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81.
W P Schrader C A West N L Strominger 《The journal of histochemistry and cytochemistry》1987,35(4):443-451
Adenosine deaminase and adenosine deaminase complexing protein have been localized in rabbit brain. Brains fixed in paraformaldehyde or in Clarke's solution were blocked coronally. Blocks from brains fixed in paraformaldehyde were either frozen in liquid nitrogen or embedded in paraffin. Tissue fixed in Clarke's solution was embedded in paraffin. Sections from each block were stained by the peroxidase-antiperoxidase method for adenosine deaminase or complexing protein using affinity-purified goat antibodies. Adenosine deaminase and complexing protein did not co-localize. Adenosine deaminase was detected in oligodendroglia and in endothelial cells lining blood vessels, whereas complexing protein was concentrated in neurons. The subcellular location and appearance of the peroxidase reaction product associated with individual cells was also quite distinctive. The cell bodies of adenosine deaminase-positive oligodendroglia were filled with intense deposits of peroxidase reaction product. In contrast to oligodendroglia, the reaction product associated with most neurons stained for complexing protein was concentrated in granular-appearing cytoplasmic deposits. In some instances, these deposits were clustered about the nuclear membrane. Staining of neurons in the granular layer of cerebellum was an exception. Granule cells were lightly outlined by peroxidase reaction product. Cerebellar islands, also referred to as glomeruli, were stained an intense uniform brown. These results raise the possibility that oligodendroglia and blood vessel endothelia, through the action of adenosine deaminase, might play a role in controlling the concentration of extracellular adenosine in brain. They do not, however, support the suggestion that complexing protein aids in adenosine metabolism by positioning adenosine deaminase on the plasma membrane. 相似文献
82.
The class II major histocompatibility complex antigens are cell-surface heterodimers consisting of an a and a chain. Cosmid cloning has shown that the three families of clas II antigens, DR, DQ, and DP, are encoded within the HLA-D region of chromosome 6 as a series of discrete gene clusters. The DP cluster contains two pairs of a and genes, one of which encodes the biochemically-defined DP antigen. In order to assess whether the other two genes, SXa and SX, are also expressed, potential coding regions have been subcloned and sequenced. The SX3 gene is shown to contain region closely homologous to all six exons of DP. A 1 bp deletion in the 2 exon, also observed for the SX4 allele, causes a translation frameshift, suggesting that SX is a pseudogene. However, all the other exons, as well as their splice sites and the putative promoter region, appear to be intact. 相似文献
83.
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85.
An unusually high-titer human anti-Epstein Barr virus (EBV) serum and its use in the study of EBV-specific proteins synthesized in vitro and in vivo 总被引:11,自引:0,他引:11
C M Edson L K Cohen W Henle J L Strominger 《Journal of immunology (Baltimore, Md. : 1950)》1983,130(2):919-924
Sera from a patient with a chronic Epstein Barr virus (EBV) infection contained unusually high anti-EBV antibody titers (1:2560 to 1:10,240 for EA(D) and 1:5,120 to 1:40,960 for VCA). One of these serum samples was shown by immunoprecipitation to recognize at least 11 EBV-specific proteins from virus producer cells labeled in vivo and 10 EBV-specific proteins from in vitro translations of producer cell mRNA. Six of the in vivo labeled proteins (135,000, 89,000, 50,000 to 55,000 doublet, 46,000, and 34,000 daltons) are "early" by their resistance to phosphonoacetic acid, and five (350,000, 220,000, 160,000, 140,000, and 85,000 daltons) are "late" membrane and capsid proteins. The EBV-specific proteins immunoprecipitated from in vitro translations had molecular masses of 150,000, 140,000, 115,000, 52,000, 50,000, 45,000, 34,000, 29,000, 17,000, and 15,000. Subcellular fractionation studies of cells labeled in vivo revealed that the 135,000-dalton protein and part of the 50,000 to 55,000 dalton protein doublet were found in both the nuclear and the cytoplasmic fractions, and thus are good candidates to be components of the EA(D) diffuse-type immunofluorescence observed with many EA-positive sera. 相似文献
86.
M S Krangel W E Biddison J L Strominger 《Journal of immunology (Baltimore, Md. : 1950)》1983,130(4):1856-1862
Multiple amino acid sequence differences distinguish individual HLA antigens. Those residues important in immune recognition events have not been defined. Recent studies have identified HLA-A2 structural variants that, although serologically indistinguishable from other HLA-A2 antigens, are recognized poorly, if at all, by HLA-A2-restricted, influenza virus-immune, or HLA-A2-specific alloimmune CTL. In this study we utilize double-label tryptic peptide comparisons performed by both reverse-phase HPLC and cation exchange chromatography, in conjunction with conventional and microsequence analysis, to characterize the HLA-A2 heavy chains derived from variant DK1. We detect a single tryptic peptide that distinguishes DK1 HLA-A2 from the predominant HLA-A2 heavy chain species. This peptide spans residues 147 to 157 in the second heavy chain domain, and carries substitutions at positions 149, 152, and 156. Residues in this segment of the polypeptide are also altered in another HLA-A2 variant, as well as one H-2Kb mutant. Thus, this segment appears to be critical in forming determinants important in CTL recognition of class I antigens in general. On the basis of these and other results, we suggest that in contrast to recognition by alloantibodies, a discrete region of class I antigens may be crucial for CTL recognition. 相似文献
87.
It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily. 相似文献
88.
A protein with a molecular weight of 60,000 (60K) constitutes approximately 20% of the envelope protein of Azotobacter vinelandii. This protein was removed from cells and purified from other proteins by a simple washing procedure that had no effect on cell viability. Anti-60K antiserum blocked azotophage A-22 adsorption and agglutinated both vegetative cells and cysts; ferritin-conjugated antibodies used in indirect labeling studies bound uniformly to the periphery of vegetative cells. We conclude that 60K is present on the outer surface of vegetative cells and cysts. The protein is similar to the surface protein alpha of Acinetobacter ssp. in molecular weight, reassociation characteristics, and high ratio of acidic to basic amino acids. We propose that 60K forms a layer external to the outer membrane of A. vinelandii. 相似文献
89.
Sedimentation characteristics of newly synthesized Epstein-Barr viral DNA in superinfected cells. 总被引:6,自引:5,他引:1 下载免费PDF全文
Replicating Epstein-Barr virus (EBV) DNA molecules isolated from superinfected Raji cells were shown to consist of 80S to 65S and 58S (mature) molecules Pulse-chase experiments showed that radioactive label of DNAS molecules with the larger sedimentation coefficients was partially chased into 58S labeled forms. Formation of large concatemers of viral DNA could not be detected at any time after superinfection. The continuous presence of the 65S viral DNA intermediate throughout the replicative cycle combined with the observed inhibition of EBV DNA synthesis by addition of nontoxic levels of ethidium bromide to the superinfected cell culture led us to propose that EBV replication proceeds via a relaxed circular DNA intermediate. 相似文献
90.