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301.
302.
Freshly dissociated myocytes from nonpregnant, pregnant, and postpartum rat uteri have been studied with the tight-seal patch-clamp method. The inward current contains both INa and ICa that are vastly different from those in tissue-cultured material. INa is abolished by Na+-free medium and by 1 μM tetrodotoxin. It first appears at ∼−40 mV, reaches maximum at 0 mV, and reverses at 84 mV. It activates with a voltage-dependent τ of 0.2 ms at 20 mV, and inactivates as a single exponential with a τ of 0.4 ms. Na+ conductance is half activated at −21.5 mV, and half inactivated at −59 mV. INa reactivates with a τ of 20 ms. ICa is abolished by Ca2+-free medium, Co2+ (5 mM), or nisoldipine (2 μM), and enhanced in 30 mM Ca2+, Ba2+, or BAY-K 8644. It first appears at ∼−30 mV and reaches maximum at +10 mV. It activates with a voltage-dependent τ of 1.5 ms at 20 mV, and inactivates in two exponential phases, with τ''s of 33 and 133 ms. Ca2+ conductance is half activated at −7.4 mV, and half inactivated at −34 mV. ICa reactivates with τ''s of 27 and 374 ms. INa and ICa are seen in myocytes from nonpregnant estrus uteri and throughout pregnancy, exhibiting complex changes. The ratio of densities of peak INa/ICa changes from 0.5 in the nonpregnant state to 1.6 at term. The enhanced role of INa, with faster kinetics, allows more frequent repetitive spike discharges to facilitate simultaneous excitation of the parturient uterus. In postpartum, both currents decrease markedly, with INa vanishing from most myocytes. Estrogen-enhanced genomic influences may account for the emergence of INa, and increased densities of INa and ICa as pregnancy progresses. Other influences may regulate varied channel expression at different stages of pregnancy. 相似文献
303.
p72 is one of the major allergens of American cockroach. The gene of p72 was cloned into pg2bVH, replacing the VH region, to yield pg2b-p72 which was then transfected into hybridoma cell Sp2/0-Ag14. Western blot analysis of the culture supernatant from Sp2/0-Ag14(p72) detected a diffused protein band (ca. 72 kDa) which appeared to contain heterogeneous glycoforms. 相似文献
304.
A comparative study of the effects of abscisic acid and methyl jasmonate on seedling growth of rice 总被引:4,自引:0,他引:4
The effects of abscisic acid (ABA) and methyl jasmonate (MJ) on growth of rice seedlings were compared. The lowest tested concentration of ABA and MJ that inhibited seedling growth was found to be 4.5 and 0.9 µM, respectively. Growth inhibition by ABA is reversible, whereas that by MJ is irreversible. GA3 was found to be more effective in reversing inhibition of shoot growth by ABA than by MJ. KCl partially relieved MJ-inhibited, but not ABA-inhibited, growth of rice seedlings. The beneficial effect of K+ on growth of rice seedlings in MJ medium could not be replaced by Li+, Na+ or Cs+. MJ treatment caused a marked release of K+ into the medium. In order to understand whether cell wall-bound peroxidase activity was inversely related to rice seedling growth, effects of ABA and MJ on cell wall-bound peroxidase activity were also examined. Results indicated that both ABA and MJ increased cell wall-bound peroxidase activity in roots and shoots of rice seedlings. Although MJ (4.5 µM) was less effective in inhibiting root growth than ABA (9 µM), MJ was found to increase more cell wall-bound peroxidase activity in roots than ABA. 相似文献
305.
The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity. 总被引:22,自引:4,他引:18
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The biochemical activities that underlie the genetically defined activator and repressor functions of the VIVIPAROUS1 (VP1) protein have resisted in vitro analysis. Here, we show that a glutathione S-transferase (GST) fusion protein, including only the highly conserved B3 domain of VP1, has a highly cooperative, sequence-specific DNA binding activity. GST fusion proteins that include larger regions of the VP1 protein have very low activity, indicating that removal of the flanking protein sequences is necessary to elicit DNA binding in vitro. DNA competition and DNase I footprinting analyses show that B3 binds specifically to the Sph element involved in VP1 activation of the C1 gene, whereas binding to the G-box-type VP1-responsive element is of low affinity and is nonspecific. Footprint analysis of the C1 promoter revealed that sequences flanking the core TCCATGCAT motif of Sph also contribute to the recognition of the Sph element in its native context. The salient features of the in vitro GST-B3 DNA interaction are in good agreement with the protein and DNA sequence requirements defined by the functional analyses of VP1 and VP1-responsive elements in maize cells. 相似文献
306.
Chien-Yuan Kao Cory S. Oakley Clifford W. Welsch Chia-Cheng Chang 《In vitro cellular & developmental biology. Animal》1997,33(4):282-288
Summary A chemically defined culture medium was developed to support the growth of two distinctly different types of normal human
breast epithelial cells (HBEC) derived from reduction mammoplasty. Type I cells expressed luminal epithelial cell markers
and were deficient in gap junctional intercellular communication (GJIC), whereas Type II cells expressed basal epithelial
cell markers and were efficient in GJIC. In this study, we examined and compared the growth factor and hormone requirements
of these two types of cells and a series of cell lines that were obtained by sequential transfection with SV40 DNA (extended
lifespan, nontumorigenic), treatment with 5-bromodeoxyuridine (BrdU)/black light (immortal and weakly tumorigenic), and infection
of a virus carrying the neu oncogene (highly tumorigenic). Growth of Type I cells was inhibited by withdrawing epidermal growth
factor (EGF), hydrocortisone (HC), or insulin (INS) from the culture media, but was enhanced by fetal bovine serum (FBS) supplementation.
Growth of Type II cells was inhibited by withdrawal of EGF, HC, or INS from the media, and was inhibited by FBS supplementation.
Withdrawal of human transferrin (HT) or 17β-estradiol (E2) from the media did not alter the growth of Type I or Type II cells. SV40 transfected Type I cell lines still required EGF,
HC, or INS for optimal growth. However, the highly tumorigenic cell line did not show a growth dependence on EGF, HC, or INS
but did appear to require HT and 3,3′,5-triiodo-D.L. thyronine (T3) for optimal growth. In addition, FBS stimulated the growth of these cell lines. Thus, this study shows that Type I HBEC
are distinctly different from Type II HBEC in growth response to FBS and that neoplastically transformed Type I cells could
become growth factor and hormone independent. 相似文献
307.
A Pruss B Baumann M Seibold M Kao K Tintelnot R von Versen H Radtke T D?rner G Pauli U B G?bel 《Biologicals》2001,29(2):59-66
Different procedures are available to inactivate bacteria and fungi, including their spores, as well as viruses in human bone transplants. The most efficient methods are considered to be gamma irradiation and thermal inactivation as well as chemical sterilization methods like the peracetic acid-ethanol treatment (PES). Following national and international standards or draft standards, the antimicrobial effectiveness of this procedure was evaluated. Due to the standardizable size as well as the clinical relevance, defatted human spongiosa cuboids (15x15x15 mm) served as model system. After treatment with PES for 2 and 4 hours, respectively, the titre of living micro-organisms was determined in the supernatant and the cuboid. A reduction in the titre of viable micro-organisms below the detection level (reduction factor >5 log10) was already achieved after an incubation time of 2 hours (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Bacillus subtilis, Clostridium sporogenes, Mycobacterium terrae, Candida albicans as well as spores of Bacillus subtilis). No viable micro-organisms could be detected in any of the PES-treated test cuboids. Spores of Aspergillus niger were also completely inactivated. The PES procedure proved to be a reliable method for the sterilization of human bone transplants derived from spongiosa. 相似文献
308.
T. Y. Lin Y. Y. Kao S. Lin R. F. Lin C. M. Chen C. H. Huang C. K. Wang Y. Z. Lin C. C. Chen 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,103(6-7):905-911
We have constructed a genetic linkage map for Nicotiana plumbaginifolia/Nicotiana longiflora (2n= 2x=20), based on the segregation of 69 RFLP and 102 RAPD loci in 99 F2 plants from the cross N. plumbaginifolia×N. longiflora. The map consists of nine major linkage groups, each containing more than nine marker loci, and spans 1062 cM. Twenty of the RFLP markers were mapped previously to Nicotiana sylvestris (2n=2x=24) chromosomes using monosomic alien addition lines. Taxonomically, N. plumbaginifolia and N. sylvestris belong to the same section, namely the Alatae; however, cytogenetic evidence indicates that they are not closely related. Comparison of the distribution of markers common to both maps suggests that genome reorganization has occurred during the evolution of these two species. Evidence is also presented that genome reorganization may be accompanied by gain and loss of specific classes of DNA sequences in their genomes. 相似文献
309.
S Saika S Saika C Y Liu M Azhar L P Sanford T Doetschman R L Gendron C W Kao W W Kao 《Developmental biology》2001,240(2):419-432
To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice. 相似文献
310.
Y C Kao T Higashiyama X Sun T Okubo C Yarborough I Choi Y Osawa F A Simmen S Chen 《European journal of biochemistry》2000,267(20):6134-6139
Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an 'in-cell' assay (a 3H-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one-thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17beta-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19-desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1, 4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2, 19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino-acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors. 相似文献