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171.
172.
The method of affinity coelectrophoresis was used to study the binding of
nine representative glycosaminoglycan (GAG)-binding proteins, all thought
to play roles in nervous system development, to GAGs and proteoglycans
isolated from developing rat brain. Binding to heparin and non-neural
heparan and chondroitin sulfates was also measured. All nine
proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease
nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth
factor-2-bound brain heparan sulfate less strongly than heparin, but the
degree of difference in affinity varied considerably. Protease nexin-1
bound brain heparan sulfate only 1.8- fold less tightly than heparin
(Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin
well (Kd approximately 140 nM) but failed to bind detectably to brain
heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin
sulfate, with affinities equal to or a few fold stronger than the same
proteins displayed toward cartilage chondroitin sulfate. Overall, the
highest affinities were observed with intact heparan sulfate proteoglycans:
laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2),
glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity
for brain heparan sulfate. In contrast, the affinities of fibroblast growth
factor-2 for cerebroglycan and for brain heparan sulfate were similar.
Interestingly, partial proteolysis of cerebroglycan resulted in a >400-
fold loss of laminin affinity. These data support the views that (1)
GAG-binding proteins can be differentially sensitive to variations in GAG
structure, and (2) core proteins can have dramatic, ligand-specific
influences on protein-proteoglycan interactions.
相似文献
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Chen S Yakunin AF Kuznetsova E Busso D Pufan R Proudfoot M Kim R Kim SH 《The Journal of biological chemistry》2004,279(30):31854-31862
Methanococcus jannaschii MJ0936 is a hypothetical protein of unknown function with over 50 homologs found in many bacteria and Archaea. To help define the molecular (biochemical and biophysical) function of MJ0936, we determined its crystal structure at 2.4-A resolution and performed a series of biochemical screens for catalytic activity. The overall fold of this single domain protein consists of a four-layered structure formed by two beta-sheets flanked by alpha-helices on both sides. The crystal structure suggested its biochemical function to be a nuclease, phosphatase, or nucleotidase, with a requirement for some metal ions. Crystallization in the presence of Ni(2+) or Mn(2+) produced a protein containing a binuclear metal center in the putative active site formed by a cluster of conserved residues. Analysis of MJ0936 against a panel of general enzymatic assays revealed catalytic activity toward bis-p-nitrophenyl phosphate, an indicator substrate for phosphodiesterases and nucleases. Significant activity was also found with two other phosphodiesterase substrates, thymidine 5'-monophosphate p-nitrophenyl ester and p-nitrophenylphosphorylcholine, but no activity was found for cAMP or cGMP. Phosphodiesterase activity of MJ0936 had an absolute requirement for divalent metal ions with Ni(2+) and Mn(2+) being most effective. Thus, our structural and enzymatic studies have identified the biochemical function of MJ0936 as that of a novel phosphodiesterase. 相似文献
176.
Ponzio MF Monfort SL Busso JM Dabbene VG Ruiz RD de Cuneo MF 《Journal of experimental zoology. Part A, Comparative experimental biology》2004,301(3):218-227
The Chinchilla is a rodent that was once abundant in the central Andes of South America. Excessive hunting for fur greatly reduced its distribution at the beginning of the twentieth century, and today Chinchilla species are nearly extinct in the wild. Although protected, wild populations of chinchilla are still declining. In general, this species has received little research attention and its biology is poorly understood. Improvements in captive breeding, husbandry, and genetic management are needed to ensure the conservation of the species. In this study, a noninvasive corticosteroid hormone monitoring technique was validated for use in Chinchilla lanigera. Two male domestic chinchillas were administered 3H-corticosterone (i.m.) to determine the time course and relative proportion of urinary and fecal steroid metabolites. Most radioactivity was detected in urine and feces 5-10 and approximately 30 h post-isotope administration, respectively. Corticosteroid immunoreactivity was assessed by corticosterone radioimmunoassay (RIA) and cortisol enzyme immunoassay (EIA). High-pressure liquid chromatography (HPLC) separation of corticosteroid metabolites in unprocessed urine revealed the presence of highly polar corticosteroid metabolites, but after enzymatic hydrolysis and diethyl ether extraction, most immunoreactivity co-eluted with unconjugated cortisol. A 'cause-and-effect' relationship between the administration of exogenous adrenocorticotrophic hormone (ACTH), and the appearance of increased urinary corticosteroid metabolites demonstrated the physiological relevance of these measures for evaluating adrenal status in male chinchillas. From a conservation perspective, these methods can aid in situ and ex situ initiatives designed to evaluate how environmental conditions and management strategies affect overall animal health, well-being and reproduction. 相似文献
177.
Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins
For structural and functional genomics programs, new high-throughput methods to characterize well-expressing and highly soluble proteins are essential. A faster and more convenient approach to screen expression conditions of recombinant proteins compared to classical in vivo systems is the Escherichia coli cell-free expression system. Here, we describe a rapid procedure to screen for expression and solubility of recombinant proteins using an E. coli cell-free extract. The results presented cover 24 open reading frames of unknown function from different micro-organisms. In order to screen different variables that may interfere with solubility, we expressed the recombinant proteins with a histidine6 tag, either N-terminal or C-terminal at two temperatures (25 degrees C and 30 degrees C). The identification of recombinant proteins is performed by the dot blot procedure using an anti-histidine tag antibody. We designed a rapid method that allows the characterization of soluble candidates from a large number of genes or from a large number of variants that is highly compatible with structural genomics expectations. 相似文献
178.
Busso D Keriel A Sandrock B Poterszman A Gileadi O Egly JM 《The Journal of biological chemistry》2000,275(30):22815-22823
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Nadja C. Wielebnowski Norah Fletchall Kathy Carlstead Juan M. Busso Janine L. Brown 《Zoo biology》2002,21(1):77-98
The North American clouded leopard (Neofelis nebulosa) population is far from self‐sustaining. Breeding success is poor and behavioral problems (i.e., fur‐plucking, tail‐chewing, excessive hiding or pacing, and intersexual aggression that results in mate killing) are common. This study was undertaken to investigate whether some of these problems may be indicators of chronic stress (as reflected by persistently elevated glucocorticoid levels) and whether they are associated with specific management factors. A fecal corticoid metabolite assay was validated to monitor adrenal activity in clouded leopards. Adrenocorticotropic hormone (ACTH) challenges conducted in four clouded leopards established the biological relevance of the assay system. Fecal corticoid concentrations increased 14‐fold above baseline within 24 hours after ACTH administration. Adrenal activity then was monitored in 72 (36 males; 36 females) clouded leopards (65% of the North American Species Survival Plan population) during a 6‐week period and compared to husbandry and behavior data. There was a significant (P < 0.01) gender difference in fecal corticoid concentrations, with females producing higher concentrations than males. Multiple regression analyses revealed negative associations (P < 0.01) between enclosure height, number of hours keepers spent with each animal per week, and corticoid concentrations. A positive correlation (P < 0.001) was found between the number of keepers caring for an individual and corticoid concentrations. Higher fecal corticoid concentrations (P ≤ 0.05) were measured in clouded leopards kept on public display or near potential predators compared to individuals maintained off exhibit or in the absence of predators. Individuals that performed self‐injuring behaviors also had elevated fecal corticoids (P < 0.01). Spearman‐rank correlation analysis of keeper ratings and hormone data revealed positive associations (P ≤ 0.05) between some behaviors (pace, sleep, hide, and fearful/tense) and fecal corticoid concentrations. Overall these results indicate that noninvasive fecal corticoid monitoring has enormous potential for investigating how management and behavioral problems are related to animal well‐being. If conducted under carefully controlled experimental paradigms, this technique could allow researchers and managers to identify problem areas of captive management for clouded leopards (e.g., enclosure height, keeper time) and evaluate the efficacy of strategies designed to promote animal welfare and increased reproductive success. Zoo Biol 21:77–98, 2002. © 2002 Wiley‐Liss, Inc. 相似文献