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81.
82.
Karlberg T Hansson MD Yengo RK Johansson R Thorvaldsen HO Ferreira GC Hansson M Al-Karadaghi S 《Journal of molecular biology》2008,378(5):1074-1083
The specific insertion of a divalent metal ion into tetrapyrrole macrocycles is catalyzed by a group of enzymes called chelatases. Distortion of the tetrapyrrole has been proposed to be an important component of the mechanism of metallation. We present the structures of two different inhibitor complexes: (1) N-methylmesoporphyrin (N-MeMP) with the His183Ala variant of Bacillus subtilis ferrochelatase; (2) the wild-type form of the same enzyme with deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP). Analysis of the structures showed that only one N-MeMP isomer out of the eight possible was bound to the protein and it was different from the isomer that was earlier found to bind to the wild-type enzyme. A comparison of the distortion of this porphyrin with other porphyrin complexes of ferrochelatase and a catalytic antibody with ferrochelatase activity using normal-coordinate structural decomposition reveals that certain types of distortion are predominant in all these complexes. On the other hand, dSDP, which binds closer to the protein surface compared to N-MeMP, does not undergo any distortion upon binding to the protein, underscoring that the position of the porphyrin within the active site pocket is crucial for generating the distortion required for metal insertion. In addition, in contrast to the wild-type enzyme, Cu2+-soaking of the His183Ala variant complex did not show any traces of porphyrin metallation. Collectively, these results provide new insights into the role of the active site residues of ferrochelatase in controlling stereospecificity, distortion and metallation. 相似文献
83.
84.
Vector Gti'IX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi'IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with Gti'IX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTi'IX were obtained. At the same time, previous normal adenoviral vector pAdSPi'IX containing viral genome and hFIX mini-gene was constructed, and then previous adenovirus (pre-Ad) AdSPi'IX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTi'IX was less than 0.8%. 3T3 cells were transfected with AdGTi'IX and AdSPi'IX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 μg /106@24 h and 1.6±0.3 μg/106@24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 1×1010pfu AdGTi'IX or AdSPi'IX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 μL to 60 μL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTi'IX was obviously weaker than that triggered by AdSPi'IX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector system prolonged the expression time of hFIX and reduced immune response, thus offering a promising result for further pre-clinical study. 相似文献
85.
The influence of 2,2′-dipyridyl (2,2′-DP) on the activity of one of the enzymes at the initial stages of chlorophyll (Chl)
biosynthesis, δ-aminolevulinic acid dehydratase (ALAD; δ-aminolevulinate hydro-lyase, EC 4.2.1.24), as well as on δ-aminolevulinic
acid (ALA) accumulation was investigated in green barley (Hordeum vulgare L.) leaves. In seven-day-old green leaves treated with 3 mM 2,2′-DP for 17 h in darkness and subsequently irradiated with "white
light" (15 W m-2) for 4, 8, and 24 h the ALAD activity was 51 % as compared to that in untreated leaves. At the same time,
the ALA forming system was most sensitive to the photodynamic processes caused by 2,2′-DP. After 8 h of irradiation, ALA synthesis
was entirely inhibited. After the treatment the leaves accumulated exceptionally high amounts of Chl precursors such as protoporphyrin
IX (Proto), Mg-protoporphyrin IX (Mg-Proto), its monomethyl ester, and protochlorophyllide (Pchlide) that are photosensitizers
of photodynamic processes in plants. A comparatively low Chl and carotenoid (Car) destruction was registered during the subsequent
4 and 8 h of irradiation. At the same time, the content of Chl precursors was negligible. The low photodestruction of Chl
and Car included in pigment-protein complexes, against the background of fast porphyrin disappearance, and fast decrease of
enzymatic activities at the initial stages of Chl production could mean that the photodynamic effect induced by porphyrins
accumulated in the presence of 2,2′-DP affected first the Chl enzymatic system and did not change the pool of already synthesized
photosynthetic pigments.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
86.
Lateral roots (LRs) play important roles in increasing the absorptive capacity of roots as well as to anchor the plant in the soil. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of methyl jasmonate (MJ) on LR formation in rice. Treatment with MJ induced LR formation and heme oxygenase (HO) activity. As well, MJ could induce OsHO1 mRNA expression. Zinc protoporphyrin IX (the specific inhibitor of HO) and hemoglobin [the carbon monoxide/nitric oxide (NO) scavenger] reduced LR formation, HO activity and OsHO1 expression. LR formation and HO activity induced by MJ was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-oxide. The effects of Ca2+ chelators, Ca2+-channel inhibitors, and calmodulin (CaM) antagonists on LR formation induced by MJ were also examined. All these inhibitors were effective in reducing the action of MJ. However, Ca2+ chelators and Ca2+ channel inhibitors induced HO activity when combining with MJ further. It is concluded that Ca2+ may regulate MJ action mainly through CaM-dependent mechanism. 相似文献
87.
Valeria Petronilli Alessandra Zulian Giulio Jori Giuseppe Tognon Paolo Bernardi 《BBA》2009,1787(7):897-172
We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, 1O2) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation. Thus, hematoporphyrin-mediated photooxidative stress can either inhibit or activate the mitochondrial permeability transition depending on the site of hematoporphyrin localization and on the nature of the substrate; and selective photomodification of different hematoporphyrin-containing pore domains can be achieved by fine regulation of the sensitizer/light doses. These findings shed new light on PTP modulation by oxidative stress. 相似文献
88.
Yukitake H Naito M Sato K Shoji M Ohara N Yoshimura M Sakai E Nakayama K 《Microbiology and immunology》2011,55(3):141-153
The oral anaerobic bacterium Porphyromonas gingivalis, which is implicated as an important pathogen for chronic periodontitis, requires heme for its growth. Non-iron metalloporphyrins, In-PPIX and Ga-PPIX, were examined for antibacterial effects on P. gingivalis. Both In-PPIX and Ga-PPIX caused retardation of P. gingivalis growth in a dose-dependent fashion. Microarray and qPCR analyses revealed that In-PPIX treatment upregulated the expression of several genes encoding proteins including ClpB and ClpC, which are members of the Clp (caseinolytic protease, Hsp100) family, and aRNR, aRNR-activating protein and thioredoxin reductase, whereas In-PPIX treatment had no effect on the expression of genes encoding proteins involved in heme uptake pathways, Hmu-mediated, Iht-mediated and Tlr-mediated pathways. P. gingivalis ihtA and ihtB mutants were more resistant to In-PPIX than was the wild-type parent, whereas hmuR and tlr mutants did not show such resistance to In-PPIX. The results suggest that In-PPIX is incorporated by the Iht-mediated heme uptake pathway and that it influences protein quality control and nucleotide metabolism and retards growth of P. gingivalis. 相似文献
89.
Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants 总被引:5,自引:0,他引:5
Xu-Xiang Zhang Tong Zhang Ming Zhang Herbert H. P. Fang Shu-Pei Cheng 《Applied microbiology and biotechnology》2009,82(6):1169-1177
Three hydrogen-based membrane biofilm reactors (MBfR) biologically reduced nitrate and perchlorate in a synthetic ion-exchange
(IX) brine. Inocula from different natural saline environments were employed to initiate the three MBfRs. Under high-salinity
(3%) conditions, bioreduction of nitrate and perchlorate occurred simultaneously, and the three MBfRs from the different inocula
exhibited similar removal fluxes for nitrate and perchlorate. Clone libraries were generated from samples of the biofilms
in the three MBfRs and compared to those of their inocula. When H2 was the sole exogenous electron donor under high-salinity conditions, MBfR-driven community shifts were observed with a similar
pattern regardless of inoculum. The following 16S rRNA gene phylogenetic analysis showed the presence of novel perchlorate-reducing
bacteria in the salt-tolerant mBfR communities. These findings suggest that autohydrogenotrophic and high-salinity conditions
provided strong selective pressure for novel perchlorate-reducing populations in the mBfRs.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
90.
This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous intron 1 sequence.hFIX minigene was obtained with middle sequence truncated intron 1 inserted into the relative site of hFIX cDNA,and plasmid vector pKG5i‘IX,retroviral vector G1NaCi‘IX were constructed.These vectors were transduced into target cells of PA317,C2C12,primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF).The expression level of mixed colonies are PA317/pKG5i‘IX,151 ng/10^6 cells/24h;PA317/G1NaCi‘IX,308 ng/10^6 cells/24 h;C2C12/G1NaCi‘IX,186 ng/10^6 cells/24 h;RSF/G1NaCi‘IX,1929 ng/10^6 cells/24 h;HSF/G1NaCi‘IX,1646 ng/10^6 cells/24 h.These results indicated that hFIX minigene with intron l is able to increase the expression level to about 3 times of that of hFIX cDNA.Meanwhile,in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from intron splicing during viral production,a retroviral vector G1NaCi‘IXR with reversely inserted hFIX minigene expression cassette was constructed.The expression level of reverse constructor in PA317 cells was 390 ng/10^6 cells/24 h with 79% of bioactivity.PCR detection of HT/G1NaCi‘IXR cells infected with PA317/G1NaCi‘IXR supernatant confirmed the existence of intron 1 sequence.These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene transfer,but when using the retroviral-mediated gene transfer system,reversely-inserted intronl-carrying hFIX expression cassette should be considered. 相似文献