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61.
The functional units of a peptostreptococcal protein L 总被引:4,自引:0,他引:4
Jonathan P. Murphy Clive J. Duggleby Max A. Atkinson † Angus R. Trowern Tony Atkinson Christopher R. Goward 《Molecular microbiology》1994,12(6):911-920
Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin-binding units have high amino acid sequence identity with the albumin-binding units of streptococcal cell-surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units. 相似文献
62.
The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the over-produced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity. 相似文献
63.
Jonathan H. A. Nugent Dugald J. Maclachlan Stephen E. J. Rigby Michael C. W. Evans 《Photosynthesis research》1993,38(3):341-346
Our recent EPR and EXAFS experiments investigating the structure of the oxygen-evolving complex of PS II are discussed. PS II treatments which affect the cofactors calcium and chloride have been used to poise samples in modified forms of the S-states, S1, S2 and S3. X-ray absorption studies indicate a similar overall structure for the manganese complex between treated and native samples although the influence of the treatments and cofactors is observed. Manganese oxidation (or oxidation of a ligand to the manganese cluster) is indicated to occur on each of the transitions S1 S2 and S2 S3 in these modified samples. The cluster appears to contain at least two inequivalent Mn-Mn pairs. In the native samples the Mn-Mn distance is 2.7 Å, but in samples where the calcium site is affected, one of the pairs has a 3.0 Å Mn-Mn distance. The intensity of the 3.3/3.6 Å interaction is reduced on sodium chloride treatment (calcium depletion) perhaps indicating calcium binding close to the manganese cluster. From EPR data we also propose that treatments which affect calcium and chloride binding cause a modification of the native S2 state, slow the reduction of Yz
and allow an S3 EPR signal to be observed following illumination. The origin of the S3 EPR signal, a modified S3 or S2 X where X is an organic radical of unknown charge, is discussed in relation to the results from the EXAFS studies.Abbreviations EPR
electron paramagnetic resonance spectroscopy
- EXAFS
extended X-ray absorption fine structure
- HTG
n-heptyl -d-thioglucoside
- MES
2(N-morpholino)ethanesulfonic acid
- OEC
oxygen evolving complex
- PPBQ
phenyl-1,4-benzoquinone
- PS II
Photosystem II
- Yz
redox active tyrosine 相似文献
64.
Beth J. DiDomenico Nathaniel H. Brown John Lupisella Jonathan R. Greene Michaela Yanko Yigal Koltin 《Molecular genetics and genomics : MGG》1994,242(6):689-698
Morphogenesis in the yeast Saccharomyes cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function. 相似文献
65.
Mortimer M. Civan Jonathan Robbins Simon Broad Enrique Rozengurt David A. Brown 《The Journal of membrane biology》1993,133(1):51-59
Summary Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (I
K) and the M-current (I
M). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E1 (PGE1) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: I
k, a small current through a T-like Ca2+ channel, and no M-current.Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22±7%. In contrast to the stimulation of I
k observed with cardiac cells, PKC activation produced only a small inhibition of I
k, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of I
k in neuroblastoma cells.Supported in part by research grants from the National Institutes of Health (DK-40145 and EY-08343) and from the U.K. Medical Research Council.We thank Dr. Peter J. Parker for his generous gift of PKC, and Yvonne Vallis for her skillful assistance with the cultures and harvesting of the NG108-15 transfected cells. 相似文献
66.
The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage. 总被引:3,自引:3,他引:0 下载免费PDF全文
The synthesis is described of oligoribonucleotides containing the modified nucleoside O6-methylguanosine. Solid-phase oligoribonucleotide assembly was carried out by use of 2'-silyl-protected nucleoside phosphoramidites, a new O6-methylguanosine-containing synthon and a mild deprotection method. The O6-methylguanosine-modified oligonucleotides were used in the study of the role of conserved residues G5, G8 and G12 in hammerhead ribozyme cleavage. Hammerheads thus substituted at any of these positions showed an approximately 75-fold reduction in kcat whereas Km was unaffected. Hammerheads with modifications at G5 or G8 showed a significant reduction in magnesium binding affinity whereas modification at G12 had no effect. The results show that the three conserved G residues play crucial but different role sin hammerhead cleavage. 相似文献
67.
Jonathan R. Sporn Michael T. Ergin Gerald R. Robbins Ritchard G. Cable Herbert Silver Bijay Mukherji 《Cancer immunology, immunotherapy : CII》1993,37(3):175-180
A clinical trial of adoptive immunotherapy was carried out with peripheral blood lymphocytes (PBL), cocultured in vitro with autologous tumor cells and interieukin-2 (IL-2), in 14 patients with advanced melanoma. PBL from these patients were cocultured with irradiated autologous tumor cells for 7 days, which was followed by expansion in IL-2-containing medium. These lymphocytes were returned to the patient along with intravenous IL-2 at doses up to 2×106 IU m–2 day–1. A dose of 300 mg/m2 cyclophosphamide was administered to each patient intravenously 4 days prior to each treatment. Following coculture, the lymphocytes were primarily CD3+ T cells and they expressed varied degrees of cytotoxicity against autologous melanoma cells. In 9 patients the activated cells were al least 80% CD4+ and in 2 cases they were mostly CD8+. Some of the activated cells exhibited suppressor or helper activity in a functional regulatory coculture assay. No major therapeutic response was observed in this study. Minor responses were observed in 2 patients. Toxicities were those expected from the IL-2 dose administered.This work has been supported by an American Cancer Society Institutional Research Grant (ACS-IRG 91-230), by the University of Connecticut Clinical Research Center (grant 0021), and by the Hartford Hospital Research Fund (grant 1017-20-018). Dr. Sporn is a recipient of American Cancer Society Clinical Oncology Career Development Award 90-230 相似文献
68.
Jonathan C. Fox Judith L. Swain 《In vitro cellular & developmental biology. Animal》1993,29(3):228-230
Summary Fibroblast growth factors (FGFs) are potent inhibitors of myogenic differentiation. The recent observation that the endogenous
expression of acidic and basic FGF by myogenic cells decreases coordinately with differentiation suggests a regulatory role
for these growth factors in myogenesis. Inasmuch as other proteins known to influence myogenesis (e.g., MyoD1 and myogenin)
activate their own expression as well as the expression of other members of their family, we hypothesized that the FGFs might
be capable of similar autoregulation. We examined the effect of exogenously supplied FGF on the abundance of the mRNAs encoding
acidic and basic FGF in Sol 8 myoblasts, and demonstrate that either acidic or basic FGF stimulate, through paracrine mechanisms,
the accumulation of the mRNAs encoding both of these FGFs. Thus FGFs can auto- and transregulate their own expression in a
manner analogous to that observed for the myogenic determination proteins. In addition, similar to that previously observed
for MyoD1, both acidic and basic FGF suppress myogenin expression in myoblasts. These results suggest two mechanisms whereby
endogenously produced FGFs participate in the maintenance of the undifferentiated state of myogenic cells. These data provide
support for paracrine, and suggest potential autocrine, roles for FGFs in the regulation of myogenic differentiation. 相似文献
69.
Summary DNA fingerprints generated by the Jeffreys' probes, 33.6 and 33.15, indicated the presence of minisatellite-like sequences
in the red clover genome. The fingerprints generated by probe 33.6 gave less background and fewer but better defined bands
than those obtained with probe 33.15. Assay of a regenerative somaclonal variant (F49R) by DNA fingerprinting with probe 33.6
detected mutation that was unlinked to the regenerative trait. The fingerprints obtained under the applied conditions also
demonstrated genetic stability of consecutive generations of the regenerants in tissue culture. DNA fingerprints of F1 plants revealed that each polymorphic band was inherited from either one or the other parent. Both probes distinguished individual-specific
genotypes in seven cultivars of red clover. Greater variability in DNA fingerprints was detected between (V=0.899) than within
(0.417≤V≤0.548) cultivars. 相似文献