全文获取类型
收费全文 | 12415篇 |
免费 | 990篇 |
国内免费 | 274篇 |
专业分类
13679篇 |
出版年
2024年 | 16篇 |
2023年 | 87篇 |
2022年 | 236篇 |
2021年 | 368篇 |
2020年 | 230篇 |
2019年 | 298篇 |
2018年 | 347篇 |
2017年 | 289篇 |
2016年 | 403篇 |
2015年 | 683篇 |
2014年 | 728篇 |
2013年 | 951篇 |
2012年 | 1162篇 |
2011年 | 1044篇 |
2010年 | 708篇 |
2009年 | 598篇 |
2008年 | 766篇 |
2007年 | 810篇 |
2006年 | 744篇 |
2005年 | 607篇 |
2004年 | 603篇 |
2003年 | 504篇 |
2002年 | 457篇 |
2001年 | 94篇 |
2000年 | 73篇 |
1999年 | 111篇 |
1998年 | 112篇 |
1997年 | 92篇 |
1996年 | 68篇 |
1995年 | 73篇 |
1994年 | 40篇 |
1993年 | 64篇 |
1992年 | 39篇 |
1991年 | 34篇 |
1990年 | 24篇 |
1989年 | 18篇 |
1988年 | 20篇 |
1987年 | 10篇 |
1986年 | 11篇 |
1985年 | 19篇 |
1984年 | 16篇 |
1983年 | 16篇 |
1982年 | 18篇 |
1981年 | 12篇 |
1980年 | 10篇 |
1979年 | 9篇 |
1977年 | 11篇 |
1976年 | 7篇 |
1975年 | 5篇 |
1974年 | 8篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
941.
942.
固定化酶作为一种绿色高效的生物催化剂,其性能远超游离酶。目前酶的固定化技术适用范围仍然较小,酶的研究范围多停留在模型酶阶段,扩大固定化酶的研究范围具有十分重要的意义。金属有机骨架材料(MOFs)作为酶固定化的载体在近些年得到了广泛的探索,但是具有生物功能的酶-MOFs复合材料的许多特性仍有待挖掘。采用仿生矿化的合成方法将5-羟甲基糠醛氧化酶(HMFO)固定到以沸石咪唑酯(ZIF-8)为代表的MOFs材料中,制备得到一种新的生物催化剂HMFO@ZIF-8,扫描电子显微镜表征其形态区别于经典的菱形十二面体。采用考马斯亮蓝法测定蛋白质浓度,计算得到酶的固定化效率达到89. 0%。HMFO@ZIF-8催化5-羟甲基糠醛的转化率达到84. 3%,收率和选择性均高于游离酶。拓展了MOFs固定化酶的研究范围,为研究其他生物大分子复合材料的生物催化剂提供一定的借鉴意义。 相似文献
943.
María de los A. Mesurado María L. Arias Cassará Rosana Misico Alicia Bardón María I. Ybarra Elena Cartagena 《化学与生物多样性》2017,14(5)
The new methylated grindelane diterpenoid, 7β ‐hydroxy‐8(17)‐dehydrogrindelic acid ( 1b ), together with the known 7α ‐hydroxy‐8(17)‐dehydrogrindelic acid ( 2a ), 6‐oxogrindelic acid ( 3a ), 4β ‐hydroxy‐6‐oxo‐19‐norgrindelic ( 4a ), 19‐hydroxygrindelic acid ( 5a ), 18‐hydroxygrindelic acid ( 6a ), 4α ‐carboxygrindelic acid ( 7a ), 17‐hydroxygrindelic acid ( 8a ), 6α ‐hydroxygrindelic acid ( 9a ), 8,17‐bisnor‐8‐oxagrindelic acid ( 10a ), 7α ,8α ‐epoxygrindelic acid ( 11a ), and strictanonic acid ( 12a ) as methyl esters were obtained from an Argentine collection of Grindelia chiloensis (Cornel .) Cabrera . Their structures and relative configurations were established on the basis of spectroscopic analysis. CHC l3 extract from the aerial parts and their pure compounds were evaluated for their antifungal and depigmenting effects. Methyl ester derivative of 10a ( 10b ) exhibited a remarkable mycelial growth inhibition against Botritis cinerea with an IC 50 of 13.5 μg ml?1. While the new grindelane 1b exerted a clear color reduction of the yellow‐orange pigment developed by Fusarium oxysporum against UV ‐induced damage. 相似文献
944.
Whole-genome experimental identification of insertion/deletion polymorphisms of interspersed repeats by a new general approach 总被引:3,自引:1,他引:3
A new experimental technique for genome-wide detection of integration sites of polymorphic retroelements (REs) is described. The technique allows one to reveal the absence of a retroelement in an individual genome provided that this retroelement is present in at least one of several other genomes under comparison. Since quite a number of genomes are compared simultaneously, the search for polymorphic REs insertions is very efficient. The technique includes two whole-genome selective PCR amplifications of sequences flanking REs: one for a particular genome and another one for a mixture of ten different genomes. A subsequent subtractive hybridization of the obtained amplicons with DNA of a particular genome as driver results in isolation of polymorphic insertions. The technique was successfully applied for identification of 41 new polymorphic human AluYa5/Ya8 insertions. Among them, 18 individual Alu elements first sequenced in this work were not found in the available human genome databases. This result suggests that significant part of polymorphic REs were not identified during genome sequencing and remain to be detected and characterized. The proposed method does not depend on preliminary knowledge of evolutionary history of retroelements and can be applied for identification of insertion/deletion polymorphic markers in genomes of different species. 相似文献
945.
Rodolphe Elie Gozlan Eva Zhorsk Emira Cherif Takashi Asaeda John Robert Britton Cha‐Ho Chang To Hong Rafael Miranda Jií Musil Meta Povz Ali Serhan Tarkan Elena Tricarico Teodora Trichkova Hugo Verreycken Andrej Weiperth Andrej Witkowski Lluis Zamora Irene Zweimueller Yahui Zhao Hamid Reza Esmaeili Marine Combe 《Ecology and evolution》2020,10(16):8623-8633
Rapid adaptation to global change can counter vulnerability of species to population declines and extinction. Theoretically, under such circumstances both genetic variation and phenotypic plasticity can maintain population fitness, but empirical support for this is currently limited. Here, we aim to characterize the role of environmental and genetic diversity, and their prior evolutionary history (via haplogroup profiles) in shaping patterns of life history traits during biological invasion. Data were derived from both genetic and life history traits including a morphological analysis of 29 native and invasive populations of topmouth gudgeon Pseudorasbora parva coupled with climatic variables from each location. General additive models were constructed to explain distribution of somatic growth rate (SGR) data across native and invasive ranges, with model selection performed using Akaike's information criteria. Genetic and environmental drivers that structured the life history of populations in their native range were less influential in their invasive populations. For some vertebrates at least, fitness‐related trait shifts do not seem to be dependent on the level of genetic diversity or haplogroup makeup of the initial introduced propagule, nor of the availability of local environmental conditions being similar to those experienced in their native range. As long as local conditions are not beyond the species physiological threshold, its local establishment and invasive potential are likely to be determined by local drivers, such as density‐dependent effects linked to resource availability or to local biotic resistance. 相似文献
946.
Identification and Characterization of a New Serotonergic Recognition Site with High Affinity for 5-Carboxamidotryptamine in Mammalian Brain 总被引:1,自引:0,他引:1
Ma. Elena Castro Tamara Romón Ma. Josefa Castillo Elena del Olmo Angel Pazos Carmen del Arco 《Journal of neurochemistry》1997,69(5):2123-2131
Abstract: We analyzed the existence of an additional serotonin (5-HT) receptor subtype, sensitive to 5-carboxamidotryptamine, in the mammalian brain. Radioligand binding studies with [3 H]5-HT were carried out in rat, guinea pig, and human brain membranes, in the presence of unlabeled drugs to mask the binding to all known 5-HT receptors, with the exception of 5-HT1E sites. Under these conditions, unlabeled 5-carboxamidotryptamine still showed a biphasic competition curve with a nanomolar affinity component. Saturation studies with 5-[3 H]carboxamidotryptamine were carried out in the presence of (±)-8-hydroxy-2-(di- n -propylamino)tetralin, mesulergine, and ergotamine, to mask the binding to all receptors known to be labeled by 5-carboxamidotryptamine. These studies showed the existence in cortex and hippocampus from guinea pig and human brain of a remaining binding site with high affinity ( pK D = 7.8–8.1) and a unique pharmacological profile. 5-HT and 5-carboxamidotryptamine showed nanomolar affinity, whereas 5-methoxytryptamine recognized this binding site with intermediate affinity. Other drugs exhibited low or very low potency in inhibiting this binding. The addition of 5'-guanylylimidodiphosphate significantly reduced the number of binding sites labeled by 5-[3 H]carboxamidotryptamine, in the presence of the masking drugs described above, indicating the interaction with a GTP-binding protein. Preliminary autoradiographic studies in human brain appear to indicate that this 5-HT binding site is present in areas such as the globus pallidus, neocortex, and hippocampus, among others. 相似文献
947.
Naeela Qureshi Harbans Bariana Kerrie Forrest Matthew Hayden Beat Keller Thomas Wicker Justin Faris Elena Salina Urmil Bansal 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2017,130(3):495-504
Key message
Fine mapping of Yr47 and Lr52 in chromosome arm 5BS of wheat identified close linkage of the marker sun180 to both genes and its robustness for marker-assisted selection was demonstrated.Abstract
The widely effective and genetically linked rust resistance genes Yr47 and Lr52 have previously been mapped in the short arm of chromosome 5B in two F3 populations (Aus28183/Aus27229 and Aus28187/Aus27229). The Aus28183/Aus27229 F3 population was advanced to generate an F6 recombinant inbred line (RIL) population to identify markers closely linked with Yr47 and Lr52. Diverse genomic resources including flow-sorted chromosome survey sequence contigs representing the orthologous region in Brachypodium distachyon, the physical map of chromosome arm 5BS, expressed sequence tags (ESTs) located in the 5BS6-0.81-1.00 deletion bin and resistance gene analog contigs of chromosome arm 5BS were used to develop markers to saturate the target region. Selective genotyping was also performed using the iSelect 90 K Infinium wheat SNP assay. A set of SSR, STS, gene-based and SNP markers were developed and genotyped on the Aus28183/Aus27229 RIL population. Yr47 and Lr52 are genetically distinct genes that mapped 0.4 cM apart in the RIL population. The SSR marker sun180 co-segregated with Lr52 and mapped 0.4 cM distal to Yr47. In a high resolution mapping population of 600 F2 genotypes Yr47 and Lr52 mapped 0.2 cM apart and marker sun180 was placed 0.4 cM distal to Lr52. The amplification of a different sun180 amplicon (195 bp) than that linked with Yr47 and Lr52 (200 bp) in 204 diverse wheat genotypes demonstrated its robustness for marker-assisted selection of these genes.948.
Mulet M Gomila M Lemaitre B Lalucat J García-Valdés E 《Systematic and applied microbiology》2012,35(3):145-149
An entomopathogenic, Gram-negative bacterium isolated from a female specimen of the fruit fly Drosophila melanogaster was taxonomically characterised. Strain L48(T) was strictly aerobic, non-fermentative, oxidase and catalase positive, rod-shaped, and motile due to a polar inserted flagellum. Phylogenetic analysis of the 16S rRNA gene and three other housekeeping genes placed strain L48 (T) in the Pseudomonas putida phylogenetic group. DNA-DNA hybridisation studies together with phenotypic metabolic tests and MALDI-TOF MS analysis justified the proposal of strain L48(T) as a representative of a novel species, for which the name Pseudomonas entomophila sp. nov. is proposed. The type strain is deposited in culture collections under accession numbers CCUG 61470(T) and CECT 7985(T). 相似文献
949.
Vladislav B. Bergo Oleg A. Sineshchekov Joel M. Kralj Ranga Partha Elena N. Spudich Kenneth J. Rothschild John L. Spudich 《The Journal of biological chemistry》2009,284(5):2836-2843
Proteorhodopsins (PRs), photoactive retinylidene membrane proteins
ubiquitous in marine eubacteria, exhibit light-driven proton transport
activity similar to that of the well studied bacteriorhodopsin from halophilic
archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved
histidine located near the photoactive site of the protein. Time-resolved
Fourier transform IR difference spectroscopy combined with visible absorption
spectroscopy, isotope labeling, and electrical measurements of light-induced
charge movements reveal participation of His-75 in the proton translocation
mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the
structure of the photoactive site and resulted in significantly shifted
visible absorption spectra. In contrast, His-75 substitution with a positively
charged Arg did not shift the visible absorption spectrum of PR. The mutation
to Arg also blocks the light-induced proton transfer from the Schiff base to
its counterion Asp-97 during the photocycle and the acid-induced protonation
of Asp-97 in the dark state of the protein. Isotope labeling of histidine
revealed that His-75 undergoes deprotonation during the photocycle in the
proton-pumping (high pH) form of PR, a reaction further supported by results
from H75E. Finally, all His-75 mutations greatly affect charge movements
within the PR and shift its pH dependence to acidic values. A model of the
proteorhodopsin proton transport process is proposed as follows: (i) in the
dark state His-75 is positively charged (protonated) over a wide pH range and
interacts directly with the Schiff base counterion Asp-97; and (ii)
photoisomerization-induced transfer of the Schiff base proton to the Asp-97
counterion disrupts its interaction with His-75 and triggers a histidine
deprotonation.A variety of unicellular microorganisms contain primary proton pumps that
convert solar energy into a transmembrane electrochemical proton gradient,
which is subsequently used by membrane ATP synthases to generate chemical
energy. Well known examples of such pumps are the haloarchaeal rhodopsins,
photoactive, seven-helix membrane proteins, which include the well studied
proton pump bacteriorhodopsin
(BR)4 from
Halobacterium salinarum and BR homologs in other haloarchaea.
Recently, a much larger new family of light-driven proton pumps, the
proteorhodopsins (PRs), was identified in marine proteobacteria throughout the
oceans
(1–3).
Despite the diverse properties of PRs, including different visible absorption
maxima and photocycle rates
(4–6),
they all share with BR several key conserved residues as well as an
all-trans-retinylidene chromophore in their unphotolyzed state, which
is covalently bound to transmembrane helix G via a protonated Schiff base
linkage.Many of the molecular events that occur in PRs following light activation
are similar to those of BR, including an initial ultrafast
all-trans→13-cis-retinal isomerization, which triggers
a sequence of protein conformational changes, including several intramolecular
proton transfer reactions. The two key carboxylate groups involved in proton
pumping in helix C of BR are conserved in PRs, and in the first found and most
commonly studied PR, the Monterey Bay variant eBAC31A08, also known as
green-absorbing proteorhodopsin (GPR), the helix C residues Asp-97 and Glu-108
undergo protonation changes during the photocycle similar to those of the
homologous carboxylate residues in BR. Initial FTIR studies on GPR identified
the role of Asp-97 as the Schiff base counterion and proton acceptor during
Schiff base deprotonation and concomitant M formation and Glu-108 as the
proton donor that reprotonates the Schiff base during N formation
(7,
8). Studies of other variants
indicate these roles of the two carboxylic acid residues are general in the
proteorhodopsin
family.5One major difference between BR and the PRs is the presence of a highly
conserved histidine residue at position 75, near the middle of transmembrane
helix B in the latter pigments. The His-75 homolog is not present in BR nor
thus far found in other microbial rhodopsins
(9). The proximity of His-75 to
the protein active site and specifically to the Schiff base counterion Asp-97
inferred from the x-ray crystal structure of BR suggests its involvement in
spectral tuning of the visible absorption
(10) and potentially PR
photochemical reactions. Because the pKa of histidine in
solution is close to neutral pH
(11), its imidazole group
often plays a major role in intramolecular proton transfers in enzymes,
including NADPH oxidase (12),
alcohol dehydrogenase (13),
carbonic anhydrase II (14),
and serine proteases (15).In this study we have used a combination of time-resolved FTIR difference
spectroscopy, visible absorption spectroscopy, isotope labeling, kinetic
charge displacement measurements, and site-directed mutagenesis to study the
role of His-75 in GPR. We report evidence that protonated His-75 interacts
directly with Asp-97 in the unphotolyzed protein and during the photocycle
undergoes a deprotonation in response to the protonation of Asp-97. 相似文献
950.
Elena Kudryashova David Heisler Andrew Zywiec Dmitri S. Kudryashov 《Molecular microbiology》2014,92(5):1056-1071
MARTX (multifunctional autoprocessing repeats‐in‐toxin) family toxins are produced by Vibrio cholerae, Vibrio vulnificus, Aeromonas hydrophila and other Gram‐negative bacteria. Effector domains of MARTX toxins cross the cytoplasmic membrane of a host cell through a putative pore formed by the toxin's glycine‐rich repeats. The structure of the pore is unknown and the translocation mechanism of the effector domains is poorly understood. We examined the thermodynamic stability of the effector domains of V. cholerae and A. hydrophila MARTX toxins to elucidate the mechanism of their translocation. We found that all but one domain in each toxin are thermodynamically unstable and several acquire a molten globule state near human physiological temperatures. Fusion of the most stable cysteine protease domain to the adjacent effector domain reduces its thermodynamic stability ~ 1.4‐fold (from 21.8 to 16.1 kJ mol?1). Precipitation of several individual domains due to thermal denaturation is reduced upon their fusion into multi‐domain constructs. We speculate that low thermostability of the MARTX effector domains correlates with that of many other membrane‐penetrating toxins and implies their unfolding for cell entry. This study extends the list of thermolabile bacterial toxins, suggesting that this quality is essential and could be susceptible for selective targeting of pathogenic toxins. 相似文献