全文获取类型
收费全文 | 3992篇 |
免费 | 286篇 |
出版年
2022年 | 24篇 |
2021年 | 57篇 |
2020年 | 28篇 |
2019年 | 37篇 |
2018年 | 48篇 |
2017年 | 50篇 |
2016年 | 79篇 |
2015年 | 133篇 |
2014年 | 136篇 |
2013年 | 224篇 |
2012年 | 205篇 |
2011年 | 224篇 |
2010年 | 140篇 |
2009年 | 154篇 |
2008年 | 223篇 |
2007年 | 220篇 |
2006年 | 219篇 |
2005年 | 199篇 |
2004年 | 213篇 |
2003年 | 191篇 |
2002年 | 178篇 |
2001年 | 117篇 |
2000年 | 142篇 |
1999年 | 100篇 |
1998年 | 33篇 |
1997年 | 37篇 |
1996年 | 49篇 |
1995年 | 30篇 |
1994年 | 43篇 |
1993年 | 36篇 |
1992年 | 71篇 |
1991年 | 72篇 |
1990年 | 57篇 |
1989年 | 45篇 |
1988年 | 47篇 |
1987年 | 36篇 |
1986年 | 40篇 |
1985年 | 42篇 |
1984年 | 39篇 |
1983年 | 27篇 |
1982年 | 22篇 |
1981年 | 26篇 |
1980年 | 19篇 |
1979年 | 15篇 |
1978年 | 25篇 |
1977年 | 15篇 |
1976年 | 17篇 |
1975年 | 10篇 |
1973年 | 13篇 |
1967年 | 10篇 |
排序方式: 共有4278条查询结果,搜索用时 307 毫秒
71.
Cloning of the macrolide antibiotic biosynthesis gene acyA, which encodes 3-O-acyltransferase, from Streptomyces thermotolerans and its use for direct fermentative production of a hybrid macrolide antibiotic. 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A Arisawa N Kawamura K Takeda H Tsunekawa K Okamura R Okamoto 《Applied microbiology》1994,60(7):2657-2660
A gene encoding the macrolide modification enzyme 3-O-acyltransferase (acyA) was cloned by chromosome walking onto the carbomycin biosynthetic region in Streptomyces thermotolerans TH475, with the 3' region of the gene encoding the macrolide modification enzyme 4"-O-acyltransferase (acyB1) as a probe. A shortened fragment (1.8 kb) containing acyA was subcloned with pIJ350. A high-level tylosin producer, Streptomyces fradiae MBBF, transformed with the plasmid could produce a hybrid macrolide, 3-O-acetyltylosin, most efficiently. 相似文献
72.
Isteaq Ahmed Shameem Hiroaki Kurisu Hideyasu Matsuyama Tomoyuki Shimabukuro Katsusuke Naito 《Cancer immunology, immunotherapy : CII》1994,38(6):353-357
Although the present experimental use of recombinant human granulocyte-colony-stimulating factor (rG-CSF) has been proven to alleviate the myelosuppression induced by antitumor chemotherapy, it is also believed to stimulate growth of some nonhematopoietic tumor cells. We investigated both the direct and indirect effects of rG-CSF on in vitro colony formation of human bladder cancer cell lines using a modified human tumor clonogenic assay. Peripheral blood mononuclear cells (PBMC) were used as feeder cells (a mixture of 5×104 monocytes/dish and 5×105 lymphocytes/dish obtained from healthy donors). Human bladder cancer cell lines KK-47, TCCSUP and T24, all derived from human transitional-cell carcinomas, were incubated continuously with various concentrations of rG-CSF ranging from 0.01 ng/ml to 10 ng/ml both with and without PBMC for 7–21 days. The concentrations of rG-CSF used were chosen as being in the range of achievable serum concentrations in patients treated with rG-CSF. At the end of incubation, colonies were counted under an inverted phase-contrast microscope, and an increase in the number of colonies in comparison with the control was used to evaluate the effects of rG-CSF. Results were expressed as a percentage of controls. rG-CSF in the upper layer at concentrations ranging from 0.1 ng/ml to 10 ng/ml stimulated the colony formation of all the cancer cell lines tested in the absence of PBMC in the feeder layer, whereas cells with PBMC in the feeder layer were significantly stimulated more than those without PBMC in the feeder layer (P<0.05) up to a certain concentration, which varied from cell line to cell line. At higher concentrations of rG-CSF, no further stimulation but, on the contrary, a decrease in colony formation was observed in cells with PBMC in the feeder layer in all the cell lines tested. Colony formation in KK-47 and T24 cell lines was significantly inhibited at 5 ng/ml and/or 10 ng/ml rG-CSF compared with cells without PBMC in the feeder layer. Our results suggest that rG-CSF may have both direct and indirect stimulatory effects on the growth of human bladder cancer cell lines in vitro. The results obtained also raise the possibility of adverse effects of rG-CSF in bladder cancer patients whose malignant cells may be directly and indirectly stimulated by this factor while it is being used clinically to alleviate the myelosuppression induced by antitumor chemotherapy. 相似文献
73.
Zhao Ying Hiromasa Tojo Takanori Komatsubara Manabu Nakagawa Masami Inada Sumio Kawata Yuji Matsuzawa Mitsuhiro Okamoto 《生物化学与生物物理学报:疾病的分子基础》1994,1226(2):201-205
Enzyme activity, protein contents, and mRNA contents of group II phospholipase A2 (PLA2) in hepatocellular carcinoma (HCC) surgically obtained from 8 patients were compared with those in either its neighboring liver tissues or control liver tissues. The PLA2 specific activity towards the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was significantly greater in the tumor tissues (6.62 ± 1.46 nmol/min/mg) than those in the surrounding liver tissues (1.33 ± 0.22 nmol/min/mg) and controls (0.43 ± 0.04 nmol/min/mg). The results of immunoblot analysis using a specific anti-human group II PLA2 antibody and of Northern blot analysis using a human group II PLA2 cDNA as a probe demonstrated that group II PLA2 was responsible for the increased enzyme activity. The contents of immunoreactive group II PLA2 in the tumor tissues (8.81 ± 1.24 ng/mg) were significantly higher than those in the surrounding liver tissues (1.77 ± 0.27 ng/mg); those in the control tissues were below the analytical range of the method used. The group II PLA2 mRNA was also significantly increased in the tumor tissues, compared with that in the surrounding liver tissues, whereas it was not detectable in th controls. This indicates that group II PLA2 in HCC is induced at the pretranslational level. 相似文献
74.
Serum-free culture of rat keratinocytes 总被引:2,自引:0,他引:2
Hirosuke Oku Chikara Kumamoto Tomoyuki Miyagi Takanori Hiyane Junichi Nagata Isao Chinen 《In vitro cellular & developmental biology. Animal》1994,30(8):496-503
Summary Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn
rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally
for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes,
bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine
serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support
rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors
and amino acids: insulin (10 μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2
mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth
of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and
were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately
the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study
focusing on lipid metabolism or hormonal regulation of rat keratinocytes. 相似文献
75.
Yoshiko Myoken Yoshinari Myoken Tetsuji Okamoto Mikio Kan J. Denry Sato Kazuaki Takada 《In vitro cellular & developmental biology. Animal》1994,30(11):790-795
Summary A squamous cell carcinoma cell line Nakata proliferated in serum-free culture and was not responsive to exogenous fibroblast
growth factor-1 (FGF-1). Immunostaining revealed that Nakata cells expressed FGF-1 in their cytoplasms and nuclei. Two molecular
mass species of FGF-1 (16 and 18 kDa) were identified in cell extracts by Western blot. These cells also expressed high-affinity
FGF-1 binding sites (Kd=360 pM, 28 000 sites/cell). The results of cross-linking with [125I]FGF-1 demonstrated the presence of two bands with molecular masses of 160 and 140 kDa. The addition of FGF-1 specific antisense
oligonucleotides at 25 μM to Nakata cells resulted in an 82% inhibition in cell growth and suppressed FGF-1 expression. This effect was dose-dependent
and specific, because sense oligonucleotides were ineffective in inhibiting cell growth. In addition, Nakata cell growth was
suppressed by an anti-FGF-1 neutralizing antibody, which resulted in a 52% inhibition at 8 μg/ml. These results demonstrate
that Nakata cells produce FGF-1, and indicate that this growth factor acts in an autocrine manner by interacting with FGF-1
binding sites on Nakata cells. 相似文献
76.
77.
Yoshio Oka Tetsuro Kobayashi Shoichi Fujita Nariaki Matsuura Shigeru Okamoto Hideki Asakawa Atsuo Murata Takesada Mori 《In vitro cellular & developmental biology. Animal》1993,29(7):537-542
Summary A human anaplastic thyroid cancer cell line K-119, derived from a 77-yr-old woman who had developed marked neutrophilia and
underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike and polygonal cells in shape are stably
proliferating since the beginning of its culture 2 yr ago. The cells grow rapidly and the population doubling time is 26 h.
The chromosomes show many abnormalities and many marker chromosomes have been observed. Heterotransplantation of the cells
into nude mice has resulted in the formation of tumors that are histologically interpreted as anaplastic cancer. The most
noteworthy characteristics of the cell line are the many Ki-67-positive cells (86.3%) and that the cell line spontaneously
secretes granulocyte colony-stimulating factor (G-CSF) and releases increased amounts of G-CSF in response to the stimulation
of tumor necrosis factor, interleukin 1α, and interleukin 1β. The conditioned medium obtained from K-119 cells contains an
autocrine factor stimulating the proliferation of themselves. 相似文献
78.
The enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction. 相似文献
79.
Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria. 相似文献
80.
Decomposition is a key process driving carbon and nutrient cycling in ecosystems worldwide. The home field advantage effect (HFA) has been found to accelerate decomposition rates when litter originates from “home” when compared to other (“away”) sites. It is still poorly known how HFA plays out in tropical, riparian forests, particularly in forests under restoration. We carried out three independent reciprocal litter transplant experiments to test how litter quality, soil nutrient concentrations, and successional stage (age) influenced HFA in tropical riparian forests. These experimental areas formed a wide gradient of soil and litter nutrients, which we used to evaluate the more general hypothesis that HFA varies with dissimilarity in soil nutrients and litter quality. We found that HFA increased with soil nutrient dissimilarity, suggesting that litter translocation uncouples relationships between decomposers and litter characteristics; and with litter N:P, indicating P limitation in this system. We also found negative HFA effects at a site under restoration that presented low decomposer ability, suggesting that forest restoration does not necessarily recover decomposer communities and nutrient cycling. Within each of the independent experiments, the occurrence of HFA effects was limited and their magnitude was not related to forest age, nor soil and litter quality. Our results imply that HFA effects in tropical ecosystems are influenced by litter nutrient limitation and soil nutrient dissimilarity between home and away sites, but to further disentangle major HFA drivers in tropical areas, a gradient of dissimilarity between litter and soil properties must be implemented in future experimental designs. 相似文献