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111.
112.
Abstract: Cytosolic malic enzyme (EC 1.1.1.40) was purified from bovine brain 5,600-fold to a specific activity of 47 U/mg. The enzyme is a homotetramer with a subunit molecular mass of 60 kDa and an isoelectric point of 6.2. Mouse monoclonal antibodies raised against this enzyme were purified and shown to be monospecific, as indicated by immunoblotting. Immunocytochemical examination of rat astroglia-rich primary cultures at the light microscopic level revealed colocalization of cytosolic malic enzyme with the astroglial marker glial fibrillary acidic protein. Also, a colocalization with the oligodendroglial marker myelin basic protein was found. Neurons in rat neuron-rich primary cultures did not show positive staining. The data suggest that cytosolic malic enzyme is a glial enzyme and is lacking in neurons. 相似文献
113.
Purification and characterization of acetylcoenzyme A: deacetylvindoline 4-O-acetyltransferase from Catharanthus roseus 总被引:1,自引:0,他引:1
The enzyme acetylcoenzyme A:deacetylvindoline 4-O-acetyltransferase (EC 2.3.1.-) (DAT), which catalyzes the final step in vindoline biosynthesis in Catharanthus roseus, was purified 3300-fold using ammonium sulfate precipitation followed by gel filtration, anion exchange, hydroxyapatite, and affinity chromatographies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified DAT showed the presence of two major proteins having Mr values of 33,000 and 21,000, whereas native PAGE showed three protein bands, and isoelectric focusing-PAGE one diffuse protein band (pI = 4.7-5.3) plus two minor protein bands (pI = 5.7 and 6.1). Purified DAT possessed Km values of 6.5 microM and 1.3 microM for acetylcoenzyme A and deacetylvindoline, respectively, and Vmax values of 12.6 pkat/microgram protein (acetylcoenzyme A) and 10.1 pkat/micrograms protein (deacetylvindoline). Inhibition of DAT by tabersonine, coenzyme A, and cations (K+, Mg2+, and Mn2+) was observed, while the pH optimum of this enzyme was determined to be 7.5 to 9. 相似文献
114.
W. Kurzątkowski W. Kuryłowicz A. Paszkiewicz 《Applied microbiology and biotechnology》1982,15(4):211-213
Summary The activity of penicillin G production was compared in a well defined medium by native vesicles as well as by calcium alginate gel immobilized vesicles isolated from the protoplasts of Penicillium chrysogenum PQ-96. The activity yield of the immobilized vesicles was 44% in comparison with native vesicles. After 60 h of storage at 4° C the native vesicles showed a rapid decrease in penicillin G production. The storage stability of these vesicles was improved after entrapment inside the calcium alginate gel. After 240 h of storage 1 mg of immobilized vesicular protein catalyzed the production of about 140 nmoles of penicillin G in 1 h. 相似文献
115.
116.
The kinetics and mechanism of the citrate synthase from a moderate thermophile, Thermoplasma acidophilum (TpCS), are compared with those of the citrate synthase from a mesophile, pig heart (PCS). All discrete steps in the mechanistic sequence of PCS can be identified in TpCS. The catalytic strategies identified in PCS, destabilization of the oxaloacetate substrate carbonyl and stabilization of the reactive species, acetyl-CoA enolate, are present in TpCS. Conformational changes, which allow the enzyme to efficiently catalyze both condensation of acetyl-CoA thioester and subsequently hydrolysis of citryl-CoA thioester within the same active site, occur in both enzymes. However, significant differences exist between the two enzymes. PCS is a characteristically efficient enzyme: no internal step is clearly rate-limiting and the condensation step is readily reversible. TpCS is a less efficient catalyst. Over a broad temperature range, inadequate stabilization of the transition state for citryl-CoA hydrolysis renders this step nearly rate-limiting for the forward reaction of TpCS. Further, excessive stabilization of the citryl-CoA intermediate renders the condensation step nearly irreversible. Values of substrate and solvent deuterium isotope effects are consistent with the kinetic model. Near its temperature optimum (70 degrees C), there is a modest increase in the reversibility of the condensation step for TpCS, but reversibility still falls short of that shown by PCS at 37 degrees C. The root cause of the catalytic inefficiency of TpCS may lie in the lack of protein flexibility imposed by the requirement for thermal stability of the protein itself or its temperature-labile substrate, oxaloacetate. 相似文献
117.
Proregions of papain-like cysteine proteases are potent and often highly selective inhibitors of their parental enzymes. The molecular basis of their selectivity is poorly understood. For two closely related members of the cathepsin L-like subfamily we established strong selectivity differences. The propeptide of cathepsin S was observed to inhibit cathepsin L with a K(i) of 0.08 nM, yet cathepsin L propeptide inhibited cathepsin S only poorly. To identify the respective structural correlates we engineered chimeric propeptides and compared their inhibitory specificity with the wild-types. Specificity resided in the N-terminal part, strongly suggesting that the backbone of the prodomain was the underlying structure. 相似文献
118.
Induction of the blood–brain barrier marker neurothelin/HT7 in endothelial cells by a variety of tumors in chick embryos 总被引:1,自引:0,他引:1
Papoutsi M Kurz H Schächtele C Marmé D Christ B Pröls F Wilting J 《Histochemistry and cell biology》2000,113(2):105-113
Neurothelin/HT7, a transmembrane glycoprotein of the immunoglobulin superfamily, is a marker of blood–brain barrier (BBB)-forming
endothelial cells. We have studied the expression of neurothelin in tumors grown on the chorioallantoic membrane (CAM) of
chick embryos. We inoculated each 3–5×106 rat C6 glioma, rat 10AS pancreatic carcinoma, human A375 melanoma, and human mammary duct adenoma cells on the CAM of 10-day-old
chick embryos. The tumors were harvested on day 17. All four tumor cell lines formed solid tumors which were supplied by vessels
of CAM origin. Foci of bleeding were regularly observed within the tumors. All four tumors induced the expression of neurothelin/HT7
(but not of glucose transporter-1) in tumor endothelial cells, whereas expression in adjacent endothelial cells of normal
CAM did not occur. Confocal laser scanning microscopy revealed that the pattern of neurothelin expression in tumor endothelial
cells was different from that in normal central nervous system (CNS) endothelium, but the relative molecular weight of neurothelin,
studied by western blot analysis, was the same in brain and in tumors. It has been shown that, with increasing malignancy,
vessels of CNS tumors lose their morphological characteristics, and BBB markers such as the glucose transporter-1 are downregulated.
Our results show that, in contrast, the BBB marker, neurothelin, is expressed de novo in tumor endothelial cells. Potential
common functions of neurothelin in endothelial cells of the CNS and tumors are discussed.
Accepted: 6 December 1999 相似文献
119.
Periosteum stimulates subchondral bone densification in autologous chondrocyte transplantation in a sheep model 总被引:5,自引:0,他引:5
Russlies M Behrens P Ehlers EM Bröhl C Vindigni C Spector M Kurz B 《Cell and tissue research》2005,319(1):133-142
In this sheep study, we have tested the hypothesis that an osteogenic response is triggered in the subchondral bone by periosteum implanted in full thickness cartilage defects and can be prevented by replacing the periosteum by a cell-free collagen type I/III membrane. Two 7-mm diameter osteochondral defects were made in the trochlea groove and in the medial femoral condyle of one of the knees in each of 15 adult sheep. The animals were divided into three groups (n=5): a control group with untreated cartilage defects, a group treated with autologous chondrocyte transplantation (ACT) and periosteum, and a group treated with ACT in combination with a collagen I/III membrane cover. Histological examination was performed 1 year later. The optical density of the subchondral bone in the histological sections was measured with digital imaging software. There was a dramatic, statistically significant (P<0.0001; power=1) increase in bone density of 45%–70% under defects that were treated with the periosteal cover, compared with the collagen membrane and control groups, which displayed the same bone density. There was no difference in the cartilaginous reparative tissue in the defects in the three groups. Periosteum thus stimulates the remodelling process in subchondral bone. Stiffening of the subchondral bone can lead to degeneration of the overlying reparative cartilaginous tissue because of an increase in the mechanical stress in the tissue. These findings warrant evaluation of subchondral bone changes in patients treated by ACT and the correlation of these changes with clinical outcome. 相似文献
120.
Joseph Donfack Daniel H Schneider Zheng Tan Thorsten Kurz Inna Dubchak Kelly A Frazer Carole Ober 《Respiratory research》2005,6(1):145