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71.
Asa Hagner-McWhirter Maria Winkvist Stephanie Bourin Rita Marouga 《Journal of visualized experiments : JoVE》2008,(21)
Surface proteins are central to the cell''s ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan DIGE technology and analysis of protein expression changes using DeCyder 2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.Open in a separate windowClick here to view.(76M, flv) 相似文献
72.
Kubli DA Quinsay MN Huang C Lee Y Gustafsson AB 《American journal of physiology. Heart and circulatory physiology》2008,295(5):H2025-H2031
Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) is a member of the Bcl-2 homology domain 3-only subfamily of proapoptotic Bcl-2 proteins and is associated with cell death in the myocardium. In this study, we investigated the potential mechanism(s) by which Bnip3 activity is regulated. We found that Bnip3 forms a DTT-sensitive homodimer that increased after myocardial ischemia-reperfusion (I/R). The presence of the antioxidant N-acetylcysteine reduced I/R-induced homodimerization of Bnip3. Overexpression of Bnip3 in cells revealed that most of exogenous Bnip3 exists as a DTT-sensitive homodimer that correlated with increased cell death. In contrast, endogenous Bnip3 existed mainly as a monomer under normal conditions in the heart. Screening of the Bnip3 protein sequence revealed a single conserved cysteine residue at position 64. Mutation of this cysteine to alanine (Bnip3C64A) or deletion of the NH2-terminus (amino acids 1-64) resulted in reduced cell death activity of Bnip3. Moreover, mutation of a histidine residue in the COOH-terminal transmembrane domain to alanine (Bnip3H173A) almost completely inhibited the cell death activity of Bnip3. Bnip3C64A had a reduced ability to interact with Bnip3, whereas Bnip3H173A was completely unable to interact with Bnip3, suggesting that homodimerization is important for Bnip3 function. A consequence of I/R is the production of reactive oxygen species and oxidation of proteins, which promotes the formation of disulfide bonds between proteins. Thus, these experiments suggest that Bnip3 functions as a redox sensor where increased oxidative stress induces homodimerization and activation of Bnip3 via cooperation of the NH2-terminal cysteine residue and the COOH-terminal transmembrane domain. 相似文献
73.
Håkan Sjunnesson Tobias Fält Erik Sturegård Waleed Abu Al-Soud sa Ljungh Torkel Wadström 《Current microbiology》2003,47(4):0278-0285
PCR-denaturing Gradient Gel Electrophoresis (PCR-DGGE), a method suitable for the detection of microbial species in complex ecosystems, was evaluated for the detection and identification of Helicobacter spp. in feces and stomach tissue of mice. Two commercially available stool antigen tests for clinical diagnostics in humans were also evaluated in the C57B1/6 mouse model of H. pylori infection. PCR-DGGE detected only Helicobacter ganmani in feces from H. pylori-infected as well as control animals, whereas in stomach specimens it demonstrated the presence of H. pylori in challenged and H. ganmani in control animals. Hence, the method detected DNA only of the predominant Helicobacter spp., which was also shown in cell dilution experiments. The Amplified IDEIA Hp StAR feces antigen test detected H. pylori in feces from all infected animals and generated no false-positive results, whereas the Premier Platinum HpSA-test also detected H. pylori in all infected animals but generated false-positive or equivocal results in 50% of the control animals. Premier Platinum HpSA, as opposed to Hp StAR, cross-reacted with non-pylori Helicobacter spp. in vitro.Received: 21 August 2002 / Accepted: 6 December 2002 相似文献
74.
Payam Pour Mohammadi Ahmad Moieni Asa Ebrahimi Farzad Javidfar 《Plant Cell, Tissue and Organ Culture》2012,108(2):251-256
An efficient method for producing doubled haploid plants of oilseed rape (Brassica
napus L.) was established using in vitro colchicine treatment of haploid embryos. Haploid embryos in the cotyledonary stage were
treated with one of four colchicine concentrations (125, 250, 500 and 1,000 mg/L); for one of three treatment durations (12,
24 and 36 h) at one of the two temperatures (8 and 25°C) and were compared to control embryos (without colchicine treatment).
The number of chromosomes, seed recovery, size and density of leaf stomata, and pollen grain size from regenerated plants
were determined. No doubled haploid plants were regenerated from control embryos; however, the doubled haploid plants were
regenerated from colchicine-treated embryos. A high doubling efficiency, 64.29 and 66.66% of regenerated plants, was obtained
from 250 mg/L colchicine treatment for 24 h and 500 mg/L colchicine treatment for 36 h, respectively, at 8°C. Following 500 mg/L
colchicine treatment for 36 h, a few plants regenerated (9 plants). At the higher colchicine concentration (1,000 mg/L), no
plant regenerated. These results indicate that the colchicine treatment of embryos derived from microspores can induce efficient
chromosome doubling for the production of doubled haploid lines of oilseed rape. 相似文献
75.
76.
Characterization of estrous cycles and pregnancy in Somali wild asses (Equus africanus somaliensis) through fecal hormone analyses
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Corinne P. Kozlowski Helen L. Clawitter Tim Thier Martha T. Fischer Cheryl S. Asa 《Zoo biology》2018,37(1):35-39
Although reproduction in the domestic horse has been well described, less is known about reproduction in wild equids. This study describes endocrine patterns associated with estrous cycles and pregnancy for Somali wild asses (Equus africanus somaliensis), an endangered African equid. Fecal samples were collected three times per week for more than 2 years from five female Somali wild asses at the Saint Louis Zoo; progestagen and estrogen metabolites were quantified using commercially available immunoassays. Progestagen analysis indicated that cycle lengths were 27.2 ± 1.2 days and females cycled throughout the year. Progestagen levels during early pregnancy were low and not sustained above baseline until approximately 40 weeks prior to partition. Concentrations increased markedly around 16 weeks prior to delivery and peaked 2–3 weeks before birth. Fecal estrogen levels also increased significantly starting 40–45 weeks before parturition and reached their maximal value approximately 20 weeks prior to birth. Neither foal heat nor lactational suppression of estrus was observed, and females cycled within 45 days after delivery. These data are the first to describe the reproductive physiology of Somali wild asses. As the species faces increasing threats in the wild, this information may support conservation efforts by assisting with ex situ breeding programs. 相似文献
77.
C. Asa P. Miller M. Agnew J. A. R. Rebolledo S. L. Lindsey M. Callahan & K. Bauman 《Animal Conservation》2007,10(3):326-331
The ultimate goal of the Mexican gray wolf Canis lupus baileyi captive management program is reintroduction of healthy individuals into wild habitats. To this end, zoo population managers work to provide not only for the physical well-being but also for the genetic health of these animals. However, the very limited genetic founder base, exacerbated by breeding within three distinct lineages, resulted in very high coefficients of inbreeding. Because support for measurable levels of inbreeding depression in the captive wolf population, as defined by reductions in common fitness measures such as juvenile survival or reproductive success, has been weak, we investigated the potential effects on male reproductive capacity. We analyzed semen samples from wolves from all three lineages and compared them with samples from subsequent lineage crosses and from generic gray wolves. We not only found a significant effect of inbreeding on sperm quality but we related both inbreeding and sperm quality to reproductive success. Samples from male offspring of lineage crosses, with inbreeding coefficients of zero were similar in quality to those from generic gray wolves. However, samples from a limited number of offspring from back-crosses were of extremely poor quality. Although it is reassuring that sperm quality was so much improved in male offspring of lineage crosses, the concomitant reduction in inbreeding coefficient does not eliminate the potentially deleterious alleles. Our results demonstrate that sperm quality is an important indicator of fertility and reproductive success in Mexican wolves. In addition, our data lend further support to the presence of inbreeding depression in this taxon. 相似文献
78.
79.
Apoptosis Induced by Infection of Primary Brain Cultures with Diverse Human Immunodeficiency Virus Type 1 Isolates: Evidence for a Role of the Envelope 总被引:8,自引:3,他引:5
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Asa Ohagen Sajal Ghosh Jianglin He Karen Huang Youzhi Chen Menglan Yuan Rapin Osathanondh Suzanne Gartner Bin Shi George Shaw Dana Gabuzda 《Journal of virology》1999,73(2):897-906
Apoptosis of neurons and astrocytes is induced by human immunodeficiency type 1 (HIV-1) infection in vitro and has been demonstrated in brain tissue from patients with AIDS. We analyzed a panel of diverse HIV-1 primary isolates for the ability to replicate and induce neuronal and astrocyte apoptosis in primary human brain cultures. Apoptosis was induced three- to eightfold by infection with the blood-derived HIV-1 isolates 89.6, SG3, and ADA. In contrast, the brain-derived HIV-1 isolates YU2, JRFL, DS-br, RC-br, and KJ-br did not induce significant levels of apoptosis. The ability of HIV-1 isolates to induce apoptosis was independent of their replication capacity. Studies of recombinant chimeras between the SG3 and YU2 viruses showed that replacement of the YU2 Env with the SG3 Env was sufficient to confer the ability to induce apoptosis to the YU2 virus. Replacement of the Env V3 regions alone largely conferred the phenotypes of the parental clones. The SG3 Env used CXCR4 and CCR3 as coreceptors for virus entry, whereas YU2 used CCR5 and CCR3. The V3 regions of SG3 and YU2 conferred the ability to use CXCR4 and CCR5, respectively. In contrast, the 3′ region of Env, particularly the C3V4 region, was required in conjunction with the V3 region for efficient use of CCR3. These results provide evidence that Env is a major determinant of neurodegenerative mechanisms associated with HIV-1 infection in vitro and raise the possibility that blood-derived viruses which emerge during the late stages of disease may affect disease progression in the central nervous system. 相似文献
80.
Emmanuel Olorunleke Adewuyi Yun Zhao Vishnu Khanal Asa Auta Lydia Babatunde Bulndi 《International breastfeeding journal》2017,12(1):51