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51.
52.
Zheng Jian Li Qinghua Zhao Hong Liang Shunlin Jiang Tom Chen Davida Grella Colleen Shearon Donald P. Bottaro B. Kim Lee Sim 《Biotechnology letters》2002,24(19):1631-1635
Endostatin is a 20 kDa carboxyl-terminal fragment of collagen XVIII that strongly inhibits angiogenesis and tumor growth. The methylotrophic yeast, Pichia pastoris, is a robust expression system that can be used to study methods to improve the yields of rhEndostatin. We expressed rhEndostatin in P. pastoris under the control of the alcohol oxidase 1 (aox 1) promoter (Mut+ phenotype) as a model, and used a cell biomass of about 50 g l–1 dry cell wt as a starting point for the induction phase and varied the methanol feed rate at 8 ml l–1 h–1, 11 ml l–1 h–1 and 15 ml l–1 h–1. While the cell growth rate was proportional to the rate of methanol delivery, protein production rate was not. These findings could be used to guide parameters for large-scale production of recombinant proteins in the P. pastoris system. 相似文献
53.
von Hunolstein C Parisi L Bottaro D 《Journal of biochemical and biophysical methods》2003,56(1-3):291-296
The molecular size of meningococcal polysaccharides is an important physico-chemical parameter which correlates with immunogenicity. This paper describes the experimental conditions for high-performance size-exclusion chromatography on a PL Aquagel-OH 60 column to follow changes in the size distribution and therefore in the distribution coefficient (K(D)) of the meningococcal polysaccharides of groups A, C, Y and W-135 used to formulate anti-Neisseria meningitidis vaccines. The experimental conditions were also found to be suitable for a rapid monitoring of the quality (no group A polysaccharide depolymerization) of the tetravalent meningococcal polysaccharide vaccine. 相似文献
54.
Ravera G Franceschini M Zanardi S Bottaro LC Colombi T Carbone M Percario GE 《The new microbiologica》2004,27(1):1-9
The genotyping of the hepatitis C virus (HCV) by viral nucleic acids sequencing allows accurate epidemiological evaluation of a cohort of patients suffering from HCV-related chronic hepatopathy. The identification of viral isolates, which can be generally associated with hepatic damage or, vice versa, which are more responsive to pharmacological treatment, might enhance clinical interest on the nature of the infecting genotypes. We, therefore, draw attention to those viral genotypes that are characterised by significantly high or altered viremic and enzymatic levels. 相似文献
55.
H Zhou J R Casas-Finet R Heath Coats J D Kaufman S J Stahl P T Wingfield J S Rubin D P Bottaro R A Byrd 《Biochemistry》1999,38(45):14793-14802
Hepatocyte growth factor (HGF) is a heparin-binding, multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. Heparin or closely related heparan sulfate has profound effects on HGF signaling. A heparin-binding site in the N-terminal (N) domain of HGF was proposed on the basis of the clustering of surface positive charges [Zhou, H., Mazzulla, M. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., Rubin, J. S., Bottaro, D. P., and Byrd, R. A. (1998) Structure 6, 109-116]. In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resonance spectroscopy, and we estimated the apparent dissociation constant (K(d)) of the heparin-protein complex by NMR and fluorescence techniques. The primary heparin-binding site is composed of Lys60, Lys62, and Arg73, with additional contributions from the adjacent Arg76, Lys78, and N-terminal basic residues. The K(d) of binding is in the micromolar range. A heparin disaccharide analogue, sucrose octasulfate, binds with similar affinity to the N domain and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. (15)N relaxation data indicate structural flexibility on a microsecond-to-millisecond time scale around the primary binding site in the N domain. This flexibility appears to be dramatically reduced by ligand binding. On the basis of the NK1 crystal structure, we propose a model in which heparin binds to the two primary binding sites and the N-terminal regions of the N domains and stabilizes an NK1 dimer. 相似文献
56.
A Brusco S Saviozzi F Cinque A Bottaro M DeMarchi 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(8):4392-4398
Human Ig heavy chain constant regions are encoded by a cluster of genes, the IGHC locus, on 14q32.3. Several forms of IGHC deletions and duplications spanning one to five genes have been described in different populations, with frequencies of 1.5-3.5% and 4.5-44%, respectively. Despite the common occurrence of these gene rearrangements, little is known about the breakpoint sites; evidence obtained from deletions in the IGHC locus and in other regions of the human genome suggests that they preferentially occur in highly homologous regions and might be favored by a variety of recombinogenic signals. We present here a detailed study of three homozygotes for the most common type of IGHC multiple gene deletion, spanning the A1-GP-G2-G4-E genes. Using a combination of Southern blotting, long-range PCR, and automated sequencing, the unequal crossover events of all of the six studied haplotypes have been mapped to a region of approximately 2 kb with almost complete homology between EP1-A1 and E-A2, flanked by two minisatellites. These results are consistent with the hypothesis that segments of complete homology may be required for efficient homologous recombination in humans. The possible role of minisatellites as recombination signals is inferred, in agreement with current knowledge. 相似文献
57.
Biodegradation of the hexahydro-1,3,5-trinitro-1,3,5-triazine ring cleavage product 4-nitro-2,4-diazabutanal by Phanerochaete chrysosporium 总被引:1,自引:0,他引:1
Fournier D Halasz A Spain J Spanggord RJ Bottaro JC Hawari J 《Applied and environmental microbiology》2004,70(2):1123-1128
Initial denitration of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 produces CO2 and the dead-end product 4-nitro-2,4-diazabutanal (NDAB), OHCNHCH2NHNO2, in high yield. Here we describe experiments to determine the biodegradability of NDAB in liquid culture and soils containing Phanerochaete chrysosporium. A soil sample taken from an ammunition plant contained RDX (342 micromol kg(-1)), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 3,057 micromol kg(-1)), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine; 155 micromol kg(-1)), and traces of NDAB (3.8 micromol kg(-1)). The detection of the last in real soil provided the first experimental evidence for the occurrence of natural attenuation that involved ring cleavage of RDX. When we incubated the soil with strain DN22, both RDX and MNX (but not HMX) degraded and produced NDAB (388 +/- 22 micromol kg(-1)) in 5 days. Subsequent incubation of the soil with the fungus led to the removal of NDAB, with the liberation of nitrous oxide (N2O). In cultures with the fungus alone NDAB degraded to give a stoichiometric amount of N2O. To determine C stoichiometry, we first generated [14C]NDAB in situ by incubating [14C]RDX with strain DN22, followed by incubation with the fungus. The production of 14CO2 increased from 30 (DN22 only) to 76% (fungus). Experiments with pure enzymes revealed that manganese-dependent peroxidase rather than lignin peroxidase was responsible for NDAB degradation. The detection of NDAB in contaminated soil and its effective mineralization by the fungus P. chrysosporium may constitute the basis for the development of bioremediation technologies. 相似文献
58.
Radaelli R Bottaro M Wilhelm EN Wagner DR Pinto RS 《Journal of strength and conditioning research / National Strength & Conditioning Association》2012,26(9):2577-2584
The purpose of this study was to evaluate the time course responses of strength, delayed-onset muscle soreness (DOMS), muscle thickness (MT), circumference (CIRC), and ultrasonography echo intensity (EI) after a traditional hypertrophic isoinertial resistance training session in young women. Ten (22.0 ± 3.2 years) healthy, untrained volunteers participated in the study. The resistance exercise session consisted of 4 sets of 10 repetitions at 80% of 1 repetition maximum (1RM) of the dominant arm elbow flexors. Maximum isometric elbow flexion peak torque (PT) at 90°, MT, and EI were recorded for both arms at baseline (PRE), immediately after exercise (0 hours) and at 24, 48, and 72 hours after exercise. Comparisons were made using a 2 × 5 mixed factor analysis of variance. There was a significant (p < 0.05) loss in PT and increase in MT at 0, 24, 48, and 72 hours. In contrast, EI increased only after 24, 48, and 72 hours, not at 0 hours. There were no significant changes in PT, DOMS, MT, and EI in the nondominant (control) arm after the exercise protocol. Our data suggest that after 4 sets of 80% of 1RM of unilateral elbow flexion resistance exercise, nonresistance trained women need >72 hours to fully recover muscle strength, MT, CIRC, and EI. Furthermore, the EI appears to be a sensitive and reliable method to assess MD. 相似文献
59.
Mungunsukh O Lee YH Marquez AP Cecchi F Bottaro DP Day RM 《American journal of physiology. Lung cellular and molecular physiology》2010,299(6):L905-L914
Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 (InlB??????) was demonstrated to bind to and partially activate the HGF receptor Met. InlB?????? has a stable β-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned InlB?????? (1×InlB??????) and engineered a head-to-tail repeat of InlB?????? with a linker peptide (2×InlB??????); 1×InlB?????? and 2×InlB?????? were purified from E. coli. Both 1× and 2×InlB?????? activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB?????? activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB?????? activated only STAT3 and ERK1/2. The 2×InlB?????? promoted improved motility compared with 1×InlB?????? and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB?????? prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2×InlB?????? to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair. 相似文献
60.
Manish A. Shah Zev A. Wainberg Daniel V. T. Catenacci Howard S. Hochster James Ford Pamela Kunz Fa-Chyi Lee Howard Kallender Fabiola Cecchi Daniel C. Rabe Harold Keer Anne-Marie Martin Yuan Liu Robert Gagnon Peter Bonate Li Liu Tona Gilmer Donald P. Bottaro 《PloS one》2013,8(3)