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931.
Antibody conjugates mimic specific B cell presentation of antigen: relationship between T and B cell specificity 总被引:12,自引:0,他引:12
We developed antibody conjugates by covalently coupling antibodies against mouse mu-chain and monoclonal antibodies against nominal antigen, myoglobin, as a tool for antigen presentation and as a model of specific presentation of antigen by antigen-specific B cells and T-B interaction. In the presence of the antibody conjugates, myoglobin-specific Iad-restricted cloned T cells proliferated at 1000-fold lower concentration of myoglobin than the stimulatory concentration without the conjugates. This enhanced presentation was observed only when Iad spleen cells were 1000 R-irradiated but not 3300 R-irradiated, consistent with B cell presentation. The simple mixture of each component of the conjugates had no enhancement effects. The conjugates per se had no mitogenic effects on either splenic B cells or the cloned T cells at concentrations employed for antigen presentation. The conjugates reduced the number of antigen-presenting cells required for the maximal response but did not change the kinetics of response. The enhanced presentation by the conjugates required a genetically restricted interaction with B cells. Antigen specificity of the enhanced presentation was confirmed by using various T cell clones or lines with different antigen specificities and different conjugates constructed with monoclonal antibodies of known epitope specificity. The enhanced presentation was significantly inhibited by competition with exogenous mouse IgM or anti-mouse mu-chain but was not significantly inhibited by monoclonal antibodies against Fc receptor. Thus, conjugate-coated B cells serve as models for myoglobin-specific B cells in that they can take up specific antigens at extremely low concentration and can present the antigen to specific T cells. This model system can be applied to any antigen and any species without the need to develop antigen-specific B cell clones, which is not yet possible for most antigens and species of experimental animals. This system allowed us to investigate the relationship between T cell epitope and B cell epitope when these cells interact with each other in an antigen-specific and Ia-restricted manner. Experiments using antibody conjugates of different monoclonal antibodies against myoglobin and various myoglobin-specific cloned T cells of known antigen specificity revealed that there are some particular combinations in which much more limited enhancement of antigen presentation is observed.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
932.
We obtained evidence that amiloride specifically potentiates 125I-labeled alpha-rat atrial natriuretic peptide (1-28) [atrial natriuretic peptide (ANP)-(99-126); rANP] binding to cerebral capillaries isolated from the rat cerebral cortex. The binding parameters, KD of 173 pM and Bmax of 159 fmol/mg of protein, became 33 pM and 88 fmol/mg of protein, respectively, when 10(-4) M amiloride was added to the incubation medium. When the effect of rANP was investigated on in vitro 22Na+ uptake into isolated cerebral capillaries, 10(-7) M rANP significantly inhibited the uptake in the presence of 1.0 mM ouabain, 1.0 mM furosemide, and 2.0 mM LiCl in the uptake buffer, a finding suggesting a specific inhibitory effect of rANP on amiloride-sensitive Na+ transport. Thus, the possibility that ANPs control amiloride-sensitive Na+ transport at the blood-brain barrier by interacting with specific receptors has to be considered. 相似文献
933.
An exoribonuclease has been purified nearly to homogeneity from rat liver microsomes and its mode of action and general properties were studied. The molecular weight values for the enzyme, as estimated by gel filtration and SDS-polyacrylamide gel electrophoresis, were 88 000 and 92 000, respectively. The enzyme produced, via a processive mechanism Ado5'P as the only product from poly(A). The results of the hydrolysis of 4 S (Ado5'P)n and (Ado3'P)n by the exoribonuclease with or without alkaline phosphatase and the inhibition of the enzymatic activity by oligonucleotides having a 3'-phosphate group in the 3'-terminus suggested that the degradation proceeds in the 3' to 5' direction. These findings were confirmed by the analysis of hydrolyzed products of various oligoadenylates and Ado3'PUrdPGuo and by the comparison of the rates of hydrolysis of (Ado3'P)2Ado by the enzyme in the presence of varying amounts of (Ado3'P)3. Mg2+ was required for the enzymatic activity, and Mn2+ partially substituted for Mg2+. The activity of the enzyme was stimulated by K+ and spermine. 相似文献
934.
S Kumagai S Namiuchi S Ozaki H Sano T Tsubata T Suginoshita H Imura 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(2):711-717
T cells that proliferate in the autologous mixed lymphocyte reaction (auto-MLR) have been shown to acquire some suppressor or regulatory activities. In the present study, we examined the suppressive effects of T cells activated in the auto-MLR on the induction of hapten-specific cytotoxic T cells. NRFT (depletion of ARFT from UT) were used as the responder cells of TNP-MLR. After primary and secondary TNP-MLR, the cells were harvested and tested for their cytotoxic activities against TNP-modified autologous cells by 51Cr-release assay. When UT cells cultured for 1 wk in auto-MLR were added to primary TNP-MLR at the beginning of culture, the cytotoxic activity tested at the end of the culture was suppressed from 15.6% +/- 2.7 to 5.8% +/- 1.1 (percent cytotoxicity, mean +/- SE). However, these auto-MLR-activated UT cells had little suppressive activity against cytotoxic T cells when they were added to the final assay of TNP-CTR. Suppressive activities of these cells on the generation of cytotoxic T cells during secondary TNP-MLR were also tested. The addition of auto-MLR-activated UT cells to the secondary TNP-MLR at the beginning of the culture reduced the cytotoxic activities of NRFT from 23.8% +/- 2.3 to 9.7% +/- 1.7 after secondary TNP-MLR. Allo-activated T cells, PHA blasts, and fresh autologous T cells were used as the controls, but none of the cells had suppressive effects on the generation of CTL. Characteristics of these suppressor cells were examined. Auto-MLR-activated cells from ARFT fractions exhibit very powerful suppressor activity. Treatment of the auto-MLR-activated T cells with mitomycin C eliminated their suppressive effects on the generation of CTL; 21.2% +/- 6.3 of UT cells became anti-Tac positive after 1 wk of auto-MLR. Treatment of auto-MLR-activated UT cells with anti-Tac antibody plus complement eliminated their suppressive activities on the induction of CTL. Thus, T cells stimulated in auto-MLR were shown to have suppressive effects on the induction of cytotoxic T cells against TNP-modified autologous cells. These cells were mitomycin C sensitive. Because anti-Tac antibody is reactive to activated T cells, activation of T cells during auto-MLR was thought to be necessary for the acquisition of the suppressive activity. 相似文献
935.
In previous studies, Mg2+ -dependent, HCO3- -activated ATPase in the brush border and carbonic anhydrase in the cytoplasm of rat duodenal and jejunal mucosa decreased after adrenalectomy. Both enzyme activities increased to near normal levels 4 h after i.p. injection of aldosterone (40 micrograms/kg). These results suggest the possibility that both enzymes in the small intestinal mucosa may be mediators of the action of aldosterone. In the present studies, therefore, the effects of actinomycin D (500 micrograms/kg, i.p.), spironolactone (50 mg/kg, s.c.) and potassium canrenoate (50 mg/kg, s.c.) on aldosterone-induced activation of both enzymes in the upper small intestinal mucosa from adrenalectomized rats were examined to clarify the mechanism of action of aldosterone in enzyme levels. Actinomycin D inhibited carbonic anhydrase activity in small intestinal mucosa from normal rats 4 h after i.p. injection but had no effect on ATPase activity, while two other drugs had no effect on either enzyme activity in normal rats up to 4 h later. Pretreatment with these 3 drugs 1 h before aldosterone administration (40 micrograms/kg, i.p.) to adrenalectomized rats blocked the aldosterone-induced activation of ATPase and carbonic anhydrase in the upper small intestine. On the other hand, adrenalectomy and administration of aldosterone and its antagonists, alone or in combination, had no effect on kidney enzyme activities. These results confirm that Mg2+ -HCO3- -ATPase and carbonic anhydrase are mediators of the action of aldosterone in the upper small intestinal mucosa. 相似文献
936.
Purification and characterization of NADP+-linked isocitrate dehydrogenase from an alkalophilic Bacillus 总被引:1,自引:0,他引:1
We have succeeded in purifying to homogeneity a very labile NADP+-linked isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) from a strain of alkalophilic Bacillus, by a simple method, with an overall yield over 76% of the original activity. The molecular weight on Sephadex G-200 was around 90,000; and that by electrophoresis on SDS-polyacrylamide gels was about 44,000. The sedimentation coefficient (s020,w) and isoelectric point of the enzyme were determined to be 3.22 S and pH 4.7, respectively. The enzyme required Mn2+ for the reaction and for stability. The optimum pH for the reaction was in the range 7.8-8.4 at 30 degrees C; the optimum temperature at pH 8.0 was 75 degrees C; the activation energy of the reaction was 6.2 kcal/mol. The Km values for threo-Ds-isocitrate, DL-isocitrate, and NADP+ were 5.4 microM, 9.9 microM, and 7.3 microM, respectively. This enzyme was inhibited by NADPH, glyceraldehyde 3-phosphate, 3-phosphoglycerate, phosphoenol pyruvate, cis-aconitate, alpha-ketoglutarate, and oxaloacetate. In addition, it was subject to a concerted inhibition by a combination of glyoxylate and oxaloacetate, and also to a cumulative inhibition by nucleoside triphosphates. 相似文献
937.
938.
Kazuaki Igarashi Yuji Hatada Hiroshi Hagihara Katsuhisa Saeki Mikio Takaiwa Takaaki Uemura Katsutoshi Ara Katsuya Ozaki Shuji Kawai Tohru Kobayashi Susumu Ito 《Applied and environmental microbiology》1998,64(9):3282-3289
A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein−1, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)8 barrel motif in domain A.α-Amylase (1,4-α-d-glucan glucanohydrolase [EC 3.2.1.1]) and pullulanase (pullulan 6-glucanohydrolase [EC 3.2.1.41]) are amylolytic enzymes of industrial importance, particularly in the food and detergent industries. We have found and characterized some unique debranching enzymes, such as a high-alkaline pullulanase (2), an alkali-resistant neopullulanase (16), and an alkaline isoamylase (3), from cultures of alkaliphilic Bacillus strains, and these enzymes can be used as effective additives in dishwashing and laundry detergents under alkaline conditions, especially when used in combination with α-amylase. We have also found the first known alkaline amylopullulanase from alkaliphilic Bacillus sp. strain KSM-1378 (4), which is very unique in that it efficiently hydrolyzes the α-1,6 linkages of pullulan, as well as the α-1,4 linkages of various carbohydrates at different active sites (1, 13).Liquefying α-amylases, particularly the Bacillus licheniformis enzyme (BLA) (35), are used widely in technical application fields, such as in bread making, production of glucose and fructose syrup and fuel ethanol from starch materials, and textile treatment. The demand for α-amylase for use in laundry and automatic dishwashing detergents has also been growing for several years (42). However, most of the Bacillus liquefying amylases, such as the enzymes from Bacillus amyloliquefaciens (BAA) and Bacillus stearothermophilus (BSA) (28), including BLA (35), have pH optima of between 5 and 7.5 (44). These neutrophilic enzymes are essentially not good for use in detergents, because the working pH range between 8 and 11 is relevant to washing in detergents (17). Since Horikoshi (15) first reported an alkaline amylase from alkaliphilic Bacillus sp. strain A-40-2, many alkaline amylases have been found in cultures of, for example, Bacillus sp. strain NRRL B-3881 (31), Bacillus sp. strain H-167 (14), Bacillus alcalothermophilus A3-8 (7), and Bacillus sp. strain GM8901 (21). The alkaline amylases from these alkaliphilic Bacillus strains reported to date are all of the saccharifying type, except for the enzymes from Bacillus sp. strain 707 (22, 41) and B. licheniformis TCRDC-B13 (5). However, very limited or no information about enzymatic properties of these two liquefying amylases is available. In this paper, we report the isolation of a novel liquefying α-amylase (LAMY) from cultures of the alkaline amylopullulanase producer Bacillus sp. strain KSM-1378 (13). This enzyme is highly active at alkaline pH compared with those of other liquefying α-amylases reported to date. Furthermore, analysis of the gene for this α-amylase (amyK) indicates that LAMY exhibits low amino acid identity to the reported liquefying α-amylases. 相似文献
939.
Enzymatic Properties of a Novel Liquefying α-Amylase from an Alkaliphilic Bacillus Isolate and Entire Nucleotide and Amino Acid Sequences
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Kazuaki Igarashi Yuji Hatada Hiroshi Hagihara Katsuhisa Saeki Mikio Takaiwa Takaaki Uemura Katsutoshi Ara Katsuya Ozaki Shuji Kawai Tohru Kobayashi Susumu Ito 《Applied microbiology》1998,64(9):3282-3289
A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein−1, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)8 barrel motif in domain A. 相似文献
940.
Higuchi Hiroyoshi Shibaev Yuri Minton Jason Ozaki Kiyoaki Surmach Sergey Fujita Go Momose Kunikazu Momose Yuria Ueta Mutsuyuki Andronov Vladimir Mita Nagahisa Kanai Yutaka 《Ecological Research》1998,13(3):273-282
Autumn migration routes of red-crowned cranes, Grus japonensis, from two continental east Asian sites were documented in detail by satellite tracking. Two routes were identified: a 2200km western route from Russias Khingansky Nature Reserve to coastal Jiangsu Province, China; and a 900km eastern route from Lake Khanka (Russia) to the Korean Peninsula and the Demilitarized Zone. The most important rest-sites were identified as Panjin Marsh (China), coastal mudflats south-east of Tangshan City (China), the Yellow River mouth (China), Tumen River mouth (North Korea/China/Russia), Kumya (North Korea) and Cholwon (Korean DMZ). Movements within the wintering range were also recorded, including complex commuting between sites by individual cranes and patterns of daily movements within sites. These data should prove useful for conservation of the flyway. 相似文献