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71.
Lipid vesicles (liposomes) containing pH-sensitive fluorophores were used as probes for the study of liposome entry and intracellular fate. Pyranine [8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS)] was entrapped in the liposome aqueous core during preparation to provide a means of detecting internalization into living cells. HPTS is highly water soluble and shows a strong pH-dependent shift in its fluorescence excitation spectrum. Fluorescence emission (FEM) is slightly pH dependent with excitation (lambda EX) at 350-415 nm but highly pH dependent with lambda EX at 450 nm. Liposomes bearing a net negative charge bound rapidly to CV-1 cells and underwent endocytosis. One hour after liposome addition, high FEM with lambda EX at 413 nm and low FEM with lambda EX at 450 nm suggest that most cell-associated liposomes had been internalized and resided at a mean pH of approximately 6.6. Collapse of cellular H+ gradients with NH4Cl or monensin treatment rapidly and reversibly increased FEM with lambda EX at 450 nm. Direct examination by fluorescence microscopy corroborates the fluorometric data on internalization; over time, FEM remained high with lambda EX at 350-405 nm but decreased with lambda EX at 450-490 nm, showing that all lipid vesicles were internalized within 40 min at 37 degrees C. Acidification of intracellular liposomes increased over 3 h, reaching a minimum value of approximately pH 5.5. HPTS persisted within acidic cellular vesicles for 2-3 days, and cytoplasmic dye was observed infrequently, suggesting that liposome fusion with cellular membranes seldom occurs. Material delivered to the endocytic pathway via lipid vesicles labeled an assortment of intracellular organelles of varying motility and morphology, including dynamic tubular structures whose lumen is acidic. 相似文献
72.
Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay 总被引:8,自引:0,他引:8
The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra- and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of cell-associated dye was determined by normalizing peak fluorescence intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with a t1/2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated liposomes with antibody protected liposomes from the initial release. The leakage of liposomal contents was monitored by co-encapsulating HPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold).(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
73.
74.
Summary The effects of various agents on active sodium transport were studied in the toad bladder in terms of the equivalent circuit comprising an active conductanceK
a, an electromotive forceE
Na, and a parallel passive conductanceK
p. For agents which affectK
a, but notE
Na orK
p, the inverse slope of the plot of total conductance against short-circuit currentI
0 evaluatesE
Na, and the intercept representsK
p. Studies employing 5×10–7
m amiloride to depressK
a indicate a changingE
Na, invalidating the use of the slope technique with this agent. An alternative suitable technique employs 10–5
m amiloride, which reducesI
0 reversibly to near zero without effect onK
p. Despite curvilinearity of the -I0 plot under these conditions,K
p may therefore be estimated fairly precisely from the residual conductance. It then becomes possible to follow the dynamic behavior ofK
a andE
Na (in the absence of 10–5
m amiloride) by frequent measurements of andI
0, utilizing the relationshipsK
a=K-K
p, andK
Na=I
O/(K-K
p). 2-deoxy-d-glucose (7.5×10–3
m) depressedK
a without affectingE
Na. Amiloride (5×10–7
m) depressedK
a and enhancedE
Na. Vasopressin (100 mU/ml) enhancedK
a markedly and depressedE
Na slightly. Ouabain (10–4
m) depressed bothK
a andE
Na. All of the above effects were noted promptly;K
p was unaffected. The electromotive force of Na transportE
Na appears not to be a pure energetic parameter, but to reflect kinetic factors as well, in accordance with thermodynamic considerations. 相似文献
75.
J P Leroux J C Marchand R Hong Tuan Ha P Cartier 《European journal of biochemistry》1975,58(2):367-373
Viable human polymorphonuclear leukocytes isolated from peripheral blood were incubated for 1 h at 37 degrees C with variable concentrations of insulin in a saline medium buffered at pH 7.4. The hormone increased glucose consumption by about 40% without influencing the permeability of the membranes to glucose, whose uptake followed a passive diffusion process. The measurement of intermediates localized activation of glycolysis by insulin, down to 0.36 nM, at the phosphofructokinase step. However, the spectrophotometric measurement showed no activation of phosphofructokinase after preincubation with insulin of either intact granulocytes or crude or ultracentrifuged homogenates. The level of cyclic AMP, which is known to activate phosphofructokinase, was not modified by insulin; cyclic GMP did not activate the enzyme in the granulocyte extracts: neither of the two nucleotides can therefore be considered as a direct messenger of the action of insulin on phosphofructokinase. An important fraction of the extra glucose consumed under the influence of insulin was recovered as neither glycogen nor lactate, nor was it oxidized in the Krebs cycle. It might be assumed to have been converted into glycerolipids. However, insulin produced no detectable accumulation of triglycerides and activated neither the pentose phosphate pathway nor oxidative decarboxylation of pyruvate. The fate of the extra glucose consumed under the influence of insulin therefore remains questionable. 相似文献
76.
A permanent human lymphoblast culture was synchronized with repetitive thymidine blocks, and the changes in the levels of activity of four X-chromosome-linked enzymes were followed during the cell cycle. The four enzymes studied were phosphoglycerate kinase (PGK), α-galactosidase (α-Gal), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and glucose-6-phosphate dehydrogenase (G6PD). The levels of PGK and α-Gal activities increased simultaneously in G1 and in S, while HGPRT and G6PD increased close together in middle and late S. Therefore, different control mechanisms may be involved in the increases of the activities of these two sets of enzymes. 相似文献
77.
Changes in active transport, intracellular adenosine 5'-triphosphate levels, macromolecular syntheses, and glycolysis in an energy-uncoupled mutant of Escherichia coli. 总被引:3,自引:0,他引:3 下载免费PDF全文
The temperature-sensitive Escherichia coli mutant ecfts metC (Lieberman and Hong, 1974), previously shown to be defective in the coupling of metabolic energy to active transport, is also altered in a wide variety of cellular activities at the nonpermissive temperature. These alterations include a lowering of intracellular adenosine 5'-triphosphate levels, an alteration of glucose metabolism such that large quantities of pyruvate and dihydroxyacetone phosphate are excreted into the medium, excretion of accumulated potassium ions, and a cessation of deoxyribonucleic acid, ribonucleic acid, and phospholipid synthesis. Since these effects closely mimic the action of colicins E1 and K on E. coli cells, the possibility that the ecf gene product is the primary biochemical target for these colicins is discussed. 相似文献
78.
Malocclusions of the premolar and molar teeth of inbred Strain 2/N and Strain 13/N and outbred Dunkin-Harley guinea pigs were examined. A higher incidence of malocclusion observed in the inbred strains suggested a genetic basis for the disorder. 相似文献
79.
Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (75NIT77 and 90NDT92) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes. 相似文献
80.
西藏东南部色季拉山主要类型森林叶片和枯落物养分含量特征 总被引:2,自引:0,他引:2
为阐明不同生长年限森林叶片和不同分解程度枯落物养分含量特征,为植物-土壤养分循环研究提供科学依据。以藏东南色季拉山几种典型森林植被(雪山杜鹃(Rhododendron aganniphum)、海拔4000 m和3900 m区域急尖长苞冷杉(Abies georgei var. smithii)、川滇高山栎(Quercus aquifolioides))为研究对象,分析了1年生和2年生植物叶片及不同分解程度枯落物有机碳(OC)、全氮(TN)、全磷(TP)和全钾(TK)含量。结果表明:色季拉山森林叶片和枯落物OC含量表现为2年生叶片1年生叶片未分解枯落物(ND)半分解枯落物(SD)完全分解枯落物(CD),即老叶片以C积累为主,而枯落物OC含量随分解程度的增加而下降,叶片OC平均含量(68.5%)显著高于中国平均水平(45.5%);叶片N、P、K含量表现为1年生2年生,即新叶以N、P、K等营养物质的吸收积累为主。枯落物TN含量低于中国森林的平均水平(12.03 g/kg),而TP含量显著高于中国森林平均水平(0.74 g/kg),枯落物TN和TP以SD最高,即分解初期表现为净固定,而后期则呈净释放,TK含量随分解程度的增加而增加,表现为K的净固定;叶片C∶N,C∶P和C∶K表现为2年生1年生,枯落物C∶N,C∶P和C∶K随着分解程度的增加而显著降低;叶片N∶P处于较低水平(6.08),显著低于全球平均水平(16.0),表现出明显的N限制营养型;研究结果为科学阐明藏东南森林生态系统植被-土壤养分循环研究提供了数据支撑。 相似文献