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2.
The activities of glycogen phosphorylases a and b from the body wall musculature of the marine worm Arenicola marina (Annelida, Polychaeta) were determined after various periods of anoxia. Already under normoxic conditions one third of the total activity was produced from the a form. During anoxia the ratio of both forms as well as the total activity did not change. The activity of soluble phosphorylase kinase was comparatively low in this tissue 4.3 +/- 1.2 nmol . min-1 . (g wet wt.)-1; the fast twitching tail muscle of shrimps, e.g., had a 10-fold higher phosphorylase kinase activity, whereas phosphorylase activities in both tissues were about the same 2.3 +/- 0.5 mumol . min-1 . (g wet wt.)-1. Glycogen phosphorylase b was purified from the body wall tissue of the marine worm in one step by 5'-AMP-Sepharose resulting in a single protein band in SDS-PAGE. This preparation was accepted as substrate by the phosphorylase kinase from rabbit muscle but a complete phosphorylation could not be achieved. The molecular mass of native phosphorylase was approximately 216 kDa, that of subunits 95 kDa indicating that the enzyme exists as a dimer. There were no isozymes in this preparation, the RF-value (0.17) of the single band in PAGE ranged between those of the isozymes from mice hearts. The activities of phosphorylases b and a were similarly dependent on pH and temperature but differed drastically in the affinities to phosphate and AMP. In presence of 1 mM AMP the app. Km of phosphorylase a for phosphate was 16 mM, that of phosphorylase b above 100 mM.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   
3.
Tryptic peptides of the ribosomal proteins S11, L9 and L29 were separated by reversed phase chromatography under conditions which enabled direct micro-sequencing with the 4-(dimethylamino)azobenzene-4'-isothiocyanate/phenylisothiocyanate double coupling method [Chang, Brauer, Wittmann-Liebold (1978) FEBS Lett. 93, 205-214]. The peptides were separated on a RP-18 column employing volatile buffers at pH 2.0, 4.1 and 7.8. Depending on the different chromatographic behaviour of the peptide mixture, the elution gradient was optimised for each hydrolysate using 20 micrograms of the hydrolysed protein. Preparative separations were made with 150-250 micrograms. At least 80% of the peptides could be isolated by these techniques and used for direct micro-sequencing without further purification or desalting. The results show that the high-performance liquid chromatographic method employed allows easy isolation and sequencing with minute amounts of peptides.  相似文献   
4.
The site of the Escherichia coli envelope of the conversion of 1-acylglycero-3-phosphoethanolamine to diacylglycerophosphoethanolamine was explored, using two K12 strains with a wild-type phospholipid-degradative apparatus and a K12 mutant lacking detectable phospholipase A1 and A2 activity.Experiments with various radioactively labeled substrates show that acylation by crude envelope preparations as well as isolated inner and outer membranes of parent and mutant strains involves neither exogenous fatty acids nor a transacylation reaction with added monoacylglycerophosphoethanolamine. Furthermore, acylation exhibits no absolute requirement for added ATP and coenzyme A.Specific activity of acylating activity is the same in inner membrane preparations of parent and mutant strain and in outer membrane preparations of the mutant deficient in phospholipase A. Although clearly evident, net diacylglycerophosphoethanolamine formation by outer membranes of the parent strain, however, was about 6-fold less. This lower conversion may be attributed to activation during incubation of phospholipases A within the outer membrane, resulting in breakdown of the diacylcompound formed.Reacylation of lysophospholipids formed in the E. coli envelope by the action of endogenous or exogenous phospholipases A provides the organism with the potential of biochemically inexpensive repair and modification of the envelope phospholipids. Moreover, major phospholipids hydrolyzed in the outer membrane of E. coli can be resynthesized in the same location, without need for the transport of the products of hydrolysis to the lipid biosynthetic apparatus associated with the cytoplasmic membrane.  相似文献   
5.
Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non-specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6-times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP-depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.  相似文献   
6.
Opposing views exist as to how unesterified fatty acids (FA) enter and leave cells. It is commonly believed that for short- and medium-chain FA free diffusion suffices whereas it is questioned whether proteins are required to facilitate transport of long-chain fatty acid (LCFA). Furthermore, it is unclear whether these proteins facilitate binding to the plasma membrane, trans-membrane movement, dissociation into the cytosol and/or transport in the cytosol. In this mini-review we approach the controversy from a different point of view by focusing on the membrane permeability constant (P) of FA with different chain length. We compare experimentally derived values of the P of short and medium-chain FA with values of apparent permeability coefficients for LCFA calculated from their dissociation rate constant (k(off)), flip-flop rate constant (k(flip)) and partition coefficient (Kp) in phospholipid bilayers. It was found that Overton's rule is valid as long as k(flip)相似文献   
7.
The polymorphic minor histocompatibility Ag HA-1 locus encodes two peptides, HA-1(H) and HA-1(R), with a single amino acid difference. Whereas the immunogenicity of the HA-1(R) allele has not yet been shown, the nonameric HA-1(H) peptide induces HLA-A2-restricted cytotoxic T cells in vivo and in vitro. It is not known whether the mHag HA-1(H) or HA-1(R) associates with other HLA class I molecules. Therefore, the polymorphic regions of both HA-1 alleles were analyzed to identify HLA class I binding peptides that are properly processed by proteasomal degradation. Peptide binding analyses were performed for all nonameric HA-1(H/R) peptides for binding to nine HLA class I molecules with >10% prevalence in the Caucasian population and for seven nonameric/decameric HA-1(H/R) peptides predicted to bind to HLA-A3, -B14, and -B60. Only the nonameric KECVL(H)/(R)DDL and decameric KECVL(H)/(R)DDLL peptides showed strong and stable binding to HLA-B60. In vitro digestion of 29-aa-long HA-1 peptides by purified 20S proteasomes revealed proper cleavage at the COOH termini of both HLA-B60 binding HA-1(H) and HA-1(R) peptides. In subsequent analyses, dendritic cells pulsed with the nonameric HA-1(R) peptide did not induce CTLs that recognize the natural HLA-B60/HA-1(R) ligand. In contrast, dendritic cells pulsed with the nonameric HA-1(H) peptide induced IFN-gamma-secreting T cells specific for the natural HLA-B60/HA-1(H) ligand in three HLA-B60(+) HA-1(RR) individuals, demonstrating the immunogenicity of the HLA-B60/HA-1(H) ligand. In conclusion, this study shows a novel HLA-B60-restricted T cell epitope of the minor histocompatibility Ag HA-1 locus.  相似文献   
8.
Skeletal muscle atrophy commonly occurs in acute and chronic disease. The expression of the muscle-specific E3 ligases atrogin-1 (MAFbx) and muscle RING finger 1 (MuRF1) is induced by atrophy stimuli such as glucocorticoids or absence of IGF-I/insulin and subsequent Akt signaling. We investigated whether glycogen synthase kinase-3β (GSK-3β), a downstream molecule in IGF-I/Akt signaling, is required for basal and atrophy stimulus-induced expression of atrogin-1 and MuRF1, and myofibrillar protein loss in C(2)C(12) skeletal myotubes. Abrogation of basal IGF-I signaling, using LY294002, resulted in a prominent induction of atrogin-1 and MuRF1 mRNA and was accompanied by a loss of myosin heavy chain fast (MyHC-f) and myosin light chains 1 (MyLC-1) and -3 (MyLC-3). The synthetic glucocorticoid dexamethasone (Dex) also induced the expression of both atrogenes and likewise resulted in the loss of myosin protein abundance. Genetic ablation of GSK-3β using small interfering RNA resulted in specific sparing of MyHC-f, MyLC-1, and MyLC-3 protein levels after Dex treatment or impaired IGF-I/Akt signaling. Interestingly, loss of endogenous GSK-3β suppressed both basal and atrophy stimulus-induced atrogin-1 and MuRF1 expression, whereas pharmacological GSK-3β inhibition, using CHIR99021 or LiCl, only reduced atrogin-1 mRNA levels in response to LY294002 or Dex. In conclusion, our data reveal that myotube atrophy and myofibrillar protein loss are GSK-3β dependent, and demonstrate for the first time that basal and atrophy stimulus-induced atrogin-1 mRNA expression requires GSK-3β enzymatic activity, whereas MuRF1 expression depends solely on the physical presence of GSK-3β.  相似文献   
9.
In industrial fermentations, Penicillium chrysogenum uses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes, sutA and sutB, whose encoded products belong to the SulP superfamily of sulfate permeases were isolated. Transformation of a sulfate uptake-negative sB3 mutant of Aspergillus nidulans with the sutB gene completely restored sulfate uptake activity. The sutA gene did not complement the A. nidulans sB3 mutation, even when expressed under control of the sutB promoter. Expression of both sutA and sutB in P. chrysogenum is induced by growth under sulfur starvation conditions. However, sutA is expressed to a much lower level than is sutB. Disruption of sutB resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate permease involved in sulfate uptake by P. chrysogenum.  相似文献   
10.
High performance liquid chromatography was applied to the separation of proteins derived from the Escherichia coli 30S ribosomal subunit. Several methods of separating this protein mixture has been tested: size-exclusion chromatography on hydrophilic phases; ion exchange and reversed phase chromatography (on C2 to C18 hydrocarbon-bonded supports). Various elution systems were examined in order to obtain pure proteins suitable for micro-sequence analysis. The resolution and yields of the proteins varied considerably, depending on the type of support and gradient system used. The best results were achieved with uniformly globular-shaped supports of large pore size, and by combining high performance size exclusion with rechromatography on reversed phase columns. Purification conditions for the individual proteins are listed. The methods employed avoid any precipitation step and allow easy identification of the proteins by one or two-dimensional gel electrophoresis, amino-acid analysis or direct manual or automatic micro-sequencing. Since the isolation time is much reduced compared with conventional purification procedures, the proteins obtained by the techniques described here are well suited for topographical and immunological studies or reconstitution assays. Ribosomal proteins of other organisms can be separated under similar conditions.  相似文献   
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