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101.
K Venkatesh Y A Abdel-Aal F B Armstrong R M Roe 《The Journal of biological chemistry》1990,265(35):21727-21732
Juvenile hormone (JH) esterase found primarily in the hemolymph and tissues of insects is a low abundance protein involved in the ester hydrolysis of insect juvenile hormones, JHs. The enzyme was purified from the larval plasma of wild-type Manduca sexta using an affinity column prepared by binding the ligand, 3-[(4'-mercapto)butylthio]-1,1,1-trifluoropropan-2-one (MBTFP), to epoxy-activated Sepharose. The purification was greater than 700-fold with a 72% recovery, and the purified enzyme appeared as a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoelectrophoresis, reverse phase high performance liquid chromatography, and amino acid sequence analysis. The molecular weight was 66,000. The plasma JH esterase in wild-type, black, and white strains of M. sexta was similar when analyzed by immunotitration, wide range (pH 3.5-9.0) isoelectric focusing, and inhibition with MBTFP and 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP). Inhibition studies revealed a sensitive and insensitive form (I50 = 10(-9) and 10(-6) M, respectively) in these three biotypes. Narrow range isoelectric focusing (pH 4.0-7.0) indicated the presence of two major isoelectric forms with pI values of 6.0 and 5.5, but their inhibition kinetics with OTFP and O,O-diisopropyl phosphorofluoridate were identical. 相似文献
102.
Increased sensitivity of the rapid hydrophobic grid membrane filter enzyme-labeled antibody procedure for Escherichia coli O157 detection in foods and bovine feces. 总被引:3,自引:3,他引:0
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Several strains of Escherichia coli O157:H7 artificially inoculated into vegetables and dairy products were recovered on hydrophobic grid membrane filters and enumerated by an enzyme-labeled antibody assay. The mean of the recoveries from 12 fresh vegetables was 108.8%, whereas that from 10 dairy products was 93.2%. Modified tryptic soy broth at 43 degrees C with shaking at 100 rpm provided optimum recovery of the organism from meat, with a sensitivity of less than or equal to 1 CFU/g, which is 10 times more sensitive than direct plating. The method performed equally well with vegetable and dairy products. Tryptic soy broth, however, under the same conditions gave the best results for fecal samples. Of 22 asymptomatic dairy cattle, reported as having positive Brucella titers when assayed with polyclonal antibodies, eight were found to contain E. coli O157 in their feces as demonstrated by the enzyme-labeled antibody assay by using monoclonal antibodies. This finding may explain some of the false-positive Brucella tests. 相似文献
103.
Rapid oxidation processes relevant to the degradation of [4Fe4S] clusters in Clostridium pasteurianum ferredoxin were studied via direct (unmediated) heterogeneous electron transfer at a pyrolytic graphite electrode. Differential-pulse voltammograms of native [4Fe4S] ferredoxin showed two well-defined oxidation peaks corresponding to apparent E-values of +793 and +1120 mV at 5°C. Direct involvement of the cluster was established through parallel experiments with the 2[4Fe4Se] derivative for which peak positions were shifted. Square-wave voltammetry showed that the product of the first electron transfer, which may correspond to the ‘super-oxidised’ [4Fe4S]3+ oxidation level, undergoes rapid degradation (t < 1.6 ms at 5°C). The second oxidation process, as characterised by a significant (?100 mV) negative shift upon selenium substitution, very likely represents oxidation of S(Se) still associated with the protein and possibly contained within the remaining FES(Se) substructure. 相似文献
104.
A. Diez de los Rios N. E. DeRose W. McD Armstrong 《The Journal of membrane biology》1981,63(1-2):25-30
Summary Open-tip and liquid ion-exchanger microelectrodes were used to study the effects of cAMP (6mm, added to the serosal medium) on apical membrane potential (E
m
) and intracellular sodium, potassium, and chloride activities (a
Na
i
,a
K
i
,a
Cl
i
) inNecturus gallbladder under open-circuit conditions. Transepithelial potential difference (E
Tr
) was also measured. In the presence of cAMP,a
Cl
i
fell from about 1.5 times its equilibrium value to a level that corresponded to electrochemical equilibrium across the apical and basolateral cell membranes. Under these conditionsa
Na
i
decreased anda
K
i
increased,E
m
was unchanged andE
Tr
increased from virtually zero to a small but significant serosal positive value. The cAMP-induced increase ina
K
i
was abolished when Cl–-free incubation media were used. Addition of the Ca++-ionophore A23187 (0.5 g/ml) to the serosal medium had no effect onE
m
,E
Tr
, ora
Cl
i
. When A23187 was added to the mucosal medium,E
m
and the basolateral membrane potential hyperpolarized by about 20 mV and an increase in the outwardly directed electrochemical driving force for Cl– was observed. These results indicate that cAMP inhibits coupled transapical Na–Cl entry into epithelial cells ofNecturus gallbladder and suggest that this inhibition may not be mediated by an increase in intracellular Ca++ concentration. 相似文献
105.
Summary Asymmetrical displacement currents are measured in the absence and in the presence of the lipophilic anion dipicrylamine (DPA) in the extracellular solution of nerve fibres of the frogRana esculenta. DPA (30nM-3 M) enhances the current by a component that has the properties expected for a translocation current of DPA ion across the lipid membrane. Analysis in terms of a single-barrier model yields the translocation rate constant (k), the total surface density of DPA absorbed to the membrane (N
t
), and the equidistribution voltage (). The value ofk of about 104 s–1 is similar to that for a solvent-free artificial bilayer formed by the Montal-Mueller method. The surface densityN
t
varies with the DPA concentration as it does in the artificial bilayer, but is about tenfold smaller at all concentrations. The DPA ions sense an intrinsic electric field that is offset by a transmembrane voltage between 0 and 30 mV (inside positive). The part of the axolemma probed by the DPA ion appears as a thin (<2.5 nm), fluid bilayer of lipids. DPA ions seem, however, to be excluded from the major part of the axolemma as if this area is occupied by integral proteins or negative charges. 相似文献
106.
Presence of antigenic determinants reacting with homogeneous IgM/kappa cold agglutinin (CA) of a new specificity, tentatively called Sa, was investigated by bithermic cytotoxicity assay and by immunofluorescence. CA Sa killed on average 38% allogeneic peripheral blood lymphocytes (PBL) and up to 74% of autologous PBL. There was preferential kill of B-PBL compared to T-PBL. Some preference toward B cells was also noted using tonsillary B and T lymphocytes. Cytotoxic activity of CA Sa against chronic lymphocytic leukemia cells of B-type was almost equal to that of potent anti-I CA and much stronger than anti-i CA. Presence of additional B-cytotoxic factor in the serum was excluded by the use of red blood cell eluate composed solely of homogeneous CA. Thymocytes and helper-type T cells from a patient with T cell chronic lymphocytic leukemia were very susceptible to the cytotoxic action of Sa. CA Sa killed 39% of monocytes, but there was almost no kill of polymorphonuclear leukocytes. Lymphocytotoxicity of CA Sa was abolished by sialyllactose and was not influenced by I-active glycoproteins. Comparison of CA Sa to CA of other specificities showed marked differences, supporting the view that Sa has new, previously unrecognized specificity. 相似文献
107.
Summary Intracellular C1, K and Na activities (a
Cl
i
,a
k
i
anda
Na
i
) and transmucosal membrane potential (E
m) in epithelial cells ofNecturus gallbladder were measured at different external Na concentrations ([Na]o), with liquid ion-exchanger and conventional microelectrodes. Bladders were mounted in a divided chamber at 23°C between identical HCO3-free Ringer solutions containing 5mm K. The pH was 7.2. Tris was substituted for Na. Measurements were made under steady-state conditions as determined by the constancy of the transepithelial potential difference. Both,a
Cl
i
anda
Na
i
increased in a saturable fashion with [Na]o.E
m did not change significantly. Average values (±sem) under normal conditions ([Na]o=100mm) fora
Cl
i
,a
Na
i
andE
m were 16.8±0.8mm (n=9), 9.7±0.6mm (n=10) and –52.6±0.6 mV (n=26), respectively. In Na-free mediaa
Cl
i
declined to its equilibrium value.a
K
i
(96±2mm;n=7) did not change when [Na]o was varied between 100 and 10mm but decreased to 80±3mm (n=4) in Na-free media.Transmembrane electrochemical potential differences,
, for Cl and Na were calculated at four different [Na]o levels. A highly significant linear relation between
and
was found, indicating that Cl and Na transport are energetically linked. The results support the view that the energy necessary for intracellular Cl accumulation is derived from the simultaneous dissipation of the chemical potential gradient of Na across the apical membrane and that the coupled entry mechanism is electroneutral. 相似文献
108.
Spherical aggregates of chick heart, sclera and skin fibroblasts were fused with tritiated thymidine-labelled aggregates of the identical cell type. After being placed in contact, the two aggregates cohered and broadened the area of contact to form a single aggregate with a planar interface between the labelled and unlabelled halves. The motility of cells in the aggregate was determined by measuring the movement of labelled cells across the boundary into the unlabelled half. Exposure to pharmacological doses of antimicrotubule agents resulted in a significant reduction in fibroblast motility in the three-dimensional aggregates. 相似文献
109.
110.
Armstrong R. B.; Marum P.; Tullson P.; Saubert C. W. th 《Journal of applied physiology》1979,46(4):835-842