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31.
32.
Henning Juhl 《Molecular and cellular biochemistry》1981,35(2):77-92
Summary Glycogen synthase I from human polymorphonuclear leukocytes was phosphorylated with cAMP dependent protein kinase, synthase
kinase or phosvitin kinase prepared from these cells. Limited tryptic hydrolysis released four phosphopeptides (t-A, t-B,
t-C, t-D). Subsequent α-chymotryptic hydrolysis of the trypsin resistant core released three phosphopeptides. (c-A, c-B, c-C).
The kinetic changes of glycogen synthase were compared with the phosphorylation of the peptides. Equivalent kinetic changes
(Kc=0.2–0.3 mM Glc-6-P) were obtained when 1 Pi/subunit was introduced by cAMP dependent protein kinase, 0.5 Pi/subunit by synthase kinase and 0.8 Pi/subunit by both kinases. Initially, cAMP dependent protein kinase phosphorylated peptides c-A and t-C in parallel and somewhat
later also t-B, whereas synthase kinase initially phosphorylated only c-A. The ultimate effect of the two kinases on c-A was
additive. It was concluded that the initial kinetic changes were dependent on phosphorylation of c-A, which contained two
sites, one for each kinase. The same kinetic changes were induced by phosphorylation on each of the sites.
In the subsequent phosphorylation the kinases, separately or together, phosphorylated peptide c-C indicating one non-specific
phosphorylatable site in this peptide. The cAMP dependent protein kinase alone phosphorylated t-C maximally, whereas both
kinases were required for an equal phosphorylation of t-A and t-B. It is suggested that the cAMP dependent protein kinase
phosphorylated t-A and t-C, whereas the data did not allow a similar suggestion for t-B. The kinetic changes occurring during
the later stages of phosphorylation were an increase in Kc for Glc. 6-P to 4–5 mM at 1.85 Pi/subunit and to 20 mM at 3.3 Pi/subunit, but the changes could not be assigned to phosphorylation of any specific peptide. Phosphorylation of the peptides
t-D and c-B were insignificant, but c-B may be phosphorylated under other experimental conditions (25).
The phosvitin kinase phosphorylated glycogen synthase extremely slowly to an extent of 0.8 Pi/subunit, mainly in peptide c-C. Glycogen synthase would appear without physiological importance as substrate for this kinase.
Phosphorylase kinase from rabbit skeletal muscle incorporated 0.7 Pi/subunit, mainly in peptide c-A causing a decrease in RI to 0.3, which upon further incubation remained constant.
The rate of decrease in RI to 0.5 was unaffected by several synthase modifiers, including Glc-6-P, but was inhibited by ADP
and Pi. The rate of phosphorylation by cAMP dependent protein kinase and synthase kinase was diversely affected in different buffers,
however, without affecting the ultimate phosphorylation pattern. 相似文献
33.
Summary Glycogen synthase I in a homogenate of human polymorphonuclear leukocytes was phosphorylated under imitated physiological
conditions utilizing the endogenous protein kinases. At subsequent steps of phosphorylation the32P-labelled synthase was purified and characterized. Limited tryptic hydrolysis of the32P-labelled synthase released four phosphopeptides (t-A, t-B, t-C, t-D) and subsequent chymotrypsinization of the trypsin resistant
core released three phosphopeptides (c-A, c-B, c-C).
One Pi/subunit was incorporated within 8–10 min and 2.2 Pi/subunit within 60 min increasing the Kc for Gle-6-P to 4–6 mM. The initial phosphorylation up to 0.8 Pi/subunit occurred mainly in peptide c-A and a linear relation between ratio of independence (RI) of glycogen synthase in the
interval RI 0.85 to RI 0.05 and phosphorylation of this peptide to 0.5 Pi was observed. Phosphorylation of this peptide is responsible for the decrease in ratio of independence.
From experiments with inhibitors and activators, the initial phosphorylation was found predominantly catalysed by the endogenous
cAMP independent synthase kinase, however, the endogenous cAMP dependent protein kinase and phosphorylase kinase also phosphorylate
endogenous glycogen synthase I to a minor degree. Circumstantial evidence for a Ca-dependent synthase kinase different from
phosphorylase kinase is presented.
The endogenous Gle-6-P dependent glycogen synthase occurring in a homogenate of leukocytes disrupted in the presence of NaF incorporated 1.07 Pi/subunit and Kc for Glc-6-P was increased from 6–8 mM to 20 mM.
From the present and previous experiments [7] a total of 8 major phosphorylatable sites have been defined, one on each of
the peptides t-A, t-B, t-C, c-B and c-C and two on peptide c-A, which in addition may contain a third site for phosphorylase
kinase. Assuming identical subunits, only 13 out of 32 sites are thus covalently modified at maximum phosphorylation. The
operational defined synthase R (Kc for Glc-6-P 0.5 mM) and D (Kc for Glc-6-P 2–8 mM) activities correspond to synthase with about 0.8 Pi and 1.8–2.3 Pi/subunit, respectively. 相似文献
34.
Summary The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h
90) strains of Schizosaccharomyces pombe. The MT region of h
90 comprises three cassette genes: the expression site mat1: 1 and two silent loci, mat2: 2 and mat3: 3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1: 1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2: 2 and mat3: 3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region. 相似文献
35.
Selenium in the central nervous system of rats exposed to 75-Sel-selenomethionine and sodium selenite 总被引:1,自引:0,他引:1
The aim of the present study is to investigate the accumulation and retention of organic and inorganic selenium in the central nervous system (CNS) of the rat. Selenium accumulation was investigated after oral treatment (3.0 mg Se/L drinking water) or ip injection (1.7 mg Se/kg body wt) of rats exposed to 75-Se L-selenomethionine (SeMeth) or sodium selenite (NaSe). Significant higher concentrations were observed after exposure to organic compared to inorganic selenium after oral as well as ip administration. Highest concentrations in both experiments were observed in cerebellum followed by the nearly identical levels in the cerebral hemisphere and spinal cord independent of the chemical form of selenium or the route of administration. The difference in concentrations observed between the different parts of the CNS investigated in each group were, however, not significant. Retention of selenium in the CNS was investigated after a single ip injection (1.7 mg Se/kg body wt) of 75-Se SeMeth or NaSe. In both groups, we observed an initial fast excretion phase followed by a slower excretion phase resembling a first-order reaction. Organic selenium disappeared much slower from all parts of the central nervous system compared to NaSe after a single injection. 相似文献
36.
Nucleotide sequence of 7 S RNA. Homology to Alu DNA and La 4.5 S RNA 总被引:20,自引:0,他引:20
W Y Li R Reddy D Henning P Epstein H Busch 《The Journal of biological chemistry》1982,257(9):5136-5142
7 S RNA, a component of normal higher eukaryotic cells and several oncornaviruses, was shown to be conserved in evolution (Erikson, E., Erikson, R. L., Henry, B., and Pace, N. R. (1973) Virology 53, 40-46). Recently, 7 S RNA was shown to be partially complementary to Alu family DNA sequences (Weiner, A. (1980) Cell 22, 209-218). In the present study the nucleotide sequence of Novikoff hepatoma 7 S RNA was determined to be: (formula, see text) Comparison of 7 S RNA, Alu and B1 family DNA, and La 4.5 S RNA sequences for homologies showed that 1) one-third of 7 S RNA, mainly the 5'-end, was homologous to Alu and B1 family sequences; 2) one 300-nucleotide long Alu family sequence contained two binding sites for 7 S RNA; and 3) the 5'-ends of 7 S RNA and La 4.5 S RNA also had extensive (60%) homologies. A model for the secondary structure of 7 S RNA based on maximal base pairing and preferential nuclease cleavage sites is also presented. 相似文献
37.
38.
The primary nucleotide sequence of U4 RNA 总被引:7,自引:0,他引:7
U4 RNA is one of the "capped" nuclear snRNAs recently found to be precipitable by anti-Sm antibodies as ribonucleoprotein particles. U4 RNA, along with other snRNAs, has been implicated in hnRNA processing, mRNA transport, or both (Lerner, M. R., Boyle, J., Mount, S., Wolin, S., and Steitz, J. A. (1980) Nature 283, 220-224). Since the proteins bound to different snRNAs appear to be the same, the functions of different snRNPs might be dependent on the RNA components. To help understand the function of U4 RNP, the nucleotide sequence of U4 RNA was determined. The sequence is (formula see text) In addition to the modified nucleotides in the "cap," U4 RNA contains Am at position 63 and m6A at position 98. It also exhibited A-C microheterogeneity at position 97. 相似文献
39.
Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ. 总被引:20,自引:9,他引:11 下载免费PDF全文
Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture. 相似文献
40.
E L Lloyd M A Gemmell C B Henning D S Gemmell B J Zabransky 《International journal of radiation biology and related studies in physics, chemistry, and medicine》1979,36(5):467-478
Mammalian cells in culture have been shown here for the first time to be transformed by alpha irradiation. Mouse embryo (C3H 10T1/2) cells were transformed with 5.6 MeV alpha particles from a Tandem Van de Graaff machine. Malignant tumours were induced following inoculation of the transformed cells into syngeneic hosts. Unirradiated control cells failed to produce tumours. The morphology of the transformed foci was similar to that obtained by X-rays and chemicals but different from virally transformed cells. The transformation frequency increased approximately as the cube of the dose to a maximum of about 4 per cent ofthe surviving cells which occurred between 1.5 and 2.5 x 10(7) alpha particles per cm2 (205-342 rad). It appears that alpha particle irradiation may exert a direct effect on the genome of the cell to produce malignancy without any external immunological or hormonal influences. 相似文献