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41.
Dependence of catalysis and CO2/O2 specificity of Rubisco on the carboxy-terminus of the large subunit at different temperatures 总被引:1,自引:1,他引:0
Zhu Genhai Jensen Richard G. Bohnert Hans J. Wildner Günter F. Schlitter Jürgen 《Photosynthesis research》1998,57(1):71-79
Variations in length and charge of the C-terminus of the Rubisco large subunit (L) can be seen in L from different phylogenetic lineages. We examined the catalytic parameters of Rubisco from higher plants and from engineered Synechococcus rbcL in relation to differences in the C-terminus. Among three selected higher plants, spinach, wheat, and Flaveria pringlei, spinach Rubisco with the shortest C-tail extension (D-473 + 2) showed the lowest temperature response. The response of Rubisco from wheat (D-473 + 4) was intermediate, and the enzyme from F. pringlei (D-473 + 12) displayed the highest temperature response in terms of Vmax for the carboxylase reaction. This observation was further investigated in a model system: the temperature-response for carboxylation was enhanced after lengthening the C-terminus of the Synechococcus large subunit protein by two amino acid residues (DK). The results point towards the length of the C-terminus as an additional factor for controlling Rubisco activity, especially as an adaptation that widens the temperature range in which the enzyme can function. Longer C-termini, we suggest, could establish additional interactions with the protein surface. 相似文献
42.
Genetic analysis of rec E activities in Bacillus subtilis 总被引:3,自引:0,他引:3
Piotr Ceglowski Gerhild Lüder Juan C. Alonso 《Molecular & general genetics : MGG》1990,222(2-3):441-445
Summary ArecE mutant (recE6) ofBacillus subtilis was constructed by insertion of a selectable marker into therecE coding region. The insertional inactivation of therecE gene renders cells very sensitive to DNA damaging agents and severely impairs intermolecular recombination, but does not markedly affect plasmid interstrand annealing and intramolecular recombination. TherecE6 allele was then introduced into a set of DNA repair-deficient strains ofB. subtilis. The removal of DNA damage by therecF,addAaddB,recH,recL andrecP gene products is strictly dependent on an activerecE gene product (recE-dependent pathway). On the other hand, the increased sensitization to purine adducts in theuvrA42recE6 andpolA5recE6 strains suggests that such lethal lesions may be removed either by therecE-dependent or by therecE-independent pathway. 相似文献
43.
44.
45.
Maria Strianese Gerhild Zauner Luigi Bubacco Thijs J. Aartsma Leandro C. Tabares 《Analytical biochemistry》2009,385(2):242-248
Fluorescently labeled hemocyanin has been previously proposed as an oxygen sensor. In this study, we explored the efficacy of this biosensor for monitoring the biological oxygen consumption of bacteria and its use in testing bacterial cell growth and viability of Escherichia coli, Pseudomonas aeruginosa, Paracoccus denitrificans, and Staphylococcus simulans. Using a microwell plate, the time courses for the complete deoxygenation of samples with different initial concentrations of cells were obtained and the doubling times were extracted. The applicability of our fluorescence-based cell growth assay as an antibacterial drug screening method was also explored. The results provide a proof-of-principle for a simple, quantitative, and sensitive method for high-throughput monitoring of prokaryotic cell growth and antibiotic susceptibility screening. 相似文献
46.
Wibke Bayer Matthias Tenbusch Ruth Lietz Lena Johrden Simone Schimmer Klaus überla Ulf Dittmer Oliver Wildner 《Journal of virology》2010,84(4):1967-1976
We present a new type of adenoviral vector that both encodes and displays a vaccine antigen on the capsid, thus combining in itself gene-based and protein vaccination; this vector resulted in an improved vaccination outcome in the Friend virus (FV) model. For presentation of the envelope protein gp70 of Friend murine leukemia virus on the adenoviral capsid, gp70 was fused to the adenovirus capsid protein IX. When compared to vaccination with conventional FV Env- and Gag-encoding adenoviral vectors, vaccination with the adenoviral vector that encodes and displays pIX-gp70 combined with an FV Gag-encoding vector resulted in significantly improved protection against systemic FV challenge infection, with highly controlled viral loads in plasma and spleen. This improved protection correlated with improved neutralizing antibody titers and stronger CD4+ T-cell responses. Using a vector that displays gp70 without encoding it, we found that while the antigen display on the capsid alone was sufficient to induce high levels of binding antibodies, in vivo expression was necessary for the induction of neutralizing antibodies. This new type of adenovirus-based vaccine could be a valuable tool for vaccination.Adenoviruses have been a focus of interest as vaccine vectors for more than a decade and have been tested in various preclinical and clinical studies for vaccination against viral and bacterial infections (reviewed in reference 38). This interest is based on the ability of adenoviral vectors to induce high antibody titers and robust cytotoxic T-lymphocyte (CTL) responses and on the high immunogenicity of the vector, which might have an adjuvant effect on vaccination (17). Adenoviral vectors have also been extensively evaluated for immunization against HIV (reviewed in reference 1), where they were used either alone or in combination with plasmid DNA or protein in prime-boost immunizations. However, vaccination with adenoviral vectors against HIV showed no effectiveness in a large phase IIb study (4), but it is conceivable that the observed lack of effectiveness was due to the choice of vaccine antigen rather than the vector itself, as the vaccine relied exclusively on the induction of CTL responses, and the outcome was unexpected given previous results from studies in nonhuman primates (33, 42). The findings of the phase IIb study brought about a shift of focus from the CTL response to a more balanced immune response, including neutralizing antibodies, that is now expected to be necessary for protection from HIV infection.Apart from adenoviral vectors that encode vaccine antigens, there have also been approaches to modify adenoviral capsid proteins to include antigenic epitopes. These were mostly inserted into external loops of the hexon protein (5, 22, 25, 26, 43), which is the main component of the adenovirus capsid, but also other components of the capsid, such as fiber, protein IX, and penton base, have been evaluated (22). These studies showed that incorporation of single epitopes into capsid proteins of adenovirus leads to induction of antibody and CD4+ T-cell responses, suggesting that incorporation of epitopes into the adenovirus capsid is a useful tool for epitope-based vaccination.Fusion of a polylysine sequence or an arginine-glycine-aspartic acid motif to adenovirus pIX has been shown to be a tool for redirection of adenovirus tropism to heparan sulfate and αvβ integrins, respectively (9, 41). By fusing green fluorescent protein and luciferase to the C terminus of pIX, it was shown that relatively large proteins can be displayed on the adenovirus capsid while maintaining the protein''s conformation and function as well as virion integrity (24, 28).Here we describe a novel vaccination approach that combines genetic and protein vaccination by using adenoviral vectors not only as gene expression vectors but also as nanoparticle carriers for a vaccine antigen to improve the vaccination efficiency through enhanced induction of antibodies. Display of the vaccine antigen on the adenovirus capsid was achieved by fusion of the antigen to the C terminus of the adenovirus capsid protein pIX. It was shown before that the presentation of antigens in ordered arrays leads to improved antibody responses by cross-linking of B-cell receptors (13). As the adenoviral capsid is highly structured, we hypothesized that fusion to pIX would result in an ordered display of the antigen, presumably facilitating antibody induction.We evaluated this vaccine approach using the Friend virus (FV) infection model. FV is an immunosuppressive retroviral complex that consists of Friend murine leukemia virus (F-MuLV) and the replication-deficient, F-MuLV-dependent spleen focus-forming virus. FV infection of susceptible mice induces rapid polyclonal erythroblast proliferation, which leads to splenic enlargement and erythroleukemia and takes a lethal course also in adult mice (14). Protection from FV infection has been shown to require complex immune responses involving antibodies as well as CD4+ and CD8+ T cells (7). FV is regarded as a useful retrovirus infection model because basic requirements for vaccine protection seem to be similar for FV and HIV infection (8). We demonstrated previously that the FV model is suitable to evaluate and improve adenoviral vectors for antiretroviral vaccination (2), as we showed that a heterologous prime-boost vaccination with adenovirus type 5 (Ad5) and fiber chimeric Ad5F35 vectors led to better protection from FV infection than homologous vaccination, which correlated with improved induction of neutralizing antibodies.For vaccination with expression/display vectors against FV we constructed a fusion protein of the adenoviral capsid protein pIX and the F-MuLV envelope protein gp70 and produced adenoviral vectors expressing the pIX-gp70 fusion protein, which was incorporated into the viral capsid. We vaccinated FV-susceptible CB6F1 hybrid mice with antigen expression/display vectors or with conventional antigen-expressing adenoviral vectors and analyzed the protection conferred by these two vaccines. Having demonstrated that the expression/display vector leads to better protection of mice from FV challenge, we constructed a panel of expression/display vectors displaying different fusion proteins containing F-MuLV Env or Gag in order to elucidate the underlying immunological mechanisms of the improved protection conferred by the adenoviral expression/display vectors. 相似文献
47.
Sphingolipids were discovered more than a century ago in the brain. Cerebrosides and sphingomyelins were named so because they were first isolated from neural tissue. Although glycosphingolipids and especially those containing sialic acid in their oligosaccharide moiety are particularly abundant in the brain, sphingolipids are ubiquitous cellular membrane components. They form cell- and species-specific profiles at the cell surfaces that characteristically change in development, differentiation, and oncogenic transformation, indicating the significance of these lipid molecules for cell-cell and cell-matrix interactions as well as for cell adhesion, modulation of membrane receptors and signal transduction. This review summarizes sphingolipid metabolism with emphasis on aspects particularly relevant in neural cell types, including neurons, oligodendrocytes and neuroblastoma cells. In addition, the reader is briefly introduced into the methodology of lipid evaluation techniques and also into the putative physiological functions of glycosphingolipids and their metabolites in neural tissue. 相似文献
48.
Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species 总被引:3,自引:0,他引:3
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Jogler C Hoffmann D Theegarten D Grunwald T Uberla K Wildner O 《Journal of virology》2006,80(7):3549-3558
Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice. 相似文献
49.
Kuznetsova S Zauner G Schmauder R Mayboroda OA Deelder AM Aartsma TJ Canters GW 《Analytical biochemistry》2006,350(1):52-60
A method for fluorescence detection of a protein's redox state based on resonance energy transfer from an attached fluorescence label to the prosthetic group of the redox protein is described and tested for proteins containing three types of prosthetic groups: a type-1 copper site (azurin, amicyanin, plastocyanin, and pseudoazurin), a heme group (cytochrome c550), and a flavin mononucleotide (flavodoxin). This method permits one to reliably distinguish between reduced and oxidized proteins and to perform potentiometric titrations at submicromolar concentrations. 相似文献
50.
Nadine Hagen Paul P. Van Veldhoven Richard L. Proia Hyejung Park Alfred H. Merrill Jr. Gerhild van Echten-Deckert 《The Journal of biological chemistry》2009,284(17):11346-11353
Cerebellar granule cells from sphingosine 1-phosphate (S1P) lyase-deficient
mice were used to study the toxicity of this potent sphingolipid metabolite in
terminally differentiated postmitotic neurons. Based on earlier findings with
the lyase-stable, semi-synthetic, cis-4-methylsphingosine phosphate,
we hypothesized that accumulation of S1P above a certain threshold induces
neuronal apoptosis. The present studies confirmed this conclusion and further
revealed that for S1P to induce apoptosis in lyase-deficient neurons it must
also be produced by sphingosine-kinase2 (SK2). These conclusions are based on
the finding that incubation of lyase-deficient neurons with either sphingosine
or S1P results in a similar elevation in cellular S1P; however, only S1P
addition to the culture medium induces apoptosis. This was not due to S1P
acting on the S1P receptor but to hydrolysis of S1P to sphingosine that was
phosphorylated by the cells, as described before for
cis-4-methylsphingosine. Although the cells produced S1P from both
exogenously added sphingosine as well as sphingosine derived from exogenous
S1P, the S1P from these two sources were not equivalent, because the former
was primarily produced by SK1, whereas the latter was mainly formed by SK2 (as
also was cis-4-methylsphingosine phosphate), based on studies in
neurons lacking SK1 or SK2 activity. Thus, these investigations show that, due
to the existence of at least two functionally distinct intracellular origins
for S1P, exogenous S1P can be neurotoxic. In this model, S1P accumulated due
to a defective lyase, however, this cause of toxicity might also be important
in other cases, as illustrated by the neurotoxicity of
cis-4-methylsphingosine phosphate.Sphingosine 1-phosphate
(S1P)2 is a potent
lipid mediator that has been shown to regulate a wide range of physiological
processes, including proliferation, differentiation, motility, cytoskeleton
rearrangements, and calcium homeostasis
(1,
2). There is convincing
experimental evidence that this bioactive sphingolipid can act both
extracellularly, as a ligand for a family of five specific G protein-coupled
receptors, and inside the cells, as a second messenger
(3,
4). In most cell types
described so far, S1P and its metabolic precursor ceramide exert antagonistic
effects on cell survival with S1P being generally regarded as a survival
signal, whereas ceramide and sphingosine are generally toxic
(5,
6). Interestingly, generation
of sphingosine and S1P is generally thought to be dependent on the
availability of ceramide (7),
however, relatively high amounts of S1P are also present in blood, lymph, and
cerebrospinal fluid (8,
9) and may serve as additional
sources for some cells.More than a decade ago, we introduced the synthetic sphingosine analog
cis-4-methylsphingosine as a tool for studies of sphingoid base
metabolism and function (10).
When added to the culture medium, this analog is taken up and mainly
phosphorylated to the respective cis-4-methylsphingosine phosphate,
which accumulates intracellularly, because it is poorly cleaved (if at all) by
S1P lyase (10). Intriguingly,
this compound promoted proliferation of quiescent Swiss 3T3 fibroblasts
(11), as does S1P
(12), but induced apoptosis in
postmitotic terminally differentiated primary cultured neurons
(13).Despite the fact that neither S1P nor sphingosine were able to induce
apoptosis in neurons, we proposed that cis-4-methylsphingosine is
phosphorylated by cells yielding a metabolically stable analog of S1P. This
prediction was based on experimental results indicating that the different
physiological effects, apoptosis in the case of the accumulating metabolically
stable synthetic compound versus no apoptosis in the case of the
short living S1P, rely only on nuances of impact
(13). Both sphingoid
phosphates affected similar pathways. However, the effect of the synthetic
accumulated compound was more pronounced and persistent when compared with the
more transient and less pronounced effect of the short living physiological
counterpart (13). We therefore
assumed that conditions that allow sufficient accumulation of S1P in primary
cultured neurons should end up in neuronal apoptosis.To explore this hypothesis, which might be relevant to neurodegenerative
processes, we attempted to elevate intracellular S1P using siRNAs directed to
S1P lyase (encoded by the Sgpl1 gene). However, suppression of lyase
by ∼70% did not result in an accumulation of endogenous S1P in primary
cultured neurons (14).The central aim of the present study was to evaluate the hypothesis that
endogenous S1P induces neuronal apoptosis when it exceeds a certain threshold
by a more effective method for lyase activity suppression. We thus used
primary cultured neurons prepared from cerebella of 6-day-old lyase-deficient
mice (15). The present studies
not only confirmed that elevation of S1P induced cell death but also revealed
that the origin of the S1P was important. Intriguingly, neuronal apoptosis was
induced only by S1P derived from exogenous S1P that was dephosphorylated and
then resynthesized to S1P by sphingosine kinase 2 (SK2). Interestingly, we
then found that this is also the kinase responsible for synthesis of
cis-4-methylsphingosine phosphate. In addition, our data document
that the pro-apoptotic effect of S1P is independent of cellular ceramide
content. 相似文献