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31.
32.
The plasmid pCI6, carrying the attP site of the temperate phage phiU, integrates into the attB site on the chromosome of Rhizobium leguminosarum biovar trifolii strain 4S. The 4 kb EcoRI-HindIII region of pCI6 involved in site-specific integration was subcloned as the attP fragment of phage phiU and sequenced. The attL fragment, one of the new DNA junctions generated from the insertion of pCI6 into the chromosome of the host Rhizobium, was used as a hybridization probe for isolation of the attB fragment of strain 4S. The nucleotide sequence of the 2 kb PstI fragment of strain 4S, which hybridized with the attL fragment, was decided and compared with that of the attP fragment. A 53 bp common sequence was expected to be the core sequence of site-specific integration between phage phiU and strain 4S. One of the ORFs on the attP fragment, which was located adjacent to the core sequence, had structural homology to the integrase family. However, the attB fragment showed high homology with the tRNA genes of Agrobacterium tumefaciens and E. coli. A 47 bp sequence of the 53 bp core sequence overlapped with this tRNA-like sequence. This indicates that the target site of phage phiU integration is the putative tRNA gene on the chromosome of the Rhizobium host. 相似文献
33.
Tomoo Kamiya A-Hon Kown Toshiki Kanemaki Yoichi Matsui Shouji Uetsuji Tadayoshi Okumura Yasuo Kamiyama 《In vitro cellular & developmental biology. Animal》1998,34(2):131-137
Summary The Anaeropack system for cell culture, which was originally designed for the growth of anaerobic bacteria, was used to produce
a hypoxic atmosphere for cultured hepatocytes. We measured changes in the oxygen and carbon dioxide concentrations and the
atmospheric temperature in an airtight jar. We also measured changes in the pH of the medium during hypoxia to assess the
accuracy of this system. Moreover, we used three durations (2, 3, and 4 h) of hypoxia and 8 h of reoxygenation in cultured
rat hepatocytes, and then measured the lactate dehydrogenase (LDH), ketone body concentration (acetoacetate + β-hydroxybutyrate),
and the ketone body ratio (KBR: acetoacetate/β-hydroxybutyrate) in the medium in order to assess the suitability of this system
as a model for reperfusion following liver ischemia. The oxygen concentration dropped to 1% or less within 1 h. The concentration
of carbon dioxide rose to about 5% at 30 min after the induction of the hypoxic conditions, and was maintained at this level
for 5 h. No effect of the reaction heat produced by the oxygen absorbent in the jar was recognized. The extent of cell injury
produced by changing the hypoxic parameters was satisfactorily reflected by the KBR, the ketone body concentration, and the
LDH activity released into the medium. Because this model can duplicate the conditions of the hepatocytes during revascularization
following ischemic liver, and the Anaeropack system for cell culture is easy to manipulate, it seems suitable for the experimental
study of hypoxic injury and revascularization in vitro. 相似文献
34.
35.
Toshiki Doi Suguru Yamamoto Takatoshi Morinaga Ken-ei Sada Noriaki Kurita Yoshihiro Onishi 《PloS one》2015,10(6)
Background
Few risk scores are available for predicting mortality in chronic kidney disease (CKD) patients undergoing predialysis nephrology care. Here, we developed a risk score using predialysis nephrology practice data to predict 1-year mortality following the initiation of haemodialysis (HD) for CKD patients.Methods
This was a multicenter cohort study involving CKD patients who started HD between April 2006 and March 2011 at 21 institutions with nephrology care services. Patients who had not received predialysis nephrology care at an estimated glomerular filtration rate (eGFR) of approximately 10 mL/min per 1.73 m2 were excluded. Twenty-nine candidate predictors were selected, and the final model for 1-year mortality was developed via multivariate logistic regression and was internally validated by a bootstrapping technique.Results
A total of 688 patients were enrolled, and 62 (9.0%) patients died within one year of HD initiation. The following variables were retained in the final model: eGFR, serum albumin, calcium, Charlson Comorbidity Index excluding diabetes and renal disease (modified CCI), performance status (PS), and usage of erythropoiesis-stimulating agent (ESA). Their β-coefficients were transformed into integer scores: three points were assigned to modified CCI≥3 and PS 3–4; two to calcium>8.5 mg/dL, modified CCI 1–2, and no use of ESA; and one to albumin<3.5 g/dL, eGFR>7 mL/min per 1.73 m2, and PS 1–2. Predicted 1-year mortality risk was 2.5% (score 0–4), 5.5% (score 5–6), 15.2% (score 7–8), and 28.9% (score 9–12). The area under the receiver operating characteristic curve was 0.83 (95% confidence interval, 0.79–0.89).Conclusions
We developed a simple 6-item risk score predicting 1-year mortality after the initiation of HD that might help nephrologists make a shared decision with patients and families regarding the initiation of HD. 相似文献36.
CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was previously reported to oxidize 2-naphthoic acid and 4-ethylbenzoic acid. In this study, we examined the substrate specificity
and regioselectivity of CYP199A2 towards indole- and quinolinecarboxylic acids. The CYP199A2 gene was coexpressed with palustrisredoxin gene from R. palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli. Following whole-cell assays, reaction products were identified by mass spectrometry and NMR spectroscopy. CYP199A2 did not
exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidized indole-2-carboxylic acid, indole-5-carboxylic
acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid was converted to 5- and 6-hydroxyindole-2-carboxylic acids at
a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generated only one product, 2-indolinone-6-carboxylic
acid, at a rate of 130 mol (mol P450)−1 min−1. Furthermore, CYP199A2 also oxidized quinoline-6-carboxylic acid, although this enzyme did not exhibit any activity towards
quinoline and its derivatives with a carboxyl group at the C-2, C-3, or C-4 positions. The oxidation product of quinoline-6-carboxylic
acid was identified to be 3-hydroxyquinoline-6-carboxylic acid, which was a novel compound. These results suggest that CYP199A2
may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids. 相似文献
37.
The purpose of this study is to extract components of the capacity which elderly people have in comprehending electrical devices and determine its relationship with the components. Initially, we proposed a hypothesis through examining previous studies. The hypothesis states that the capacity which elderly people have mainly consists of four components, i.e., motivation, working memory, logical thinking and experience with personal computers (PC) or mobile phones. Then, some tests were conducted to examine the hypothesis. In this research, elderly people were interviewed about their impressions and experience with electrical devices. Moreover, three tests were conducted including, card sorting, tasks using digital video cameras and a test to measure working memory. As analysis methods, the Quantification 1 was used to see which component was important. In addition, Boolean algebra was conducted to simplify the components and understand some relationships. As a result, a relationship with the components in electrical devices was revealed. Furthermore, the use of Boolean algebra and the Quantification 1 suggested that the experience with PCs or mobile phones was the most important component for elderly people. 相似文献
38.
Ariko Miyake Takaomi Ishida Makoto Yamagishi Takuma Hara Kazuo Umezawa Toshiki Watanabe Ryouichi Horie 《Microbes and infection / Institut Pasteur》2010,12(5):400-408
Previous reports indicate that nuclear factor (NF)-κB regulates induction of human immunodeficiency virus type 1 (HIV-1) gene expression in latently infected cells. However, the role of NF-κB in cells with active HIV-1 replication is not well understood. In this study, we examined the effect of a new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on HIV-1 replication in a human T cell line and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMCs). We further explored the mechanism of DHMEQ-mediated inhibition of HIV-1 replication. DHMEQ inhibited HIV-1 replication in HIV-1-infected Molt-4 and PHA-PBMCs. DHMEQ inhibited constitutive NF-κB activity in HIV-1-infected PHA-PBMCs and HIV long terminal repeat promoter activity driven by tumor necrosis factor (TNF)-α and the trans-activator Tat. The single-round assay using vesicular stomatitis virus-pseudotyped virus in the human T cell line M8166 indicated that DHMEQ treatment resulted in decreased integration of HIV-1 provirus into the host genome and decreased HIV-1 expression. These results indicate that NF-κB regulates early events as well as the initial and accelerated expression of HIV-1 in its life cycle. Therefore, we conclude that NF-κB is a molecular target for controlling active HIV-1 replication. 相似文献
39.
Yamada T Konno N Matsuda K Uchiyama M 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2007,177(5):503-508
In our previous study, it was suggested that ANP and cGMP may increase Na+ absorption in the urinary bladder of the Japanese tree frog, Hyla japonica. Thus, Na+ transport activated by ANP was investigated electrophysiologically by using a cell-attached patch-clamp technique in freshly
isolated cells from the urinary bladder. A predominant channel expressed was a low conductance Na+ channel in the epithelial cells. The channel exhibited conductance for inward currents of 4.9 ± 0.2 pS, long open and closed
times (c.a. 190 ms), and positive reversal potential. The channel activity was decreased under the pipette solution including
10−6 M amiloride. These characteristics were similar to those of amiloride-sensitive Na+ channels (ENaC). Addition of 10−9 M ANP activated and significantly increased the ENaC activity from 0.58 ± 0.09 to 1.47 ± 0.34. On the other hand, mean amplitudes
and conductance of single channel did not change significantly after the addition of ANP. Addition of 10−5 M 8-Br-cGMP also activated the ENaC and significantly increased the channel activity from 0.56 ± 0.10 to 2.00 ± 0.33. The
addition of ANP failed to activate the ENaC in the presence of 10−6 M amiloride. These results suggested that ANP and cGMP activate Na+ transport via ENaC in the epithelial cells of frog urinary bladder. 相似文献
40.
Shingai M Ebihara T Begum NA Kato A Honma T Matsumoto K Saito H Ogura H Matsumoto M Seya T 《Journal of immunology (Baltimore, Md. : 1950)》2007,179(9):6123-6133
Laboratory adapted and vaccine strains of measles virus (MV) induced type I IFN in infected cells. The wild-type strains in contrast induced it to a far lesser extent. We have investigated the mechanism for this differential type I IFN induction in monocyte-derived dendritic cells infected with representative MV strains. Laboratory adapted strains Nagahata and Edmonston infected monocyte-derived dendritic cells and activated IRF-3 followed by IFN-beta production, while wild-type MS failed to activate IRF-3. The viral IRF-3 activation is induced within 2 h, an early response occurring before protein synthesis. Receptor usage of CD46 or CD150 and nucleocapsid (N) protein variations barely affected the strain-to-strain difference in IFN-inducing abilities. Strikingly, most of the IFN-inducing strains possessed defective interference (DI) RNAs of varying sizes. In addition, an artificially produced DI RNA consisting of stem (the leader and trailer of MV) and loop (the GFP sequence) exhibited potential IFN-inducing ability. In this case, however, cytoplasmic introduction was needed for DI RNA to induce type I IFN in target cells. By gene-silencing analysis, DI RNA activated the RIG-I/MDA5-mitochondria antiviral signaling pathway, but not the TLR3-TICAM-1 pathway. DI RNA-containing strains induced IFN-beta mRNA within 2 h while the same recombinant strains with no DI RNA required >12 h postinfection to attain similar levels of IFN-beta mRNA. Thus, the stem-loop structure, rather than full genome replication or specific internal sequences of the MV genome, is required for an early phase of type I IFN induction by MV in host cells. 相似文献