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121.
Dido Vassilacopoulou James A. Ripellino Nikolaos Tezapsidis Vivian Y. H. Hook Nikolaos K. Robakis 《Journal of neurochemistry》1995,64(5):2140-2146
Abstract: The amyloid β peptide (Aβ) of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APPs), which are considered type I transmembrane proteins. Here we report that the soluble fraction of isolated adrenal medullary chromaffin granules (CG), a model neuronal secretory vesicle system, contains an antigen that immunochemically and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from full-length APP. A truncated APP fragment with intact Aβ sequence was also detected in the soluble fraction of CG. In vitro experiments showed that full-length APP was solubilized from CG membranes at 37°C as a function of pH, with a peak of activity between pH 8.5 and pH 9.0. Solubilization of full-length APP was inhibited by several protease inhibitors, including aprotinin, cystatin, and iodoacetamide, by the divalent cations Ca2+ and Zn2+ , and by preheating of the membranes. These results are consistent with and suggest the involvement of an enzymatic mechanism in the solubilization of potentially amyloidogenic full-length APP. Production of Aβ from a transmembrane APP predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Thus, the detected soluble, potentially amyloidogenic, full-length APP may be a substrate for the proteases producing Aβ. The detection of soluble APP with intact Aβ sequence in secretory vesicles is consistent with the extracellular topology of amyloid depositions. 相似文献
122.
Donald R. Gehlert Douglas A. Schober Susan L. Gackenheimer 《Journal of neurochemistry》1995,64(6):2792-2800
Abstract: ( R )-[3 H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower-affinity site. The goal of the present study was to identify the nature of this low-affinity site by comparing the binding properties of ( R )-[3 H]tomoxetine with those of ( R/S )-[3 H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas ( R )-[3 H]tomoxetine also bound to a second, lower-affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE-containing neurons. However, low levels of ( R )-[3 H]-tomoxetine binding were seen in the caudate-putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where ( R/S )-[3 H]nisoxetine binding was almost absent. In homogenates of the caudate-putamen, the NE uptake inhibitors desipramine and ( R )-nisoxetine and the serotonin (5-HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 n M ( R )-nisoxetine to mask the binding of ( R )-[3 H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [3 H]citalopram. Therefore, ( R )-[3 H]tomoxetine binds to the NE uptake site with high affinity and the 5-HT uptake site with somewhat lower affinity. 相似文献
123.
H+-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H+-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K+ for 40 min under various conditions. The rate of efflux of K+ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H+-ATPase of the plasma membrane, increased the efflux of K+. Al repressed this efflux at low pH, probably through an effect on K+ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K+ irrespective of the presence of vanadate. Ca2+ also had a repressive effect on the efflux of K+ at low pH. The effect of Ca2+, greater in Scout, might be related to the regulation of the net influx of H+, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H+, which in turn is counteracted by the efflux of K+ and H+. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H+ and the efflux of K+ seem to participate in the mechanism of tolerance to Al stress under acidic conditions. 相似文献
124.
The primary free polyamines identified during growth and development of strawberry (Fragaria × ananassa Duch.) microcuttings cultivated in vitro were putrescine, spermidine and spermine. Polyamine composition differed according to tissue and stages of development; putrescine was predominant in aerial green tissues and roots. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), strongly inhibited growth and development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition, indicating that polyamines are involved in regulating the growth and development of strawberry microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis by ornithine decarboxylase, promoted growth and development. We propose that ADC regulates putrescine biosynthesis during microcutting development. The application of exogenous polyamines (agmatine, putrescine, spermidine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these polyamines can be growth limiting.Abbreviations ADC
arginine decarboxylase
- ODC
ornithine decarboxylase
- DFMA
-difluoromethylarginine
- DFMO
-difluoromethylornithine
- Put
putrescine
- Spd
spermidine
- Sp
spermine
- DW
dry weight
- PA
polyamine
- PPF
photosynthetic photon flux 相似文献
125.
M. A. C. Demeulemeester W. Rademacher A. Van de Mierop M. P. De Proft 《Plant Growth Regulation》1995,17(1):47-52
Root explants of chicory (Cichorium intybus L.) were cultured in vitro under continuous light or darkness. On a standard medium (no plant growth regulators added), flowering-stems were initiated under continuous light while under continuous dark, vegetative-stems were formed. Different types of GA (gibberellin) biosynthesis inhibitors were added to the culture medium. Paclobutrazol and compounds belonging to the group of cyclohexanetriones clearly reduced flowering-stem growth under light conditions and vegetative-stem growth under dark conditions. Under light conditions, flower bud initiation was not affected. These and other results suggest that GA1 may be synthesized during the in vitro culture period and that it controls flowering-stem growth but not floral initiation.Abbreviations CCC
chlormequat chloride
- GA
gibberellin
- LAB 198 999
3,5-dioxo-4-butyryl-cyclohexane carboxylic acid ethyl ester
- BAS 111..W
1-phenoxy-3-(1H-1,2,4-triazol-1-yl)-4-hydroxy-5,5-dimethylhexane 相似文献
126.
N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography
Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies. Surprisingly, pseudo-peptide bond inhibitors of the atrial enzyme [Damodaran and Harris (1995),J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R, L]GNPVP[F, G]R[Q, I]XY[G, E]XR(N, A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.Abbreviations AGSP
atrial granule serine proteinase
- ANF
atrial natriuretic factor
- BSA
bovine serum albumin
-
Bz
benzoyl
- EACA
6()-aminocaproic acid
- HEPES
N-2-hydroxyethylpiperazine-N'-propanesulfonic acid
- HPLC
high-performance liquid chromatography
- PEG
polyethylene glycol-3350
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single-letter abbreviations are used to denote amino acids 相似文献
127.
Pseudo-peptide bond inhibitors (-bond inhibitors) and peptide-aldehyde inhibitors of atrial granule serine proteinase, the candidate processing enzyme of pro-atrial natrieuretic factor, are prepared in high yield and purity by novel synthetic routes. The -bond compounds retain essential residues for enzyme binding, but place the enzyme inhibition site in the midst of the peptide sequence. Thus, Bz-APR--LR and Bz-APR--SLRR can be considered readthrough inhibitors of atrial granule serine proteinase. The most potent -peptide, Bz-APR--SLRR (IC50=250 M), is about fivefold less potent than the best peptide-aldehyde inhibitor (EACA-APR-CHO), and both the -bond and peptide-aldehyde compounds are competitive, reversible inhibitors of the enzyme. The -bond peptides containing two C-terminal Arg residues are three-to tenfold more potent than the analogous compounds containing only one C-terminal Arg residue, confirming the importance of both Arg residues in the enzyme processing recognition site. As expected, because of their moderate potencies, the -peptides are not useful affinity ligands for purification of atrial granule serine proteinase, but both peptide aldehydes are effective affinity ligands [Damodaran and Harris (1995),J. Protein Chem., this issue].Abbreviations AGSP
atrial granule serine proteinase
- ANF
atrial natriuretic factor
- Bz
benzoyl
- DIEA
diisopropylethylamine
- DIPCDI
diisopropylcarbodiimide
- DMF
dimethylformamide
- DMSO
dimethylsulfoxide
- EACA
6(e)-aminocaproic acid
- EtOAc
ethyl acetate
- HEPES
N-2-hydroxyethylpiperazine-N-propanesulfonic acid
- HOBt
N-hydroxybenzotriazole
- HPLC
high-performance liquid chrornatography
- NMR
nuclear magnetic resonance
- PEG
polyethylene glycol-3350
- PyBOP
benzotriazole-1-yl-oxy-trispyrrolidino-phosphonium-hexafluorophospate
- TEA
triethylamine
- TFA
trifluoroacetic acid
- THF
tetrahydrofuran
- TLC
thin-layer chromatography
- UV
ultraviolet
-
pseudo-peptide bond -CH2-NH-. Single-letter abbreviations are used to denote amino acids 相似文献
128.
Mordechai Manoach Dalia Varon Mordechai Erez 《Molecular and cellular biochemistry》1995,147(1-2):181-185
Ventricular fibrillation (VF) is one of the most life threatening events. Although in humans VF is generally sustained (SVF) requiring artificial defibrillation, in various mammals and in some cases in humans VF terminates by itself, reverting spontaneously into sinus rhythm. Since VF is one of the main causes of sudden death, one of the important clinical problems today is if and how we can transform the fatal SVF into a self limited transient one (TVF).From electrophysiological studies carried out on anaesthetized open chest animals, we have found that TVF requires a high degree of intercellular coupling and synchronization.Cardiac myocytes are electrically coupled with adjacent cells. The intercellular coupling is a focus of low electrical resistance which allows rapid transmission of electrical impulses between cells. Any decrease in intercellular coupling decreases the ability of the heart for self defibrillation. The cell-to-cell coupling decreases with age, ischemia, VF and variations in physiological conditions probably due to an increase in intercellular resistance (Ri), widening in the internexal gaps, decrease in electrotonic space constant () etc. All of these factors are known to be affected by intracellular concentration of free Ca++ ([Ca++]).On the basis of studies carried out on various mammals at different ages, we hypothesized that the ability of the heart to defibrillate depends on the cardiac catecholamine level [CA], during VF. This hypothesis is supported by the facts, known from the literature, that increase in [CA] decreases intracellular free Ca++ concentration, decreases Ri and increases . By these effects, increase in [CA] enhances intercellular coupling and intercellular synchronization, and thereby, according to our hypothesis, leads to spontaneous ventricular defibrillation — TVF.During VF the sympathetic activity is enhanced but in some cases the [CA] does not reach the level needed for TVF. In order to help the heart in its effort to elevate the [CA] during VF, we proposed to treat these cases with drugs which inhibit the reuptake of [CA]. The facts that administration of [CA] reuptake inhibitors, before the induction of VF, and/or intracoronary infusion of adrenaline, during VF, transforms SVF into TVF, emphasized the validity of our hypothesis. 相似文献
129.
During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic
cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it
was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium.
The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of 35S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By
pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the
protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding
to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released
from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and
nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic
proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease
inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII
against cleavage, probably by binding to rFVIII.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
130.
Christelle David Laurent Bischoff Hervé Meudal Catherine Llorens-Cortes Bernard Pierre Roques Marie-Claude Fournié-Zaluski 《Letters in Peptide Science》1997,4(4-6):411-414
In order to study the physiological role of aminopeptidase A (APA),several -mercapto--amino acyl dipeptides were synthesized toobtain compounds having a high affinity for APA and a high selectivityversus aminopeptidase N (APN). Sulfonamide and carboxylate moieties whichhave been shown to be recognized by the S1 subsite of theenzyme were introduced on the side chain of the -mercapto--aminoacyl sub-unit, the latter being coupled to dipeptides optimized to interactwith the S1 andS2 subsites by means of combinatorialchemistry. Good affinities (16 nM) were obtained, the selectivity factorsbeing up to 160-fold versus APN. 相似文献