomum on the other hand, comprised an array of discretely emerging necks of at least two gland types including one with rhabdiform (rhammite) and one with globular mucous secretion granules neither of which emerge at the apical pole. In neither species did the organ appear to be sensory. Our findings indicate a low probability of homology between the frontal glands of the Acoela and Macrostomida. 相似文献