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71.
Rafael Apitz-Castro Alicia De Murciano 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,544(3):529-539
32P phosphorylation of plasma membranes from human blood platelets, under conditions that closely resemble physiological ones (endogeneous phosphate donors and intact platelets in homologous plasma), result in the incorporation of the label mainly in a membrane glycoprotein of apparently high molecular weight (greater than 400 000). Dibutyryl cyclic AMP, an inhibitor of platelet aggregation, specifically increases the degree of phosphorylation of this glycoprotein. Moreover, it has been found that prostaglandin E1 one of the most potent inhibitors of platelet aggregation which also increases phosphorylation of the same glycoprotein, is significantly more effective than cyclic AMP.Cyclic GMP does not have any apparent effect on platelet aggregation. However, incubation of platelet-rich plasma with both cyclic GMP and cyclic AMP results in a partial recovery of the platelet responsiveness towards ADP-induced aggregation. Coincidently, the degree of phosphorylation of the high molecular weight glycoprotein under these conditions, although still higher than in controls (no nucleotides added), is significantly decreased as compared with cyclic AMP-treated cells. Furthermore, cyclic GMP inhibits the cyclic AMP-dependent protein kinase activity in isolated platelet plasma membranes.These results suggest a central role for this membrane phosphoglycoprotein in the triggering of platelet aggregation and, furthermore, suggest that modulation of its degree of phosphorylation may be exerted through some cyclic AMP/cyclic GMP relationship, which in the basal state might be critical for platelet responsiveness. 相似文献
72.
The subcellular site of -amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA3). Using [35S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA3 was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker -amylase. This protein was identified as authentic -amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized -amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled -amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of -amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA3 were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA3-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA3 only three peaks of activity were found. In incubation media, four isoenzymes were found after GA3 treatment and two were found after incubation without GA3. We conclude that at least five -amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.Abbreviations CHA
cyclohepataamylose
- DEAE-cellulose
diethylaminoethyl-cellulose
- ER
endoplasmic reticulum
- GA3
gibberellic acid
- SDS-PAGE
sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis 相似文献
73.
The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion.Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of 125I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on 125I-labelled LDL metabolism.Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of 125I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes. 相似文献
74.
An adoptive transfer system is described to measure serum helper activity in the primary antibody response to sheep red blood cells (SRBC). Mice injected with a high dose of cyclophosphamide and reconstituted with rabbit anti-thymocyte serum-treated spleen cells were used as recipients. Serum obtained 9 hr after ip injection of normal mice with 2 × 108 SRBC (S(SRBC)) injected i.v. in the recipients caused a significant enhancement of the antibody response to 2 × 107 SRBC. The serum helper activity was not generated in thymectomized animals and could be absorbed from S(SRBC) by normal and formalinized SRBC. The SRBC-specific serum helper activity (SSHA) is heat labile (30 min 56 °C) and shows allogeneic restriction. Another test system described in literature for measuring T-cell help in vivo was less suited to measure SSHA in the response to 2 × 107 SRBC. A system using normal mice injected with 105 SRBC for determining specific immune response-enhancing factor (SIREF), demonstrated SIREF activity in S(SRBC). It did, however, not measure SSHA, as absorption of S(SRBC) with formalinized SRBC did not abolish the activity in that system. 相似文献
75.
During chloroplast development in the primary leaves of Phaseolus vulgaris, the extractable activity of adenosine 5-phosphosulfate sulfotransferase increased ten-fold. When chloroplast development took place in air enriched with 3.5 l H2S·l-1 there was a decrease in adenosine 5-phosphosulfate sulfotransferase activity. Cyst(e)ine in concentrations up to 1 mM (in the external medium) did not affect the increase in adenosine 5-phosphosulfate sulfotransferase activity in intact plants. In plants with excised roots, 0.75 mM cyst(e)ine inhibited this increase. In green primary leaves, H2S or cyst(e)ine treatment resulted in a decrease of extractable adenosine 5-phosphosulfate sulfotransferase activity. In intact plants, this effect of cyst(e)ine was observed at a concentration of 1 mM, and in plants with excised roots, 0.25 mM had a comparable effect.In developing plants, the extractable activities of O-acetyl-L-serine sulfhydrylase (EC 4.2.99.9) and ribulosebisphosphate carboxylase (EC 4.1.1.39.) were not affected by H2S or cyst(e)ine.Abbreviations APS
adenosine 5-phosphosulfate
- APSSTase
adenosine 5phosphosulfate sulfotransferase
- BSA
bovine serum albumin
- DTE
dithioerythritol
- EDTA
ethylenediaminetetra-acetic acid
- OASSase
O-acetyl-L-serine sulfhydrylase
- PAPS
adenosine 3-phosphate 5-phosphosulfate
- POPOP
1,4 Di 2-(5-phenyloxazolyl)-benzene
- PPO
2,5-diphenyloxazol
- RubP
ribulose-bisphosphate
- RubPCase
ribulosebiphosphate carboxylase
This is no. 8 in the series Regulation of Sulfate Assimilation in Plants. The term cysteine is used when it is clear that cystine is not involved; cyst(e)ine is used for an undefined mixture of cysteine and cystine. The concentrations are expressed in all cases relative to cysteine 相似文献
76.
The involvement of the endomenbrane system of barley (Hordeum vulgare L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40–60% of the -amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent -amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of -amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the -amylase. The membrane fraction with which the -amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound -amylase is prepared in buffers containing 3 mM MgCl2 two fractions from a sucrose step gradient contain most of the -amylase activity. These fractions are enriched in the ER marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminete-traacetic-acid-treated membranes, -amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with -amylase being associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated -amylases, as well as the results from pulsechase experiments using L-[3H]leucine for labeling of -amylase, are all consistent with the hypothesis that membrane-associated -amylase is an intermediate in the secretory process.Abbreviations CNTPE
N-carbobenzoxy-L-tyrosine p-nitrophenylester
- Cyt oxidase
Cytochrome oxidase
- ER
endoplasmic reticulum
- EDTA
ethylenediaminetetraacetic acid
- GA3
gibberellic acid
- IDPase
inosine diphosphatase
- K+-ATPase
pH 6.5 K+-stimulated adenosine triphosphatase
- MES
2-(N-morpholino)ethanesulfonic acid
- MOPS
3-(N-morpholino)propanesulfonic acid
- NADH: Cyt c reductase
cyanide-insensitive NADH-linked cytochrome-c reductase
- RER
rough endoplasmic reticulum
- Tris
tris-(hydroxymethyl)-aminomethane 相似文献
77.
Summary Vesicles of inner mitochondrial membrane, mitoplasts, from rat brown adipose tissue were prepared by osmotic swelling and studied using the patch-clamp technique. Current events of a 107.8±8.7 pS (n=16, 21°C) channel were recorded in the mitoplast-attached mode. This channel was selective for anions and its kinetics resembled those of channels previously found in liver and heart mitochondria of mouse and ox. In whole-mitoplast mode each of five purine nucleotides (20 m) blocked the channel. This is the first demonstration of pharmacological blockade of this type of channel. Although a similar anion channel in mouse and ox mitochondria was suggested to be the uncoupling protein (UCP) associated with nonshivering thermogenesis, we present several arguments against this possibility. Thus we describe a high-conductance, purine-nucleotide-binding, anion selective mitochondrial channel, that is not the UCP. 相似文献
78.
Brian K. Davis 《Journal of molecular evolution》1991,33(4):343-356
Summary A rapidly acquired partial resistance to the replicase antagonist, ethidium bromide (EB), seen by Spiegelman and coresearchers in Q RNA variants competitively replicating under defined conditions in vitro, reflected existence of a pool of mutant RNA molecules, preadapted to EB, and their cross-propagation from the pre-EB optimum species, MDV-1, and from other kindred variants, some of which remained undetected, according to this quantitative analysis of midivariant RNA replication kinetics. DNAlike features of their evolution, such as the cloning of variants from an MDV-1 subtype and a complicance with the fundamental theorem of natural selection, resulted from the suppression, both real and apparent, of intrinsic RNA heterogeneity through sampling and detection methods, and also by the ascendency of self-propagation over cross-propagation with advancement of a superior variant. The deficit in mean polymer fitness, compared with optimum levels, determines the lower limit of this heterogeneity. Stability conditions for frequency equilibrium and strategies for counteracting viral drug resistance have been considered. 相似文献
79.
A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.Abbreviations FR
far-red light
- R
red light
- WL
white light
- WL + FR
white light supplemented with FR
- HIR
high-irradiance response
- PAR
photosynthetically active radiation
- Pr, Pfr
R- and FR-absorbing forms of phytochrome
- Ptot
total phytochrome
-
phyA (PhyA)
gene (encoded protein) for phytochrome
- WT
wild type
This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.M.; J.R. Cherry and R.D. Vierstra, (Department of Horticulture, University of Wisconsin-Madison, USA) are thanked for the provision of the transgenic tobacco line. 相似文献
80.
Olga N. Kulaeva Anastasiya B. Fedina Emiliya A. Burkhanova Natalya N. Karavaiko Marat Ya. Karpeisky Igor B. Kaplan Michael E. Taliansky Joseph G. Atabekov 《Plant molecular biology》1992,20(3):383-393
Exogenous human interferon 2 (IFN) and 2–5 oligoadenylates (2–5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1–1 u/ml and 2–5A at 1–10 nM. 相似文献