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61.
Dopaminergic neurons from embryonic rat mesencephalon were grown in simple serum-free media. The cells develop over a period of several weeks in vitro, particularly between day 14 and day 23. Removing the culture medium and replacing it with fresh medium during this interval caused severe damage to the cultures; this damage is mediated by excitatory amino acids acting through glutamate receptors. Damage could be completely prevented by antagonists of the N-methyl-D-aspartate subtype of glutamate receptor. As expected, medium that contains glutamate (i.e., Ham's F-12 medium) caused damage; however, medium that contains no glutamate or aspartate (i.e., Dulbecco's modified Eagle medium) also caused severe damage, and most of the damage was dependent on the presence of glutamine in the medium. The presence of the antibiotics penicillin and streptomycin greatly enhanced damage caused by medium change. 相似文献
62.
Vitellogenin and vitellin of Manduca sexta and some other insect species were purified by immobilized metal ion affinity chromatography. Ferric ion was chosen as the immobilized metal ion. Agarose-bound carboxymethylpicolylamine was used as the chelating adsorbent for the ferric ion. Vitellogenin and vitellin, both phosphorylated lipoproteins, were shown to bind specifically to the iron. The general applicability of immobilized ferric ion affinity chromatography for the purification of insect vitellogenin and vitellin is suggested. 相似文献
63.
Expression and characterization of the N-terminal half of antistasin, an anticoagulant protein derived from the leech Haementeria officinalis 总被引:1,自引:0,他引:1
L O Palladino J S Tung C Dunwiddie K Alves A B Lenny C Przysiecki D Lehman E Nutt G C Cuca S W Law 《Protein expression and purification》1991,2(1):37-42
Antistasin, a 15-kDa anticoagulant protein isolated from the salivary glands of the Mexican leech Haementeria officinalis, has been shown to be a potent inhibitor of factor Xa in the blood coagulation cascade. Antistasin possesses a twofold internal homology between the N- and C-terminal halves of the molecule, suggesting a gene duplication event in the evolution of the antistasin gene. This structural feature also suggests that either or both halves of the protein may possess biological activity if expressed as separate domains. Because the N-terminal domain contains a factor Xa P1-reactive site, we chose to express this domain in an insect cell baculovirus expression system. Characterization of this recombinant half antistasin molecule reveals that the N-terminal domain inhibits factor Xa in vitro, with a K(i) of 1.7 nM. 相似文献
64.
R O Law 《Biochimica et biophysica acta》1992,1107(1):186-192
When the osmolality of the bathing medium was increased from 710 to 2000 mosmol/kg H2O, cells in incubated slices of rat renal inner medulla lost water and K+, and the rate of efflux of preloaded 86Rb+ (a tracer for K+) was significantly depressed. Addition of 2-aminoisobutyric acid (AIB, 10 mmol/l) partly restored cell water content but without re-accumulation of K+; the rate of 86Rb+ efflux was greatly increased. The presence of Ba2+ (1 mmol/l) or trifluoperazine (50 mumol/l) led to complete recovery of cell volume and K+ contents, with markedly reduced efflux of 86Rb+. Neither additive had any significant effect upon these variables in the absence of AIB or in media of 710 mosmol/kg. Efflux of 86Rb+ was pH-sensitive within the physiological range, and was depressed when external AIB was reduced below approx. 5 mmol/l. When external Na+ was increased from 145 to 500 mmol/l (total osmolality 350 to 2500 mosmol/kg) efflux was retarded only slightly if AIB was present, but markedly if AIB was omitted. Inner medullary cells may contain a class of Ba(2+)-inhibitable, calmodulin-dependent K+ conductive pathway which is activated in strongly hyperosmotic media by the operation of an inwardly-directed Na(+)-amino acid symport (cf. Law, R.O. (1988) Pflügers Arch. 413, 43-50) and which serves to moderate the volume-restorative effect of this membrane mechanism. 相似文献
65.
Requirement of ADP-ribosylation for the pertussis toxin-induced alteration in electrophoretic mobility of G-proteins. 总被引:1,自引:0,他引:1
S C Roerig H H Loh P Y Law 《Biochemical and biophysical research communications》1991,180(3):1227-1232
Pertussis toxin (PTX) catalyzes the ADP-ribosylation of the alpha-subunit of GTP-binding proteins (G-proteins) in the presence of NAD+. Pertussis toxin also decreases the electrophoretic mobility of the alpha-subunit on urea SDS PAGE. This effect of PTX has been suggested to be a property of the toxin different from its ability to catalyze ADP-ribosylation. However, the present report provides evidence to the contrary; ie, this mobility shift required the ADP-ribosylation of alpha-subunits. This conclusion was based on: (1) in the presence of increasing concentrations of NAD+ (0.026-1.3 microM), there was a linear increase in the formation of the slower migrating alpha-subunit as measured by immunoblotting with selective antisera, (2) addition of NADase to the incubation mixture completely eliminated the formation of this protein, and (3) increasing concentrations of nicotinamide (50-250 mM), which inhibits ADP-ribosylation, decreased the amount of the slower migrating alpha-subunit. Thus, in addition to PTX, NAD+ was required for the mobility shift and the slower migrating alpha-subunit is likely the ADP-ribosylated form. 相似文献
66.
Isolation and molecular cloning of transferrin from the tobacco hornworm, Manduca sexta. Sequence similarity to the vertebrate transferrins 总被引:5,自引:0,他引:5
An iron-binding glycoprotein of Mr = 77,000 has been isolated from hemolymph of the adult sphinx moth Manduca sexta. Since this protein binds ferric ion both in vivo and in vitro and has a secondary structure similar to that of human serum transferrin and human lactoferrin as judged by CD spectra, we decided to clone its cDNA in order to determine its relationship to the vertebrate transferrins. Antiserum generated against this protein was used to screen a larval fat body cDNA library. A 2.0 kilobase clone was isolated that selects an mRNA which, when translated in vitro, produces an immunoprecipitable 77-kDa protein. When the library was rescreened using the 2.0-kilobase clone as a probe, three full-length clones were isolated, and the complete nucleotide sequence of one 2,183-base pair insert was determined. The deduced protein sequence contains an 18-amino acid signal sequence and a mature protein sequence of 663 amino acids with a calculated Mr of 73,436. The sequence was used to search the National Biomedical Research Foundation (NBRF) protein database, revealing significant similarity to the vertebrate transferrins, a family of 80-kDa glycoproteins which transport and sequester iron in the blood and other body fluids. A multiple sequence alignament shows the greatest areas of similarity to be around the two iron binding sites, although the insect protein seems to contain only one such functional site. Moreover, 23 of the 24 cysteine residues in the insect protein occupy identical positions as compared with the other transferrins, indicating a similar overall tertiary structure. Comparison of the two halves of the insect sequence indicates that the protein may have arisen as a result of gene duplication. The similarity of the M. sexta sequence to the vertebrate transferrins may provide important clues to transferrin evolution. 相似文献
67.
Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin 总被引:14,自引:2,他引:12
P. I. Payne L. M. Holt C. N. Law 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1981,60(4):229-236
Summary The high-molecular-weight (HMW) subunits of glutenin from about 185 varieties were fractionated by sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE). About 20 different, major subunits were distinguished by this technique although
each variety contained, with only a few exceptions, between 3 and 5 subunits. Further inter-varietal substitution lines to
those already described (Payne et al. 1980) were analysed and the results indicate that all the HMW subunits are controlled
by the homoeologous group 1 chromosomes. All hexaploid varieties studied except ‘NapHal’ contained two major subunits controlled
by chromosome 1D. Their genes were shown to be tightly linked genetically for only four different types of banding patterns
were observed. The nominal molecular weights determined after fractionation in 10% polyacrylamide gels were between 110,000
and 115,000 for the larger of the two subunits and between 82,000 and 84,000 for the smaller. One quarter of the varieties
contained only one major HMW subunit controlled by chromosome 1B whereas the rest had two. The chromosome 1B subunits were
the most varied and nine different banding patterns were detected. All the subunits had mobilities which were intermediate
between those of the two chromosome 1D-controlled subunits. Only two types of HMW subunit controlled by chromosome 1A were
detected in all the varieties examined; a single variety never contained both of these subunits and 40% of varieties contained
neither. The chromosome 1A-controlled subunits had slightly slower mobilities in 10% gels than the largest HMW subunit controlled
by chromosome 1D. About 100 single grains were analysed from each of five different crosses of the type (F1 of variety A × variety B) × variety C. The results indicate that the genes on chromosome 1B which control the synthesis of
subunits 6, 7, 13, 14 and 17 are allelic, as are the genes of the chromosome 1A-controlled subunits, 1 and 2. 相似文献
68.
Douglas L. Currell Bernice Law Michael Stevens Paul Murata Carmela Ioppolo Filippo Martini 《Biochemical and biophysical research communications》1981,102(1):348-354
Human hemoglobin reacts with 4-Isothiocyanatobenzene sulfonic acid at the four amino groups of the N-terminal valines. The modified protein shows a decreased oxygen affinity over a wide pH range, a reduced alkaline Bohr effect, decreased co-operativity, and a reduced effect of inositol hexasulfate on the oxygen affinity. 相似文献
69.
70.
Control of Endosperm Proteins in TRITICUM AESTIVUM (Var. Chinese Spring) and AEGILOPS UMBELLULATA by Homoeologous Group 1 Chromosomes 总被引:1,自引:0,他引:1
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The genetic control of major wheat endosperm proteins by homoeologous group 1 chromosomes has been studied by two-dimensional polyacrylamide gel electrophoresis. The control of at least 15 distinct protein subunits or groups of protein subunits has been allocated to chromosomes 1A, 1B and 1D of Chinese Spring wheat from the analysis of grains of aneuploid genotypes. In addition, six protein subunits have been shown to be controlled by chromosome 1Cu of the related species, Aegilops umbellulata, from studies of wheat lines carrying disomic substitutions of 1Cu chromosomes. On the basis of protein subunit patterns, chromosome 1Cu is more closely related to chromosome 1D of wheat than to chromosomes 1A or 1B. 相似文献