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31.
Micheli L Radoi A Guarrina R Massaud R Bala C Moscone D Palleschi G 《Biosensors & bioelectronics》2004,20(2):190-196
The construction of an electrochemical immunosensor coupled to differential pulse voltammetry (DPV) for the detection of domoic acid (DA), a neurotoxic aminoacid responsible for the human syndrome known as "Amnesic Shellfish Poisoning" (ASP), is proposed here. The method involves the use of disposable screen-printed electrodes (SPEs) for the immunosensor development based on a "competitive indirect test". Domoic acid conjugated to bovine serum albumin (BSA-DA) was coated onto the working electrode of the SPE, followed by incubation with sample (or standard toxin) and anti-DA antibody. An anti-goat IgG-alkaline phosphatase (AP) conjugate was used for signal generation. A spectrophotometric enzyme-linked immunosorbent assay (ELISA) was used in a preliminary phase of development, prior to transferring the assay to the SPEs. Results showed a detection limit equal to 5 ng/ml of toxin. The electrochemical system is simple and cost-effective due to the disposable nature of the SPEs, and the analysis time is 150 min, shorter than that for the spectrophotometric method. The suitability of the assay for DA quantification in mussels was also evaluated. Samples were spiked with DA before and after the sample treatment to study the extraction efficiency and the matrix effect, respectively. After treatment, samples were analysed using a 1:250 v/v dilution in PBS-M (phosphate saline buffer pH 7.4 + CH3OH 10%) to minimise the matrix effect and allow for the detection of 20 microg/g of DA in mussel tissue. This represents the maximum acceptable limit defined by the Food and Drug Administration [Compliance Programme 7303.842. Guidance Levels, Table 3, p. 248, http://www.fda.org]. The optimised ELISA systems were then used, in parallel with a conventional HPLC method, to detect and confirm DA in shellfish extract in order to verify the performance of the electrochemical system. Very good recoveries were obtained, demonstrating the suitability of the proposed assay for accurate determination of the DA concentration in mussel samples. 相似文献
32.
Takahashi TT Bala AD Spitzer MW Euston DR Spezio ML Keller CH 《Biological cybernetics》2003,89(5):378-387
The barn owl (Tyto alba) is capable of capturing prey by passive hearing alone, guided by a topographic map of auditory space in the external nucleus of its inferior colliculus. The neurons of this auditory space map have discrete spatial receptive fields that result from the computation of interaural differences in the level (ILD) and time-of-arrival (ITD) of sounds. Below we review the synthesis of the spatial receptive fields from the frequency-specific ITDs and ILDs to which the neurons are tuned, concentrating on recent studies exploiting virtual auditory space techniques to analyze the contribution of ILD. We then compared the owls spatial discrimination, assessed behaviorally, with that of its space map neurons. Spatial discrimination was assessed using a novel paradigm involving the pupillary dilation response (PDR), and neuronal acuity was assessed by measuring the changes in firing rate resulting from changes in source location, scaled to the variance. This signal-detection-based approach revealed that the change in the position of the neural image on this map best explains the spatial discrimination measured using the PDR. We compare this result to recent studies in mammalian systems. 相似文献
33.
Inactivation of Cyanobacterial Nitrogenase After Exposure to Ultraviolet-B Radiation 总被引:2,自引:0,他引:2
Exposure of the N2-fixing cyanobacterium Anabaena BT2 to ultraviolet-B radiation (2.5 W m−2) for 30 min resulted in complete loss of nitrogenase activity but 100% cell killing occurred only after a 90-min exposure.
Inactivation of nitrogenase activity was not specific to Anabaena BT2; other species also showed a similar effect. The time required for 100% killing and inactivation of nitrogenase activity
differed in various species, and this difference may be ascribed to the presence of different levels of UV-B protection mechanisms
in individual species. Inhibition of nitrogenase activity was immediate, since exposure of cultures to UV-B for as little
as 5 min elicited some inhibition of activity. The activity of UV-B-inhibited nitrogenase did not recover upon transfer of
exposed cells to fluorescent light, suggesting that the inhibition may be due to specific inactivation of the enzyme. By employment
of inhibitors of protein synthesis and PS-II activity, it was demonstrated that restoration of nitrogenase activity in a UV-B-treated
culture occurred by fresh synthesis of nitrogenase polypeptide. Our findings suggest that estimation of nitrogenase activity
in diazotrophic species may be used as a marker enzyme for assessing the impact of UV-B radiation.
Received: 13 June 2002 / Accepted: 22 July 2002 相似文献
34.
Three-dimensional localization of pORF65 in Kaposi's sarcoma-associated herpesvirus capsid
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Of the six herpesvirus capsid proteins, the smallest capsid proteins (SCPs) share the least sequence homology among herpesvirus family members and have been implicated in virus specificity during infection. The herpes simplex virus-1 (HSV-1) SCP was shown to be horn shaped and to specifically bind the upper domain of each major capsid protein in hexons but not in pentons. In Kaposi's sarcoma-associated herpesvirus (KSHV), the protein encoded by the ORF65 gene (pORF65) is the putative SCP but its location remains controversial due to the absence of such horn-shaped densities from both the pentons and hexons of the KSHV capsid reconstructions. To directly locate the KSHV SCP, we have used electron cryomicroscopy and three-dimensional reconstruction techniques to compare the three-dimensional structure of KSHV capsids to that of anti-pORF65 antibody-labeled capsids. Our difference map shows prominent antibody densities bound to the tips of the hexons but not to pentons, indicating that KSHV SCP is attached to the upper domain of the major capsid protein in hexons but not to that in pentons, similar to HSV-1 SCP. The lack of horn-shaped densities on the hexons indicates that KSHV SCP exhibits structural features that are substantially different from those of HSV-1 SCP. The location of SCP at the outermost regions of the capsid suggests a possible role in mediating capsid interactions with the tegument and cytoskeletal proteins during infection. 相似文献
35.
Beaumont NJ Skinner VO Tan TM Ramesh BS Byrne DJ MacColl GS Keen JN Bouloux PM Mikhailidis DP Bruckdorfer KR Vanderpump MP Srai KS 《The Journal of biological chemistry》2003,278(11):8877-8880
Ghrelin is a 28-residue peptide hormone that is principally released from the stomach during fasting and prior to eating. Two forms are present in human plasma: the unmodified peptide and a less abundant acylated version, in which octanoic acid is attached to the third residue, a serine, via an ester linkage. The acylated form of ghrelin acts as a ligand for the growth hormone secretagogue receptor and can stimulate the release of growth hormone from the pituitary gland. It also initiates behavioral and metabolic adaptations to fasting. Here we show that an immobilized form of ghrelin specifically binds a species of high density lipoprotein associated with the plasma esterase, paraoxonase, and clusterin. Both free ghrelin and paraoxon, a substrate for paraoxonase, can inhibit this interaction. An endogenous species of ghrelin is found to co-purify with high density lipoprotein during density gradient centrifugation and subsequent gel filtration. This interaction links the orexigenic peptide hormone ghrelin to lipid transport and metabolism. Furthermore, the interaction of the esterified hormone ghrelin with a species of HDL containing an esterase suggests a possible mechanism for the conversion of ghrelin to des-acyl ghrelin. 相似文献
36.
Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b. 相似文献
37.
Effect of prostaglandin F2alpha (PGF2alpha) on carbohydrate accumulation in gonads of the multivoltine silkworm (hybrid: Xinhang x Keming) has been studied by means of topical application to larvae of the silkworm. Increased weights of larvae and reproduction organs, as well as carbohydrate metabolism in gonads of the silkworm, Bombyx mori L. was found after treatment with prostaglandin F2alpha. The increase in weight (larvae 21.9%, testis 28.9%, and ovary 33.3%) was associated with increases in the biomolecules (20-30%) and LDH and aldolase activity (18-25%). The results suggest that the accumulation of carbohydrates denotes a higher extent of utility of the energy sources in function of the testes and ovaries, and the routine application of prostaglandin F2alpha would be helpful in improving the reproductivity and egg quality of the silkworm. 相似文献
38.
Dhingra G Kumari R Bala S Majumdar S Malhotra S Sharma P Lal S Cullum J Lal R 《Journal of industrial microbiology & biotechnology》2003,30(4):195-204
The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow.
The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes,
cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have
led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have
broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has
been completely sequenced, while only the minimal replicon (pA-rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable
methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains.
Electronic Publication 相似文献
39.
In this study a simple electrophoresis approach has been proposed for assessing DNA damage per chromosome in vitro. Novel procedures of gel casting, sample loading, electrophoresis and quantification of damage have been suggested. Sets of Saccharomyces cerevisiae chromosomes subjected to DNA damage by Bleomycin, Co60--radiation alone and in combination with Hoechst were studied in detail. Statistical analyses showed that damage induced by Bleomycin bore linear positive correlation with %GA (r=0.97) and %GT (r=0.61) contents of chromosomes. Samples pre-treated with Hoechst showed much less damage by Co60--irradiation as compared to samples not treated with Hoechst but exposed to Co60--irradiation. The `protective effect of Hoechst' bore linear positive correlation (r=0.8) with %TAT content of chromosomes. 相似文献
40.