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991.
992.
Communication between the oocyte and its somatic cells has been shown to be important in oocyte development. Here we examined how the oocyte may be involved in bovine cumulus cell expansion. Intact bovine cumulus oocyte complexes (COC) were obtained by puncturing antral follicles. From the intact COC, oocytectomised complexes (OOX) were produced by micro surgical removal of the oocyte. Clumps of cumulus cells (CC) were obtained by micro-dissection. Intact or OOX complexes or CC were matured in the presence of fetal calf serum and hFSH (6 mlU/ml) for 24 hr and the degree of expansion measured. The presence of the oocyte is not essential to allow bovine cumulus expansion to occur as expansion occurred in all groups. Murine OOX complexes from eCG primed 35–40-day-old C57BL6/CBA F1 hybrids (known to require the presence of an oocyte secreted factor for cumulus expansion) were cultured with or without denuded bovine oocytes (1 oocyte/μl). Murine OOX complexes expanded only in the presence of denuded bovine oocytes. Thus some factor produced by bovine oocytes enabled expansion of murine OOX complexes. To determine whether the factor is secreted by bovine oocytes, murine OOX were cultured with or without media conditioned by bovine oocytes (1 oocyte/μl for 4 hr). Significant expansion of murine OOX occurred in media conditioned by bovine oocytes. This shows that the cumulus expansion enabling effect of bovine oocytes is released into the surrounding media. Media conditioned by bovine oocytes and then frozen for up to 1 month showed that the activity by the factor can withstand freezing. © 1995 wiley-Liss, Inc. 相似文献
993.
Roberto E. Izquierdo Kimberly Breese Shalini Jain Daniel Carestio Lawrence Jung James Figge 《In vitro cellular & developmental biology. Animal》1995,31(1):71-76
Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH),
that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered
C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard,
a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture.
A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were
positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression
from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components
of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation
of transferred genes in animals. 相似文献
994.
An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 and Val19 of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe13, Tyr19] -MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmocstrategy and Polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [125I]-[Phe13, Tyr19]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD ) was 1.18 × 10?10 M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as α-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors. 相似文献
995.
Iciar Martinez Bent Dreyer Aasta Agersborg Annick Leroux Gilles Boeuf 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,112(4)
Rearing of 1-year-old Arctic charr (Salvelinus alpinus) at 12°C, as well as the administration of 50 or 75 mgT3/kg feed, accelerated the neonatal to adult fast myosin heavy chain transition, but the effect of temperature was more dramatic than the effect of T3 administration. The endogenous plasma levels of T3 in charrs reared at 12°C were higher than those of analogous groups reared at natural temperature, which in the period under study was between 0.5 and 12°C. As in other species, T3 seemed to play a role in the regulation of the neonatal to adult fast myosin isoform transition by down-regulating the levels of the neonatal and increasing the levels of an adult fast myosin heavy chain. Temperature seemed to accelerate this transition at least, but not only, by inducing an increase in the endogenous levels of T3 in the Arctic charr. 相似文献
996.
Zoospores at various developmental stages in Hydrodictyon reticulatum were isolated from parent cells and cultured in Waris medium. Isolated zoospores grew to mature vegetative cells, and were able to reproduce zoo-spores that formed daughter hexagonal nets. Three types of shape appeared in cells 24 h after isolation: cylindrical, Y-shaped and 4-armed type. Protrusions of Y-shaped or 4-armed cells were formed at an angle of about 120° to the long axis of the cell. When cells were isolated at later stages, more cells became cylindrical in shape and fewer ceils became Y-shaped or 4-armed, Direction of cell growth also seemed to depend largely on the developmental stages of the zoospores. The later the isolated stages were, the more the cells elongated along the long axis of the zoospores. 相似文献
997.
Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy 总被引:8,自引:0,他引:8
The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate
cell type (“gene therapy”), has become a distinct possibility in recent years. In order for gene therapy to be a practical
alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that
a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among
the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes
closest to fulfilling this crucial requirement.
The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a
cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic
adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken
up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken
to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine
during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal
“break out” capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies
of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza
virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape.
Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in
vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive,
easily applicable, widespread technology. 相似文献
998.
Direct shoot formation in spontaneously occurring root pseudonodules of alfalfa (Medicago sativa L.)
P. Sarul M. Vlahova A. Ivanova A. Atanassov 《In vitro cellular & developmental biology. Plant》1995,31(1):21-25
Summary A simple and rapid procedure for direct organogenesis from root nodulelike structures of alfalfa (Medicago sativa L.) line SGg, spontaneously induced on growth regulator-free Gamborg (B5) medium, was developed. Prolific adventitious shoot initiation was obtained using a combination of 1.0 mg/liter TIBA and
0.5 mg/liter 2iP. Transfer of shoots to a medium containing 0.5 mg/liter ABA and reduced concentration of TIBA (0.5 mg/liter)
before rooting markedly stimulated shoot development. Regenerated shoots rooted easily and revealed the early appearance of
nodulelike structures on basal medium (B5) lacking growth regulators. Analysis of endogenous growth regulator levels of SGg roots maintained on growth regulators free
media, showed that spontaneous shoot appearances was correlated with high cytokinin-to-auxin ratios. 相似文献
999.
Abstract. The co-occurrence of Larix olgensis var. changpaiensis, Picea jezoensis and Abies nephrolepis in the coniferous forest of Mount Changbai, northeastern China, is discussed, and the regeneration pattern of these taxa compared on the basis of the analysis of the age structure and the age-height relationship of the three conifers. The presence of tall individuals (ca. 30 m in height) of Larix olgensis var. changpaiensis, which does not show any regeneration, was related to the large eruption of Mount Changbai up to ca. 400 yr ago. Picea jezoensis compensates its small recruitment by a large stem size and long life span together with a continuous height growth. Abies nephrolepis recruits well, but its small stem size and short life span do not result in its dominance in the forest. 相似文献
1000.
E. Fabbri A. Capuzzo A. Gambarotta T.W. Moon 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,112(4):643-651
Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α1-adrenoceptors in EPI action, at least in some fish species. The role of the α1- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist 3H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors (Kd0.36 nM, Bmax 8.61 fmol · mg−1 protein). We provide the first demonstration of α1-adrenoceptors in these same membranes; analysis of binding data with the α1-adrenergic antagonist 3H-prazosin demonstrated a single class of binding sites with a Kdof 15.4 nM and a Bmax of 75.2 fmol · mg−1 protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP3) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP3 (Kd 6.03 nM, Bmax 90.2 fmol · mg−1 protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α1-adrenergic agonists are able to increase intracellular concentrations of IP3 and Ca2+ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α1-binding sites affinity and of α1-adrenoceptor/IP3/Ca2+ transduction systems. 相似文献