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181.
John E. Fox 《Molecular biotechnology》1995,3(3):249-258
The synthesis of large numbers of peptides can be very labor intensive and, if a conventional peptide synthesizer is used,
only small numbers of peptides can be produced within a reasonable time. The techniques described below can make large numbers
of different peptides simultaneously with varying degrees of mechanization, ranging from the wholly manual methods, to those
involving complete mechanization of the whole synthesis process. Most of the multiple synthesis methods are primarily intended
for small scale production ranging from microgram amounts up to a few tens of milligrams. All of the systems are economical
in use of solvents and reagents, enabling cost-effective synthesis. The techniques described can also be used to prepare peptide
libraries, containing several millions of peptide sequences, to enable the rapid screening of all possible permutations of
amino acids within short peptides. However, it is considered that multiple synthesis methods are not particularly suited where
extreme high purity or very long peptides are required. 相似文献
182.
小麦条锈菌毒性小种及其无毒性突变型侵染初期,是不亲和反应的小麦叶片内可翻译mRNA水平迅速增加,而呈亲和反应叶片的增加幅度小且滞后。同时前者的Poly(A+)-RNA水平高于未接种对照,后者低于对照。32P标记实验证实不亲和反应叶片Poly(A+)-RNA的合成增加早于亲和反应叶片。Poly(A+)-RNA体外翻译产物经SDS-PAGE分离后,放射自显影图谱显示一些多肽条带的35S-Met相对掺入量有定量差异。 相似文献
183.
Joseph Cain 《Biology & philosophy》1994,9(3):387-427
Ernst Mayr's contributions to 20th century biology extend far beyond his defense of certain elements in evolutionary theory. At the center of mid-century efforts in American evolutionary studies to build large research communities, Mayr spearheaded campaigns to create a Society for the Study of Evolution and a dedicated journal,Evolution, in 1946. Begun to offset the prominence ofDrosophila biology and evolutionary genetics, these campaigns changed course repeatedly, as impediments appeared, tactics shifted, and compromises built a growing coalition of support. Preserved, however, were designs to balance the community and journal with careful equation of status and explicit partitioning of responsibilities within the working coalition. Choice terms such as cooperation and unity carried a strong political message. Mayr's editorship ofEvolution provides a superb example of these balancing efforts. The mid-century infrastructural activities described herein also represented aggressive attempts to leverage control across several layers of community. Leaders of these campaigns sought: (1) to promote evolutionary studies as a modernized research discipline and place it at the center of American biology, (2) to promote evolutionary studies within existing disciplines — e.g. systematics, genetics, and paleontology, (3) to foster certain research styles within evolutionary studies, and (4) to emphasize certain solutions to prominent research questions. Throughout, Mayr interjected his priorities, tactics and energy. 相似文献
184.
D. R. Joanisse K. B. Storey 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1994,164(3):247-255
The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH
6-Phosphogluconate dehydrogenase
- DHAP
dihydroxy acetone phosphate
- F6P
fructose-6-phosphate
- F6Pase
fructose-6-phospha-tase
- FBPase
fructose-bisphosphatase
- G3P
glycerol-3-phosphate
- G3Pase
glycerol-3-phosphate phophatase
- G3PDH
glycerol-3-phosphate dehydrogenase
- G6P
glucose-6-phosphate
- G6Pase
glucose-6-phosphatase
- G6PDH
glucose-6-phosphate dehydrogenase
- GAK
glyceraldehyde kinase
- GAP
glyceraldehyde-3-phosphate
- GAPase
glyceraldehyde-3-phosphatase
- GAPDH
glyceraldehyde-3-phosphate dehydrogenase
- GDH
glycerol dehydrogenase
- GPase
glycogen phosphorylase
- HMS
hexose monophosphate shunt
- LDH
lactate dehydrogenase
- NADP-IDH
NADP+-dependent isocitrate dehydrogenase
- PDHald
polyol dehydrogenase, glyceraldehyde activity
- PDHgluc
polyol dehydrogenase, glucose activity
- PFK
phosphofructokinase
- PGI
phosphoglucoisomerase
- PGK
phosphoglycerate kinase
- PGM
phosphoglucomutase
- PK
pyruvate kinase
- PMSF
phenylmethylsulfonylfluoride
- SoDH
sorbitol dehydrogenase
-
V
max
maximal enzyme activity
- ww
wet weight 相似文献
185.
The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO2 incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10–7–10–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10–7–10–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells. 相似文献
186.
Since the positive charge on the lysine residues plays an important role in the receptor recognition ability of oLH, the hormonotoxin has been synthesised with the use of 2-iminothiolane HC1 (2IT) and N-Succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The oLH activated with 2IT (oLH-10) was then mixed with SPDP activated gelonin (gelonin-30) in order to obtain a oLH-S-S-gelonin hormonotoxin. The conjugation mixture containing hormonotoxin was purified by gel-filtration chromatography according to the molecular weight and a complete physico-chemical, immunochemical and biochemical analysis were performed. The linkage occured through the -NH2 groups of -subunit of oLH as judged from RP-HPLC analysis. A 11 (oLH:gelonin) molar ratio was obtained when determined with the use of several techniques. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity. The competitive displacement analysis indicate that the binding occurs via the hormone part leaving the gelonin free which was probed with the gelonin antibodies. The presently described (C150A-02, C160A-02 and C170A-02) hormonotoxins exhibited higher receptor binding and toxicity to the target cells than the hormonotoxins prepared with the use of SPDP only. Therefore it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH which was preserved during the conjugation process.Abbreviations BSA
Bovine Serum Albumin
- CMC
Carboxy methyl Cellulose
- DTT
Dithiothreitol
- DMEM
Dulbeco's Modified Eagle's Medium
- DTNB
Ellman's reagent [5,5-dithio-bis-(2-nitrobenzoic acid)]
- EDTA
Ethylenediaminetetraacetic acid
- FPLC
Fast Protein Liquid Chromatography
- FCA
Freund's Complete Adjuvant
- FCS
Fetal Calf Serum
- Gelonin-30
Gelonin modified by SPDP
- GnRH
Gonadotropin-Releasing Hormone
- Gelonin-SPDP
SPDP modified derivative of gelonin
- HEPES
(N-[2-hydroxyethyl] piperazine-N-[-2-ethanesulphonic acid])
- IFA
Incomplete Freund's Adjuvant
- 2IT
2-Iminothiolane
- IODOGEN
1,3,4,6-tetrachloro 3,6-diphenylglycouril
- oLH
Ovine Luteinizing Hormone
- oLH-SPDP
SPDP modified derivative of oLH
- oLH-10
oLH modified by 2IT
- oLH2IT
Molar ratio of oLH and 2IT
- PDP
2-Pyridyl-dithiopropionate
- PAP
Pokeweed Antiviral Protein
- RIP
Ribosome Inactivating Protein
- RP-HPLC
Reverse-Phase High Performance Liquid Chromatography
- RPMI
Roswell Park Memorial Institute
- RIA
Radioimmunoassay
- RRA
Radioreceptor Assay
- SPDP
N-Succinimidyl-3(2-pyridyldithio)propionate
- SDS-PAGE
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
- TCA
Trichloroacetic acid
- TFA
Trifluroacetic acid 相似文献
187.
José L. Sanz Gertrud Huber Harald Huber Ricardo Amils 《Journal of molecular evolution》1994,39(5):528-532
The sensitivity of the cell-free protein synthesis systems from Acidanus brierleyi, Acidianus infernus, and Metallosphaera sedula, members of the archaeal order Sulfolobales, to 40 antibiotics with different specificities has been studied. The sensitivity patterns were compared to those of Sulfolobus solfataricus and other archaeal, bacterial, and eukaryotic systems. The comparative analysis shows that ribosomes from the sulfolobales are the most refractory to inhibitors of protein synthesis described so far. The sensitivity results have been used to ascertain in phylogenetic relationships among the members of the order Sulfolobales. The evolutionary significance of these results are analyzed in the context of the phylogenetic position of this group of extreme thermophilic microorganisms.
Correspondence to: R. Amils 相似文献
188.
Nagayasu T Miyanaga M Tanaka T Sakiyama T Nakanishi K 《Biotechnology and bioengineering》1994,43(11):1108-1117
N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc. 相似文献
189.
190.