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排序方式: 共有201条查询结果,搜索用时 546 毫秒
101.
Brigitte Hertel Sascha Tayefeh Thomas Kloss Jennifer Hewing Manuela Gebhardt Dirk Baumeister Anna Moroni Gerhard Thiel Stefan M. Kast 《European biophysics journal : EBJ》2010,39(7):1057-1068
The viral potassium channel Kcv comprises only 94 amino acids, which represent the pore module of more complex K+ channels. As for Kir-type channels, Kcv also has a short N-terminal helix exposed to the cytoplasm, upstream of the first
transmembrane domain. Here we show that this helix is relevant for Kcv function. The presence of charged amino acids, which
form dynamic inter- and intra-subunit salt bridges is crucial. Electrophysiological measurements, yeast rescue experiments
and molecular dynamics simulations show that mutants in which the critical salt bridge formation is impaired have no or reduced
channel activity. We conclude that these salt bridges destabilise the complexation of K+ ions by negative charges on the inner transmembrane domain at the entrance into the cavity. This feature facilitates a continuous
and coordinated transfer of ions between the cavity and the cytoplasm for channels without the canonical bundle crossing. 相似文献
102.
Prephenate dehydratase (PDT) is an important but poorly characterized enzyme that is involved in the production of L-phenylalanine. Multiple-sequence alignments and a phylogenetic tree suggest that the PDT family has a common structural fold. On the basis of its sequence, the PDT from the extreme thermophile Methanocaldococcus jannaschii (MjPDT) was chosen as a promising representative of this family for pursuing structural and functional studies. The corresponding pheA gene was cloned and expressed in Escherichia coli. It encodes a monofunctional and thermostable enzyme with an N-terminal catalytic domain and a C-terminal regulatory ACT domain. Biophysical characterization suggests a dimeric (62 kDa) protein with mixed alpha/beta secondary structure elements. MjPDT unfolds in a two-state manner (Tm = 94 degrees C), and its free energy of unfolding [DeltaGU(H2O)] is 32.0 kcal/mol. The purified enzyme catalyzes the conversion of prephenate to phenylpyruvate according to Michaelis-Menten kinetics (kcat = 12.3 s-1 and Km = 22 microM at 30 degrees C), and its activity is pH-independent over the range of pH 5-10. It is feedback-inhibited by L-phenylalanine (Ki = 0.5 microM), but not by L-tyrosine or L-tryptophan. Comparison of its activation parameters (DeltaH(++)= 15 kcal/mol and DeltaS(++)= -3 cal mol-1 K-1) with those for the spontaneous reaction (DeltaH(++) = 17 kcal/mol and DeltaS(++)= -28 cal mol-1 K-1) suggests that MjPDT functions largely as an entropy trap. By providing a highly preorganized microenvironment for the dehydration-decarboxylation sequence, the enzyme may avoid the extensive solvent reorganization that accompanies formation of the carbocationic intermediate in the uncatalyzed reaction. 相似文献
103.
Chiriva-Internati M Grizzi F Pinkston J Morrow KJ D'Cunha N Frezza EE Muzzio PC Kast WM Cobos E 《In vitro cellular & developmental biology. Animal》2006,42(3-4):89-95
Summary The γ-irradiation of normal cells causes an increased synthesis of specific proteins. However, few studies have described
the effects of high doses of irradiation on the expression of cell surface antigens in tumor cells. This study analyzed the
effects of high doses of γ-irradiation on the surface antigen expression of Major Histocompatability Complex (MHC) class I/II
and intercellular adhesion molecule-1 (ICAM-I) in human multiple myeloma (MM) cell lines ARP-1, ARK-RS, and 10 MM primary
tumors. The expression of surface antigens was evaluated by fluorescence-activated cell sorter analysis at different time
points, following the exposure to high doses of γ-irradiation. Doses of 10,000 and 15,000 cGy were no0105 sufficient to totally
block cell replication in both cell lines and primary tumors; cell replication was able to be inhibited completely only at
18,000 cGy. Lower doses (10,000 cGy) and lethal doses of irradiation (i.e., 15,000 and 18,000 cGy) increased the expression
of all surface antigens present on the cells before irradiation. Essentially, such upregulation was shown to be dose dependent,
with higher radiation doses resulting in higher antigen expression. Furthermore, when the kinetics of this upregulation were
studied 3 and 6 d after irradiation, there was a constant increase in antigen expression in MM cells. These findings suggest
that upregulation of costimulatory molecules, such as of MHC class I/II antigens and ICAM-1 molecules in MM patients treated
by γ-radiation, can increase the immunogenicity of the tumor cells. In light of these findings, radiotherapy combined with
immunotherapy might be considered in relapsing patients after receiving the standard treatment. 相似文献
104.
Le Poole IC ElMasri WM Denman CJ Kroll TM Bommiasamy H Lyons Eiben G Kast WM 《Cancer immunology, immunotherapy : CII》2008,57(6):789-797
Dendritic cells (DC) can be cytotoxic towards tumor cells by means of TNF family molecules expressed on the cell surface of
activated DCs. Tumor cells expressing appropriate receptors are killed by DC, generating a source of antigen to be presented
to the immune system. It has not been investigated whether Langerhans cells (LC) are selectively cytotoxic to tumor cells.
This is of particular interest for epithelial tumor cells that physically interact with LC in vivo. Among epithelial tumors,
the oncogenic process of cervical tumors is relatively well defined by their Human Papillomavirus (HPV) mediated etiology.
To study whether HPV16 E6 and E7 expressions, otherwise observed in cervical tumor cells, can sensitize normal cervical epithelial
cells to DC and LC mediated killing, the E6 and E7 genes were introduced by retroviral transfection, and cells were subsequently
used as targets in cytotoxicity assays. Expression of cytotoxic molecules by effector cells was measured in response to the
pro-inflammatory cytokine IFN-γ; cytotoxicity was established and concomitant expression of receptor molecules was assessed
on target cells. A correlation between the shrinkage of HPV16 E6 and E7+ tumors versus DC and LC infiltration was evaluated
in a murine model of cervical cancer. DC and LC proved to be equally cytotoxic towards E6 and E7 expressing cervical epithelial
cells. IFN-γ induced TRAIL expression by DC and LC, and inhibition of TRAIL partially blocked cytotoxic effects. Expression
of TRAIL decoy receptors was reduced following introduction of E6 and E7 into host cells. Shrinkage of HPV16 E6 and E7 expressing
tumors correlated with infiltration by S100+ DC and LC, co-localizing with apoptotic mouse tumor cells. In conclusion, DC
and LC mediated killing may be exploitable for anti-tumor treatment.
I. Caroline Le Poole and W.M. ElMasri have contributed equally to this paper. 相似文献
105.
Janetzki S Panageas KS Ben-Porat L Boyer J Britten CM Clay TM Kalos M Maecker HT Romero P Yuan J Kast WM Hoos A;Elispot Proficiency Panel of the CVC Immune Assay Working Group 《Cancer immunology, immunotherapy : CII》2008,57(3):303-315
The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels. 相似文献
106.
107.
Application of ultrasound for pregnancy diagnosis has been tested and evaluated in 15 Iranian camels (Camelus dromedarius), all of which ultimately calved. Transabdominal examinations were unsuccessful, while intrapelvic application resulted in the reception of sounds characteristic for foetal life, similar to those found in other domestic animals. Signals of foetal heart, pulse of umbilical vessels and uterine artery as well as foetal movement could be recognized as distinct sounds and have been recorded for further studies. An attempt was made to verify the findings of the ultrasonic diagnosis through rectal palpation. The ultrasonic technique resulted in 12 correct and three incorrect diagnoses. 相似文献
108.
Ribosomal protein S14 genes (RPS14) in eukaryotic species from protozoa to
primates exhibit dramatically different intron-exon structures yet share
homologous polypeptide-coding sequences. To recognize common features of
RPS14 gene architectures in closely related mammalian species and to
evaluate similarities in their noncoding DNA sequences, we isolated the
intron-containing S14 locus from Chinese hamster ovary (CHO) cell DNA by
using a PCR strategy and compared it with human RPS14. We found that rodent
and primate S14 genes are composed of identical protein-coding exons
interrupted by introns at four conserved DNA sites. However, the structures
of corresponding CHO and human RPS14 introns differ significantly.
Nonetheless, individual intron splice donor, splice acceptor, and upstream
flanking motifs have been conserved within mammalian S14 homologues as well
as within RPS14 gene fragments PCR amplified from other vertebrate genera
(birds and bony fish). Our data indicate that noncoding, intronic DNA
sequences within highly conserved, single-copy ribosomal protein genes are
useful molecular landmarks for phylogenetic analysis of closely related
vertebrate species.
相似文献
109.
George?M?WarimweEmail author Gema?Lorenzo Elena?Lopez-Gil Arturo?Reyes-Sandoval Matthew?G?Cottingham Alexandra?J?Spencer Katharine?A?Collins Matthew?DJ?Dicks Anita?Milicic Amar?Lall Julie?Furze Alison?V?Turner Adrian?VS?Hill Alejandro?Brun Sarah?C?Gilbert 《Virology journal》2013,10(1):349
Background
Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens.Methods
Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice.Results
A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8+ T cell response.Conclusions
Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.110.
An N-terminal protein degradation tag enables robust selection of highly active enzymes 总被引:1,自引:0,他引:1
Degradation tags are short peptide sequences that target proteins for destruction by housekeeping proteases. We previously utilized the C-terminal SsrA tag in directed evolution experiments to decrease the intracellular lifetime of a growth-limiting enzyme and thereby facilitate selection of highly active variants. In this study, we examine the N-terminal RepA tag as an alternative degradation signal for laboratory evolution. Although RepA proved to be less effective than SsrA at lowering protein concentrations in the cell, its N-terminal location dramatically reduced the occurrence of truncation and frameshift artifacts in selection experiments. We exploited this improvement to evolve a topologically redesigned chorismate mutase that is intrinsically disordered but already highly active for the conversion of chorismate to prephenate. After three rounds of mutagenesis and high-stringency selection, a robust and more nativelike variant was obtained that exhibited a catalytic efficiency (k(cat)/K(M) = 84000 M(-1) s(-1)) comparable to that of a natural dimeric chorismate mutase. Because of concomitant increases in catalyst yield, the level of intracellular prephenate production increased approximately 30-fold overall over the course of evolution. 相似文献