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31.
Experimental support for the use of fluid aqueous organic solvent systems and subzero temperatures in mechanistic studies of -galactosidase is presented. The enzyme was stable and retained catalytic activity and structural integrity in 50% aqueous dimethyl sulfoxide and 60% aqueous methanol at 0°C; at lower temperatures higher concentrations of cosolvent may be successfully used. The effects of dimethyl sulfoxide on the catalytic and structural properties of the enzyme were investigated in detail. For the -galactoside-catalyzed h ydrolysis ofo-nitrophenyl--D-galactoside the value ofk
cat decreased in a linear manner with increasing cosolvent concentration, whereasK
m increased exponentially. The decrease ink
cat paralleled the decrease in water concentration, consistent with rate-limiting hydrolysis of a galactosylenzyme intermediate. The increase inK
m is attributed to less favorable partitioning of the substrate to the active site in the cryosolvent compared to aqueous solution. ThepH*-rate profile for this reaction at 0°C in 50% dimethyl sulfoxide was similar to that in aqueous solution, withpK*1=5.8 andpK*2=8.0. Linear Arrhenius plots, with energies of activation of 13.9 and 16.0 kcal mol–1, respectively, were obtained for the -galactosidase-catalyzed hydrolysis ofo-nitrophenyl- andp-nitrophenyl--D-galactosides in 50% dimethyl sulfoxide at temperatures to –57°C. Examination of the intrinsic fluorescence and ultraviolet spectra of the enzyme as a function of increasing cosolvent concentration showed no evidence for structural perturbation up to and including 50% dimethyl sulfoxide at 0°C. We conclude that these cryosolvent systems are suitable for mechanistic investigations of -galactosidase, in particular for trapping intermediates at subzero temperatures. 相似文献
32.
R N Bergman 《Federation proceedings》1977,36(2):265-270
The rates of storage and release of carbohydrate by the liver are determined by the plasma concentrations of several blood-borne signals; most important are the concentrations of glucose, and of the hormones insulin and glucagon. To understand the complex control relationships of these three signals as they affect the liver, their individual dynamic influences have been determined experimentally, and the findings have been integrated by means of a computer simulation of the pathways of hepatic glycogen metabolism. The simulation studies have led to specific hypotheses about the biochemical effects of glucose and insulin on the liver. The simulation studies have also led to the conclusion that glucose exerts a rapid moment-to-moment influence on the rate of uptake of glucose by the liver. Insulin, however, by exerting a slower influence on the sensitivity of the liver to glucose, is very effective in "optimizing" the amount of glycogen which the liver stores during food intake. Thus, integrated experimental and simulation studies can lead to a view of a physiological regulating system which does not emerge from either approach used alone.- 相似文献
33.
Ronald Levy Yehudit Bergman Olivera Finn 《In vitro cellular & developmental biology. Plant》1981,17(12):1051-1057
Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined
by monoclonal antibodies: Leu-1 and HLA-DR.
Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington,
D.C., June 7–11, 1981.
This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302.
This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech
Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England
Nuclear Corporation, and Ortho Pharmaceutical Corporation. 相似文献
34.
A "random-hit" matrix model is proposed to account for the dynamic and steady state relationship between occupation of bovine renal medullary membrane receptors by [Lys8]vasopressin (LVP) and neurohypophyseal hormones (NHH) and the associated activation of membrane-bound adenylate cyclase. The model was developed by systematic introduction of specific rules concerning receptor coupling into a general structural model which consists of two square matrices of identical size, one composed of homogeneous R ("receptor") units, the second of homogeneous C ("cyclase") units. R units are either occupied (RO) or unoccupied (RU); C units are either active (CA) or inactive (CI). Hormone molecules are envisioned to "collide" with R units randomly; collision with RU leads to "binding", and occupation is maintained for a characteristic mean occupancy time, TO. In this structure, each R unit has an "interaction field" which consists of the "twin" unit in the "C" matrix, and the 4 nearest neighbor C units surrounding the twin. Occupation of an R unit leads to activation of all CI units in the interaction field of that R; CA units in the interaction field are refractory. Thus binding at a given R may "recruit" a variable number of inactive neighboring C units (5, 4, 3, 2, 1, or 0). The model requires that there be individual coupling delays between the moment of binding at a given R and subsequent activation of CI units (mean coupling delay (Td) approximately 10% To). Activation of C units persists as long as the "parent" R is occupied and is maintained for an additional short time interval (Tp) after RO reverts to RU, corresponding to hormone dissociation from receptor. The model accounts for the following previously demonstrated relations between LVP occupation of receptors and adenylate cyclase activation in bovine renal medullary membranes: 1) the shape of the nonlinear steady state relation between normalized (percentage maximal) receptor occupation (O) and cyclase activation (A), uniformly observed in different membrane preparations: 2) variable hormone concentration-dependent trajectories of approach to the final steady state A:O value (A:Oss) which may be either monophasic or biphasic; 3) the loss of intrinsic adenylate cyclase activity observed in bovine membranes for a series of NHH analogs with progressively diminishing affinity for receptors. The model represents an explicit theory of coupling where a successive series of temporal events are quantitatively related to each other and privide major constraints to any interpretation of the molecular organization of receptors and adenylate cyclase units in membranes. The model excludes a number of mechanistic proposals and suggests a new hypothesis for membrane coupling with features which may be generally applicable to other hormone-sensitive adenylate cyclase systems. 相似文献
35.
A quantitative fluorometric assay for detection and characterization of Fc receptors 总被引:1,自引:0,他引:1
A B Schreiber J Hoebeke Y Bergman J Haimovich A D Strosberg 《Journal of immunology (Baltimore, Md. : 1950)》1978,121(1):19-23
A new quantitative fluorometric binding assay that uses fluoresceinated aggregated IgG is proposed for the study of Fc receptors. The method was compared with a radiolabeling binding assay on three well characterized murine cell lines (38C-13, EL4, and BW). The apparent association constant of the binding and the amount of aggregated IgG bound per cell at saturation were calculated. The fluorometric assay enables the detection of 5 X 10(-10) M bound aggregated IgG. Inhibition studies with monomeric IgG, reduced and alkylated aggregated IgG, and aggregated F(ab')2 fragments of IgG confirmed the specificity of the assay. Staphylococcal protein A inhibited the binding of the aggregated IgG to Fc receptors. 相似文献
36.
Twelve of 21 human, hemolytic, fecal isolates ofEscherichia coli produced type 1 hemolysin (HLY1), an extracellular, heat-labile molecule (alpha-hemolysin). Although no common plasmid species was apparent, 11 of 12 HLY1 strains possessed a plasmid60 megadaltons (Mdal); 5 of 9 strains with other hemolysins possessed a plasmid of comparable molecular mass (Fisher's exact probability=0.0805). One derivative of an HLY1+strain, which contained a 125 Mdal plasmid, no longer expressed HLY1 and contained a single 102 Mdal plasmid. The presence of large plasmids of varying size and an apparent deletion mutation in HLY1 strains suggest that HLY1 determinants are located on a small, unstable genetic element. In an initial survey of 224 human fecal isolates ofE. coli, the predominant hemolytic serotype was 06:H-, and conversely most (85%) 06:H-isolates were HLY1+. Serotype appears to play an important role in HLY1 expression. 相似文献
37.
Rune Bergman Lennart Larsson Göran Odham Gunilla Westerdahl 《Journal of microbiological methods》1983,1(1):19-22
Gas chromatography/mass spectrometry adapted for selected ion monitoring was used to detect C32 mycocerosic acid in short-term incubated cultures of procineand canine strains of mycobacteria. The method can be employed for rapid differentiation of Mycobacterium tuberculosis from M. avium-intracellulare. 相似文献
38.
Cation specificity and modes of the Na+:CO3(2-):HCO3- cotransporter in renal basolateral membrane vesicles 总被引:1,自引:0,他引:1
M Soleimani G A Lesoine J A Bergman P S Aronson 《The Journal of biological chemistry》1991,266(14):8706-8710
The cation specificity and possible exchange modes of the Na+:CO3(2-):HCO3- cotransporter were evaluated by use of basolateral membrane vesicles isolated from rabbit renal cortex. External Li+ inhibited HCO3- gradient-stimulated 22Na uptake, indicating that Li+ interacts with the Na+:CO3(2-):HCO3- cotransporter. No interaction with K+, choline, Rb+, Cs+, or NH4+ could be similarly detected. Imposing an outward Li+ gradient caused quenching of acridine orange fluorescence in the presence but not in the absence of HCO3-, suggesting that Li+:base cotransport takes place via the Na+:CO3(2-):HCO3- cotransporter. Imposing an outward gradient of unlabeled Na+ stimulated the initial rate of 22Na uptake and induced its transient uphill accumulation, indicating Na(+)-Na+ exchange. Na(+)-Na+ exchange was observed in the presence but not in the absence of HCO3- and was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), suggesting that it occurs via the Na+:CO3(2-):HCO3- cotransporter. Similarly, an outward Li+ gradient stimulated uphill 22Na accumulation, indicating Na(+)-Li+ exchange. Na(+)-Li+ exchange was observed in the presence but not in the absence of HCO3-, and was inhibited by DIDS, suggesting that it also occurs via the Na+:CO3(2-):HCO3- cotransporter. Both Na(+)-Na+ and Li(+)-Na+ exchange modes were sensitive to inhibition by harmaline but not by amiloride. We conclude that Li+ is an alternative substrate for the renal Na+:CO3(2-):HCO3- cotransporter. Transport modes of the system include cation:base cotransport and HCO3-dependent cation-cation exchange. 相似文献
39.
Analysis of nascent heavy chains isolated from MPC11 (gamma 2b heavy chains) and MOPC 21 (gamma 1 heavy chains) mouse myeloma cells demonstrates an accumulation of nascent heavy chains which are slightly smaller in mass (approximately 35,000 daltons) than nascent heavy chains which have just been glycosylated (approximately 38,000 daltons). The accumulation of 35,000-dalton nascent heavy chain appears to be a consequence of the glycosylation process since tunicamycin, an inhibitor of glycosylation, abolishes the apparent translational block manifested by the accumulation of 35,000-dalton nascent chains. Tunicamycin also causes a 15 to 25% increase n the relative rate of synthesis of heavy chain compared to the corresponding rate of synthesis of the nonglycosylated light chain synthesized by the same cell. These results suggest that the translation block, caused by the glycosylation process, of heavy chain synthesis contributes to the imbalance of heavy chain and light chain biosynthesis observed in malignant and normal lymphoid cells. 相似文献
40.