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1.
Van Lint Johan Van Damme Jo Billiau Alfons Merlevede Wilfried Vandenheede Jackie R. 《Molecular and cellular biochemistry》1993,127(1):171-177
The signal transduction initiated by the human cytokine interleukin-8 (IL-8), the main chemotactic cytokine for neutrophils, was investigated and found to encompass the stimulation of protein kinases. More specifically, IL-8 caused a transient, dose and time dependent activation of a Ser/Thr kinase activity towards myelin basic protein (MBP) and the MBP-derived peptide APRTPGGRR patterned after the specific concensus sequence in MBP for ERK enzymes. The activated MBP kinase was furthermore identified as an extracellular signal regulated kinase (ERK1) based on several criteria such as substrate specificity, molecular weight, activation-dependent mobility shift, and recognition by anti-ERK antibodies. For comparison, the chemotactic response of neutrophils to a stimulus of bacterial origin (fMet-Leu-Phe or fMLP) was also examined and found to involve the activation of a similar ERK enzyme. The present data clearly indicate that in terminally differentiated, non-proliferating human cells, the MBP kinase/ERK activity can serve other purposes than mitogenic signaling, and that processes such as chemotaxis, induced by bacterial peptides as well as by human cytokines like IL-8, involve the regulation of ERK enzyme.Abbreviations IL-8
interleukin-8
- fMLP
fMet-Leu-Phe
- MBP
myelin basic protein
- ERK
extracellular signal regulated kinase
- MAP2
microtubule-associated protein 2
- PK-A
cAMP dependent protein kinase
- PKI
protein kinase inhibitor
- PMSF
phenyl-methanesulfonyl fluoride
- PVDF
poly-vinylidene difluoride
- HBSF
Hank's buffered salt solution
- DAB
3,3-diaminobenzidine tetrahydrochloride
- PNPP
p-nitrophenyl-phosphate
- HSA
human serum albumin
- EGTA
[ethylenebis (oxyethylenenitrilo)]tetraacetic acid
- SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis 相似文献
2.
The wild-type (dam+) and mutant (damh) forms of the bacteriophage T2 DNA adenine methylase have been partially purified; these enzymes methylate the sequence, 5/t' … G-A-Py … 3′ (Hattman et al., 1978a). However, in vitro methylation studies using phage λ DNA revealed the following: (1) T2 dam+ and damh enzymes differ in their ability to methylate λ DNA; under identical reaction conditions the T2 damh enzyme methylated λ DNA to a higher level than did the dam+ enzyme. However, the respective methylation sites are equally distributed on the l and r strands. (2) Methylation with T2 damh, but not T2 dam+ protected λ against P1 restriction. This was demonstrated by transfection of Escherichia coli (P1) spheroplasts and by cleavage with R·EcoP1. (3) T2 dam+ and damh were similarly capable of methylating G-A-T-C sequences on λ DNA; e.g. λ·dam3 DNA (contains no N6-methyladonine) methylated with either enzyme was made resistant to cleavage by R·DpnII. In contrast, only the T2 damh modified DNA was resistant to further methylation by M·EcoP1 (which methylates the sequence 5′ … A-G-A-C-Py … 3′; Hattman et al., 1978b). (4) λ·dam3 DNA was partially methylated to the same level with T2 dam+ or T2 damh; the two enzymes produced different patterns of G-A-C versus G-A-T methylation. We propose that the T2 dam+ enzyme methylates G-A-C sequences less efficiently than the T2 damh methylase; this property does not entirely account for the large difference in methylation levels produced by the two enzymes. 相似文献
3.
An improved Enzyme Immunoassay for Myelin Basic Protein is described. Myelin Basic Protein covalently attached to glass balls, and Myelin Basic Protein in samples compete with each other for binding of a peroxidase conjugated anti Myelin Basic Protein antibody. The peroxidase activity on the balls is then inversely proportional to the amount of Myelin Basic Protein in the sample. A detection limit of 0.6 ng/ml is demonstrated for diluent or spinal fluid. For plasma a dilution step increases this to 1.8 ng/ml. Both the coated balls and the peroxidase conjugate are stable for long periods. The assay requires no expensive equipment. Although the assay appears to be valid for subcellular fractions spinal fluid and plasma, successful detection of Myelin Basic Protection peptides in clinical samples may require careful selection of suitable antisera. The assay would be very suitable for eventual use with an appropriate monoclonal antibody. 相似文献
4.
Katsuhiko Mikoshiba Kimio Akagawa Ken Takamatsu Yasuzo Tsukada 《Journal of neurochemistry》1982,39(4):1028-1032
The cerebellar hypoplasia induced by hereditary hyperbilirubinemia in the Gunn rat was analyzed neurochemically and immunohistochemically. The antiserum against myelin basic protein was used to visualize the arborization of the fibers in the cerebellum. Arborization was very scarce in the affected lobes of the homozygous (jj) cerebellum. Na,K-ATPase activity did not show significant differences between the jj and the control (Jj) cerebellum. The concentration of norepinephrine in the jj cerebellum was about 1.5 times that of the control. However, the activation ratio of the Na,K-ATPase by norepinephrine and other catecholamines such as dopamine and isoproterenol was about twice as high as the basal activity, and no significant difference was observed between the jj and the Jj cerebella. The glutamic acid decarboxylase activity of the jj cerebellum did not differ significantly from that of the control. 相似文献
5.
Stereospecific binding of human β-endorphin to rat membrane preparations is described for the first time using as the ligand. The binding is time dependent and saturable with respect to βh-endorphin with an apparent dissociation constant of 0.3 nM. Sodium ion (100 mM) elevates this value to 2.5 nM but has no effect on the total number of binding sites present in the membrane preparation. The ability of certain β-endorphin analogs, opiate agonists as well as antagonists to inhibit the binding of βh-endorphin, is presented. 相似文献
6.
Silvia C. Locatelli-Hoops Inna Gorshkova Klaus Gawrisch Alexei A. Yeliseev 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(10):2045-2056
Human peripheral cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB2 and immobilization at solid interfaces. Expression of CB2 as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB2 was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB2 was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB2 from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB2 and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB2. 相似文献
7.
Yuanqing Hu Yuwei Shang Jinlin Huang Yan Wang Fangzhe Ren Yang Jiao Zhiming Pan Xin-an Jiao 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.Methods
In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.Results
Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI–TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.Conclusion
We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.General significance
This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines. 相似文献8.
Zachary A. Bornholdt Andrew S. Herbert Chad E. Mire Shihua He Robert W. Cross Anna Z. Wec Dafna M. Abelson Joan B. Geisbert Rebekah M. James Md Niaz Rahim Wenjun Zhu Viktoriya Borisevich Logan Banadyga Bronwyn M. Gunn Krystle N. Agans Ariel S. Wirchnianski Eileen Goodwin Kevin Tierney John M. Dye 《Cell host & microbe》2019,25(1):49-58.e5
9.
Chitooligosaccharide oxidase (ChitO) catalyzes the oxidation of C1 hydroxyl moieties on chitooligosaccharides and in this way displays a different substrate preference as compared to other known oligosaccharide oxidases. ChitO was identified in the genome of Fusarium graminearum and a structural model revealed that one active site residue (Q268) was likely to be involved in the recognition of the N-acetyl moiety on the chitooligosaccharide substrates. The substrate specificity of wild type ChitO and the Q268R mutant were examined and confirmed that Q268 is indeed involved in N-acetyl recognition. 相似文献
10.
摘要 目的:观察核心稳定性训练联合高压氧对脑梗死恢复期患者血液流变学、脑能量代谢和血清神经生长因子(NGF)、神经元特异性烯醇化酶(NSE)、髓鞘碱性蛋白(MBP)的影响。方法:选择我院2018年3月~2021年3月期间收治的脑梗死恢复期患者103例。采用随机数字表法将患者分为对照组和观察组两组,例数分别为51例和52例。对照组患者接受核心稳定性训练,观察组患者接受核心稳定性训练联合高压氧治疗,对比两组疗效、血液流变学、脑能量代谢指标和血清NGF、NSE、MBP水平及相关量表评分。结果:观察组的临床总有效率高于对照组(P<0.05)。治疗后,两组上肢/下肢Lovett肌力评分升高,美国国立卫生研究院卒中量表(NIHSS)评分下降,且观察组的改变程度大于对照组(P<0.05)。治疗后,两组全血高切黏度、血细胞比容、全血低切黏度、血浆黏度下降,且观察组的改变程度大于对照组(P<0.05)。治疗后,两组血氧饱和度升高,空腹血糖、乳酸水平下降,且观察组的改变程度大于对照组(P<0.05)。治疗后,两组NGF水平升高,NSE、MBP水平下降,且观察组的改变程度大于对照组(P<0.05)。结论:核心稳定性训练联合高压氧应用于脑梗死恢复期患者,可促进其血液流变学、脑能量代谢改善,有效调节血清NGF、NSE、MBP水平,疗效明确。 相似文献