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101.
Improved production and stability ofE. coli recombinants expressing transketolase for large scale biotransformation 总被引:1,自引:0,他引:1
Summary TheE.coli tkt gene has been subcloned into high copy number vectors. In fed batch fermentations up to 4gL–1 of soluble intracellular transketolase was produced representing 43% of the total cell protein. Increased plasmid stability during fed-batch fermentations was obtained by using kanamycin resistant pBGS vectors rather than the ampicillin resistant pUC vectors. Plasmid stability was maintained throughout growth in a complex medium without any selective pressure by incorporating thecer region fromColE1 into the expression construct. 相似文献
102.
Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant. 总被引:4,自引:4,他引:0
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Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis. 相似文献
103.
Amphiphysin II (SH3P9; BIN1), a Member of the Amphiphysin/Rvs Family, Is Concentrated in the Cortical Cytomatrix of Axon Initial Segments and Nodes of Ranvier in Brain and around T Tubules in Skeletal Muscle 总被引:11,自引:2,他引:9
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Margaret Husta Butler Carol David Gian-Carlo Ochoa Zachary Freyberg Laurie Daniell Detlev Grabs Ottavio Cremona Pietro De Camilli 《The Journal of cell biology》1997,137(6):1355-1367
Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homologue of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. We report here the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasmamembrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrinG), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function. 相似文献
104.
Characterization of Myelin Basic Protein Charge Microheterogeneity in Developing Mouse Brain and in the Transgenic Shiverer Mutant 总被引:2,自引:0,他引:2
Anthony E. Palma Phillip Owh Christopher Fredric Carol Readhead Mario A. Moscarello 《Journal of neurochemistry》1997,69(4):1753-1762
Abstract: Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled-down version of the preparative CM-52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline-urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5- and 14-kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C-1), the most cationic isomer, had a molecular weight of 14,140.38 ± 0.79. C-2 consisted of two 14-kDa species, 14,136.37 ± 0.74 and 14,204.45 ± 0.70. Two variants, 14,215.57 ± 0.94 and 18,413.57 ± 0.76, constituted C-3. C-4, C-5, and C-8 (the least cationic isomer) each consisted of both 14- and 18.5-kDa isoforms. During myelinogenesis, the 18.5-kDa isoform appeared first (day 4); the 14-kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5-kDa isoform, but none of the 14-kDa isoform, similar to the 4-day-old mouse. We concluded that the trangenic shiverer was able to initiate myelinogenesis with the 18.5-kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14-kDa isoform. 相似文献
105.
Carol A. Sheppard Peter B. Simpson Alan H. Sharp Frederick C. Nucifora Christopher A. Ross †G. David Lange James T. Russell 《Journal of neurochemistry》1997,68(6):2317-2327
Abstract: We have examined the mechanisms that underlie Ca2+ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca2+ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca2+ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca2+ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP3 R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP3 Rs with Ca2+ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP3 Rs (InsP3 R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca2+ responses and InsP3 R2 immunofluorescence were compared in the same cell, domains of elevated Ca2+ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP3 R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca2+ release sites that support regenerative wave propagation include increased levels of InsP3 R2 expression. 相似文献
106.
Synopsis Two groups of coho salmon,Oncorhynchus kisutch, were raised under identical regimes to test the hypothesis that the group from a stream with lower and less variable temperatures would have a lower and less variable preferred temperature than would the group from a stream with warmer and more variable temperatures. The preferred (modal) temperatures in an electronic shuttlebox of coho salmon young from a relatively cool, groundwater-fed stream were slightly lower and less variable than those of young from a warmer and more heterothermal stream (mean = 9.6° C, range: 6–16° C vs. mean = 11.6° C, range: 7–21° C). However, there was a great deal of variation within and among individual fish. While some genetic variation in thermal preference may exist, the species seems best characterized as tolerant of relatively large temperature fluctuations. 相似文献
107.
Ronald A. Venters Chih-Chin Huang Bennett T. Farmer II Ronald Trolard Leonard D. Spicer Carol A. Fierke 《Journal of biomolecular NMR》1995,5(4):339-344
Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with 2H, 13C and 15N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments, 2H has been incorporated into HCA II in order to decrease the rates of 13C and 1HN T2 relaxation. NMR quantities of protein with essentially complete aliphatic 2H incorporation have been obtained by growth of E. coli in defined media containing D2O, [1,2-13C2, 99%] sodium acetate, and [15N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for 13C and 15N backbone NMR assignment studies, since the 13C and 1HN T2 relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential 2H isotopic shift observed for partially deuterated CHnDm moieties. 相似文献
108.
J Campbell W F Bibb M A Lambert S Eng A G Steigerwalt J Allard C W Moss D J Brenner 《Applied and environmental microbiology》1984,47(2):369-373
Six strains of a new species, Legionella sainthelensi, were isolated from freshwater in areas affected by the volcanic eruptions of Mt. St. Helens in the state of Washington. Strains of L. sainthelensi are culturally and biochemically similar to other legionellae. They grow on buffered charcoal yeast agar but not on media that lack cysteine. They are gram-negative, nonsporeforming, motile rods that are positive in reactions for catalase, oxidase, gelatin liquefaction, and beta-lactamase. They are negative in reactions for urease, hydrolysis of hippurate, reduction of nitrates, fermentation of glucose, and blue-white autofluorescence. Their cell wall fatty acid composition is qualitatively similar to those of other legionellae, with 50 to 62% branched-chain fatty acids. They contain the isobranched-chain 14- and 16-carbon acids and anteisobranched-chain 15- and 17-carbon acids and relatively large amounts of straight-chain 16-carbon acid. All strains of L. sainthelensi contain approximately equal amounts of ubiquinones Q9, Q10, Q11, and Q12, a pattern similar to those of Legionella bozemanii, Legionella dumoffi, and Legionella longbeachae. Serological cross-reactions were observed between L. sainthelensi, both serogroups of L. longbeachae, and Legionella oakridgensis. Three strains of L. sainthelensi were greater than 90% related by DNA hybridization. The type strain of L. sainthelensi, Mt. St. Helens 4, was 36% related to the type strain of L. longbeachae and 3 to 14% related to the other nine described Legionella species. 相似文献
109.
Lynn R. Trusal Albert W. Guzman Carol J. Baker 《In vitro cellular & developmental biology. Plant》1984,20(4):353-364
Summary The pathophysiology of endothelial cells is important to a variety of vascular conditions including coagulation and hemostasis
resulting from clinical frostbite. Use of an in vitro model system demonstrated that when bovine endothelial cells were frozen
at 1°C or 20°C/min and thawed immediately (20°C/min), a variety of ultrastructural alterations occurred. Membraneous structures
were most extensively damaged, with mitochondria the most sensitive organelle. Low amplitude mitochondrial swelling, first
evident at 0°C, progressed to high amplitude swelling by −10°C (frozen). In addition, the rough endoplasmic reticulum was
dilated and formed large vesicles with a homogeneous matrix. Nuclear changes first occurred at −15°C. These included separation
and distortion of the nuclear membrane, changes in chromatin distribution, and disruption of the nucleolus. Scanning electron
microscopy revealed perforated plasma membranes in some cells at −10°C (frozen) and in most cells by −20°C. Cultures frozen
at 20°C/min revealed mostly the same ultrastructural damage noted at 1°C/min except a higher percentage of cells exhibited
alterations. Data from the recovery index and lactic dehydrogenase (LDH) release correlated well with observed ultrastructural
changes. Early swelling of mitochondria and dilation of rough endoplasmic reticulum was not lethal in the absence of freezing.
Increased swelling in cytoplasmic organelles coupled with nuclear alterations at −15°C resulted in a decreased survival rate
and release of significant quantities of LDH by −20°C. No unique morphological changes were temperature specific, but the
total number of cells that displayed alterations increased as temperature decreased.
The views, opinions or findings, or both, contained in this report are those of the authros and should not be construed as
indicative of an official Department of the Army position, policy, or decision unless so designated by other official documentation. 相似文献
110.