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51.
Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na+-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of Mr ? 165000 which is apparently a dimer of Mr ? 85 000. When reconstituted and tested for transport, this protein showed Na+-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals. 相似文献
52.
A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1-10µMolar region could be accurately and reproducibly measured using this technique.
The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen. 相似文献
The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen. 相似文献
53.
Andreas Schmid Gerhard Burckhardt Heinz Gögelein 《The Journal of membrane biology》1989,111(3):265-275
Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H+ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl–=Br–=I–>SO
4
2–
F–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl–-flux measurements and studies on the H+ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H+ uptake into the endosomes. 相似文献
54.
Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50,DBDS,MCD (0.5±0.1 m) for the H2-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of DBDS is in agreement with the ID50,Cl
–,MCD (0.94±0.07 m) for H2-DIDS inhibition of MCD cell Cl– flux, thus relating DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl– exchange by a factor of two, from Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (K
I,K
+,rbc=0.017 m). These experiments indicate that the Na+,K–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton. 相似文献
55.
Hiroaki Kataoka Kazuki Nabeshima Naoto Komada Masashi Koono 《Virchows Archiv. B, Cell pathology including molecular pathology》1989,57(1):157-165
Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines
were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated
in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free
conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1
inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons
(Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human α1-antitrypsin in double immunodiffusion. PI-1 corresponding to α1
- antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin,
kallikrein and plasmin weakly. It had higher molecular weight (200–300 Kd) than that of PI-1, and did not crossreact with
antisera against human α1-antitrypsin, α2-macroglobulin, α1-antichymotrypsin, α2-plasmin inhibitor, inter-α-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma
cell lines that secrete functionally active trypsin inhibitors, including α1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors. 相似文献
56.
Bovine kidneys were found to contain about 78 ppm Zn and 0.78 ppm Cd. Approximately 45% of Zn and 60% of Cd were present in
the cytosol fraction. More than 95% of these two metals were bound to macromolecules. Both Zn- and Cd-protein complexes were
observed to be stable between pH 7 and 10.5. Separation and characterization of these proteins were carried out using several
chromatographic and electrophoretic techniques in conjunction with instrumental neutron activation analysis (INAA). The results
showed the presence of at least four Zn-binding proteins with mol wt>300,000, 260,000, 89,000, and 27,000 and at least three
Cd-binding proteins of mol wt>300,000, 32,000, and 13,000. 相似文献
57.
58.
59.
Koichi Rikimaru Hitomi Toda Noriko Tachikawa Nobuyuki Kamata Shoji Enomoto 《In vitro cellular & developmental biology. Plant》1990,26(9):849-856
Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium,
designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free,
chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1
exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly
decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the
implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of
oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration,
where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth
of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant
human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells
or on the differences of growth mechanisms between normal and neoplastic human squamous cells. 相似文献
60.
Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A 总被引:4,自引:0,他引:4
Peter Ragnhammar Maria Liljefors Anna-Lena Hjelm Håkan Mellstedt Jan-Erik Frödin J. Fagersberg 《Cancer immunology, immunotherapy : CII》1996,42(2):81-87
A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable
regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2.
Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab2). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%)
antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab3), were induced by the mAb17-1A therapy. Patients with detectable ab3 after treatment had significantly higher ab2 levels than those not developing ab3. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction
of ab2 as well as induction of anti-anti-idiotypic antibodies (ab3), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab3) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab3 (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in
mAb therapy.
Received: 20 October 1995 / Accepted: 18 December 1995 相似文献