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101.
Mark Akeson Joshua Scharff Celia M. Sharp David M. Neville Jr. 《The Journal of membrane biology》1992,125(1):81-91
Summary We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (pm) and infectivity of vesicular stomatitis virus (VSV). Complete substitution of K+ for extracellular Na+blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells. When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K+ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K+ applied apically had no effect on VSV infection. This morphological specificity correlates with a large decrease in pm of MDCK cells when high K+ buffer is perfused across the basal-lateral surface. Depolarization of the plasma membrane by 130 mm basal K+ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF. Depolarization also causes a transient increase of cytosol Ca2+ from 70 to 300 nm as reported by the dye Fura-2. Neither increase could explain the block of VSV infectivity by plasma membrane depolarization. One alternative hypothesis is that pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli.We thank Kenneth Spring for many helpful discussions concerning fluorescence digitized imaging systems, James Russell for his collaboration in the design of our imaging system, Herbert Chase for suggestions on dye loading into MDCK cells, and Manfred Schubert and George Harmison for providing expertise on VSV. 相似文献
102.
Complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation. 总被引:12,自引:7,他引:5 下载免费PDF全文
Y Li H Hui C J Burgess R W Price P M Sharp B H Hahn G M Shaw 《Journal of virology》1992,66(11):6587-6600
Previous studies of the genetic and biologic characteristics of human immunodeficiency virus type 1 (HIV-1) have by necessity used tissue culture-derived virus. We recently reported the molecular cloning of four full-length HIV-1 genomes directly from uncultured human brain tissue (Y. Li, J. C. Kappes, J. A. Conway, R. W. Price, G. M. Shaw, and B. H. Hahn, J. Virol. 65:3973-3985, 1991). In this report, we describe the biologic properties of these four clones and the complete nucleotide sequences and genome organization of two of them. Clones HIV-1YU-2 and HIV-1YU-10 were 9,174 and 9,176 nucleotides in length, differed by 0.26% in nucleotide sequence, and except for a frameshift mutation in the pol gene in HIV-1YU-10, contained open reading frames corresponding to 5'-gag-pol-vif-vpr-tat-rev-vpu-env-nef-3' flanked by long terminal repeats. HIV-1YU-2 was fully replication competent, while HIV-1YU-10 and two other clones, HIV-1YU-21 and HIV-1YU-32, were defective. All three defective clones, however, when transfected into Cos-1 cells in any pairwise combination, yielded virions that were replication competent and transmissible by cell-free passage. The cellular host range of HIV-1YU-2 was strictly limited to primary T lymphocytes and monocyte-macrophages, a property conferred by its external envelope glycoprotein. Phylogenetic analyses of HIV-1YU-2 gene sequences revealed this virus to be a member of the North American/European HIV-1 subgroup, with specific similarity to other monocyte-tropic viruses in its V3 envelope amino acid sequence. These results indicate that HIV-1 infection of brain is characterized by the persistence of mixtures of fully competent, minimally defective, and more substantially altered viral forms and that complementation among them is readily attainable. In addition, the limited degree of genotypic heterogeneity observed among HIV-1YU and other brain-derived viruses and their preferential tropism for monocyte-macrophages suggest that viral replication within the central nervous system may differ from that within the peripheral lymphoid compartment in significant and clinically important ways. The availability of genetically and biologically well characterized HIV-1 clones from uncultured human tissue should facilitate future studies of virus-cell interactions relevant to viral pathogenesis and drug and vaccine development. 相似文献
103.
Peroxidase activity in the leaf elongation zone of tall fescue : I. Spatial distribution of ionically bound peroxidase activity in genotypes differing in length of the elongation zone 总被引:8,自引:5,他引:3 下载免费PDF全文
Cessation of cell expansion has been associated with cell wall cross-linking reactions catalyzed by peroxidase. This study utilized two genotypes of tall fescue (Festuca arundinacea Schreb.) that differ in length of the leaf elongation zone to investigate the relationship between ionically bound peroxidase activity and the spatial distribution of leaf elongation. Peroxidase activity was also localized histochemically in transverse sections of the leaf blade using 3,3′ -diaminobenzidine. Soluble or soluble plus ionically bound peroxidase activities were extracted from homogenized segments of the elongating leaf blade and assayed spectrophotometrically. Activity of the ionically bound fraction, expressed per milligram fresh weight or per microgram protein, increased as cells were displaced through the distal half of the elongation zone, corresponding to the region in which the elongation rate declined. In both genotypes, the initial increase in activity preceded the onset of growth deceleration by about 10 hours. In the basal region where elongation began, histochemical localization showed that peroxidase activity was found only in vascular tissues. As cells were displaced farther through the elongation zone, peroxidase activity appeared in walls of other longitudinally continuous tissues such as the epidermis and bundle sheaths. Increase in ionically bound peroxidase activity and changes in localization of peroxidase activity occurred at comparable developmental stages in the two genotypes. The results indicate that cessation of elongation followed an increase in cell wall peroxidase activity. 相似文献
104.
105.
106.
D Hillaire J C Chomel F Lesure M Renouil C Musenger F Pierson M Berthelon G Lenoir G Gérard E Bois 《Annales de génétique》1991,34(1):5-7
The frequencies of the delta F 508 mutation and haplotypes linked to the cystic fibrosis (CF) gene and detected with DNA probes XV-2C and KM-19 have been studied in the population of Reunion Island, a French province located in the Indian Ocean. The deletion was present in 41.3% of CF chromosomes, whereas this proportion is about 70% in the French population. The delta F 508 mutation was associated with the haplotype B defined by the DNA markers XV-2C (allele 1) and KM-19 (allele 2) in 76.4% of CF chromosomes, while this proportion is over 90% in the French population. Founder effect, genetic drift and admixture can explain these differences. 相似文献
107.
Linkage of thioredoxin stability to titration of ionizable groups with perturbed pKa 总被引:2,自引:0,他引:2
The highly conserved, buried, Asp 26 in Escherichia coli thioredoxin has a pKa = 7.5, and its titration is associated with a sizable destabilization of the protein [Langsetmo, K., Fuchs, J., & Woodward, C. (1991) Biochemistry (preceding paper in this issue)]. A fit of the experimental pH dependence of thioredoxin stability to a theoretical expression for the pH/stability relation in proteins agrees closely with a pKa value of 7.5 for Asp 26. The agreement between the experimental and theoretical changes in protein stability due to substitution of Asp 26 by alanine is also good. The local structure in the vicinity of Asp 26 in the low-pH crystal structure (with uncharged Asp 26) is hydrophobic, indicating that the aspartate would be highly destabilized. In theoretical calculations, the desolvation penalty for deprotonating Asp 26 in this environment is similar to the total protein folding energy. As a consequence, the Asp 26 pKa would be much greater than 7.5, and/or the protein might not fold. This suggests that a compensating process partially stabilizes the Asp 26 carboxyl group when it is charged. A simple model for this proposed, whereby the Lys 57 side chain rotates to form a salt bridge with Asp 26 when it is deprotonated. 相似文献
108.
A cDNA encoding the nucleic acid-binding domain of the hnRNP C-type protein has been cloned by DNA-affinity screening of pituitary-derived expression libraries. An analysis revealed sequence identity with the human C-type cDNA and demonstrated the presence of a peptide sequence contained within the single-stranded DNA-binding protein, UP2, which was absent from the human cDNA. Structural analysis of the protein encoded by the rat cDNA demonstrated a net charge of +15 with 14.56% and 6.33% lysines and arginines, respectively, and an amino acid sequence that is consistent with an extensive helix-loop-helix-turn-helix structure. 相似文献
109.
Expression and secretion in Aspergillus nidulans and Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal amdS promoter system. 下载免费PDF全文
A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels. 相似文献
110.
John W. Swann C. Nelson Sinback Martha G. Pierson David O. Carpenter 《Cellular and molecular neurobiology》1982,2(4):291-308
1. Dopamine has been reported to exist in unusually large quantities in Aplysia gill. The physiological role of this neurotransmitter in this organ was examined. 2. The addition of dopamine to a gill perfusate results in the contractions of the lateral and medial external pinnule muscles, the circular and longitudinal muscles of the afferent vessel, and the circular muscles of the efferent vessel. 3. Dopamine-induced contractions persist after chemical synaptic transmission is eliminated in the gill. This suggests that excitatory dopamine receptors are present on gill smooth muscle fibers themselves. 4. Dopamine also potentiates the gill response to action potentials in single identified gill motoneurons. Evidence presented suggests that muscle contractions and modulation of motoneuron contractions are independent phenomena. 5. While modulation may in part be mediated by increases in excitatory junction potential (EJP) amplitude, in many cases large increases in muscle contractions occur while the enhancement of EJPs is disproportionately small. 6. Dopamine's ability to produce muscle contractions suggests that there may be dopaminergic motoneuron innervation of the gill. We suggest that dopamine's modulatory actions may be mediated via modification of excitation-contraction coupling in smooth muscle fibers. 相似文献