全文获取类型
收费全文 | 12429篇 |
免费 | 953篇 |
国内免费 | 6篇 |
专业分类
13388篇 |
出版年
2024年 | 15篇 |
2023年 | 74篇 |
2022年 | 213篇 |
2021年 | 353篇 |
2020年 | 199篇 |
2019年 | 247篇 |
2018年 | 299篇 |
2017年 | 246篇 |
2016年 | 491篇 |
2015年 | 772篇 |
2014年 | 836篇 |
2013年 | 866篇 |
2012年 | 1238篇 |
2011年 | 1066篇 |
2010年 | 641篇 |
2009年 | 603篇 |
2008年 | 824篇 |
2007年 | 680篇 |
2006年 | 631篇 |
2005年 | 507篇 |
2004年 | 478篇 |
2003年 | 431篇 |
2002年 | 385篇 |
2001年 | 179篇 |
2000年 | 153篇 |
1999年 | 150篇 |
1998年 | 98篇 |
1997年 | 57篇 |
1996年 | 42篇 |
1995年 | 48篇 |
1994年 | 43篇 |
1993年 | 44篇 |
1992年 | 49篇 |
1991年 | 42篇 |
1990年 | 28篇 |
1989年 | 32篇 |
1988年 | 20篇 |
1987年 | 13篇 |
1986年 | 19篇 |
1985年 | 15篇 |
1984年 | 19篇 |
1983年 | 27篇 |
1982年 | 12篇 |
1981年 | 19篇 |
1980年 | 15篇 |
1979年 | 12篇 |
1978年 | 17篇 |
1977年 | 14篇 |
1976年 | 13篇 |
1974年 | 15篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
51.
R. Ashton Lavoie Jeffrey T. Zugates Andrew T. Cheeseman Matt A. Teten Srivatsan Ramesh Julia M. Freeman Summer Swango Jeremy Fitzpatrick Amod Joshi Bradley Hollers Zufan Debebe Tyler K. Lindgren Amber N. Kozak Vinay K. Kondeti Mary K. Bright Eric J. Yearley Alexander Tracy Jacob A. Irwin Michael Guerrero 《Biotechnology and bioengineering》2023,120(10):2953-2968
Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%. 相似文献
52.
Julia Schollmeyer Saskia Waldburger Kendra Njo Heba Yehia Anke Kurreck Peter Neubauer Sebastian L. Riedel 《Biotechnology and bioengineering》2023,120(11):3322-3334
Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L−1 were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg−1 (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli. 相似文献
53.
So Young Jeong Paul W. Gabrielson Jeffery R. Hughey Andrew S. Hoey Tae Oh Cho Muhammad A. Abdul Wahab Guillermo Diaz-Pulido 《Journal of phycology》2023,59(6):1179-1201
Porolithon is one of the most ecologically important genera of tropical and subtropical crustose (non-geniculate) coralline algae growing abundantly along the shallow margins of coral reefs and functioning to cement reef frameworks. Thalli of branched, fruticose Porolithon specimens from the Indo-Pacific Ocean traditionally have been called P. gardineri, while massive, columnar forms have been called P. craspedium. Sequence comparisons of the rbcL gene both from type specimens of P. gardineri and P. craspedium and from field-collected specimens demonstrate that neither species is present in east Australia and instead resolve into four unique genetic lineages. Porolithon howensis sp. nov. forms columnar protuberances and loosely attached margins and occurs predominantly at Lord Howe Island; P. lobulatum sp. nov. has fruticose to clavate forms and free margins that are lobed and occurs in the Coral Sea and on the Great Barrier Reef (GBR); P. parvulum sp. nov. has short (<2 cm), unbranched protuberances and attached margins and is restricted to the central and southern GBR; and P. pinnaculum sp. nov. has a mountain-like, columnar morphology and occurs on oceanic Coral Sea reefs. A rbcL gene sequence of the isotype of P. castellum demonstrates it is a different species from other columnar species. In addition to the diagnostic rbcL and psbA marker sequences, the four new species may be distinguished by a combination of features including thallus growth form, margin shape (attached or unattached), and medullary system (coaxial or plumose). Porolithon species, because of their ecological importance and sensitivity to ocean acidification, need urgent documentation of their taxonomic diversity. 相似文献
54.
55.
Cosmo G. MacKenzie Julia B. MacKenzie Oscar K. Reiss 《The Journal of cell biology》1962,14(2):269-279
A cell has been isolated from explanted rabbit liver which contains, during all phases of its growth in culture, hundreds of lipid-rich particles with a distinct limiting membrane. The cell grows logarithmically with a generation time of 19 to 20 hours and during mitosis the particles are distributed between the daughter cells. Associated with the particles is the high total lipid content of the rabbit liver cell as compared with a rat liver cell, which contains few, if any, lipid-rich particles. This difference in lipid content between the two cells is due primarily to an increase in the triglyceride fraction, in contradistinction to small differences in the polar lipid and sterol ester fractions. The lipid-rich particles have been isolated and found to contain 90 per cent triglyceride on a dry weight basis. The "genetic" factors responsible for the high concentration of lipid-rich particles and triglycerides in the rabbit liver cell require for their full expression one or more factors which are present in much higher effective concentrations in rabbit serum than in horse serum. The hypothesis is advanced that the lipid-rich particles represent a normal state of the non-structural cell lipid. A procedure is described for the quantitative isolation of the lipid of cultured cells. 相似文献
56.
Julia C. McNaughton Craig J. Marshall Judith E. Broom Gillian Hughes Wyn A. Jones Peter A. Stockwell George B. Petersen 《Journal of molecular evolution》1995,40(2):127-135
A THE-1 sequence in intron 7 of the human dystrophin gene has been found to represent a new subfamily of THE-1 elements. The sequence is closely related to the MstII family of repetitive sequences and is more like single-copy sequences found in the galago genome than any other THE-1 sequence previously reported. This new THE-1 sequence has been compared with two other complete THE-1 sequences and three related long-terminal repeat elements that we have previously found in intron 7 of the dystrophin gene, and with members of the same family from elsewhere in the primate genome. Parsimony and deletion analysis show that the cluster of THE-1 sequences in intron 7 of the dystrophin gene has arisen from at least three individual insertion events, rather than from the insertion and duplication of a single progenitor sequence.
Correspondence to: G.B. Petersen 相似文献
57.
To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch-type shake flask cultures, spore mutants' ability to produce subtilisin can be well pronounced in fed-batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed-batch culture as well as enable us to use very low dilution rates (<0.1 h(-1)) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h(-1) the spollG mutant of Bacillus subtilis DB104 (Deltanpr Deltaapr) (Em(r)) spollG (Bim(r)):: pMK101 (Cm(r)) showed a subtilisin yield about ten times higher than that from wild-type DB104 (Deltanpr Deltaapr)::pMK101 (Cm(r)). (c) 1995 John Wiley & Sons, Inc. 相似文献
58.
Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate. 相似文献
59.
Lash, Julia M., and H. Glenn Bohlen. Time- andorder-dependent changes in functional and NO-mediated dilation during exercise training. J. Appl. Physiol.82(2): 460-468, 1997.Arterial vessel responses to sodiumnitroprusside (SNP) and acetylcholine (ACh) were measured in thespinotrapezius muscle of sedentary (Sed) and treadmill-trained (Tr)rats to determine whether these endothelium-dependent (ACh) and-independent (SNP) mechanisms contribute to thetraining-induced increase in functional vasodilation previouslyobserved. Control and maximal vessel diameters were similar between Sedand Tr. After 8 wk of training, functional dilation (2-, 4-, and 8-Hzcontractions) was enhanced in all orders of vessels studied[terminal feed artery (FA), largest arterioles (1A), andintermediate-sized arterioles (2A)], but responses to SNP wereincreased only in FA. Responses to ACh were not significantly increasedin any vessel order. After 16 wk of training, functional dilation hadregressed in Tr such that only the FA response to 4 Hz wassignificantly elevated relative to Sed. However, the FA and 1Aresponses to SNP were significantly greater in Tr than in Sed, as werethe 1A and 2A responses to ACh. These results show a dissociation offunctional dilation and SNP- or ACh-mediated responses, as well asage-dependent interactions, a time-dependent progression, and vesselorder specificity in the adaptations to training. 相似文献
60.
O. Julia J. Vives-Rego M. Vilamú R. López-Amorós F. Utzet 《Antonie van Leeuwenhoek》1995,67(4):371-375
A kinetic model of colony formation was proposed by Hattori, based on a count of the colonies that appear on a plate in successive short intervals of time. In this model, three parameters (,t
r and N) are defined, which reflect the ability of a bacterium to yield colonies and allow us to described the dynamics of bacterial populations in soil and ofE. coli at different growth phases. In this paper we report a reparametrization of the kinetic model of colony formation, with the aim of facilitating more accurate calculation of andt
r. Moreover, we observed that during the starvation ofE. coli andK. pneumoniae in urine, can be used to assess survival, since this parameter clearly decreases during starvation. Retardation time values (t
r) were similar inE. coli andK. pneumoniae throughout the starvation experimental period. 相似文献