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121.
Whitehouse DB; Tomkins J; Lovegrove JU; Hopkinson DA; McMillan WO 《Molecular biology and evolution》1998,15(4):456-462
The expanding molecular database provides unparalleled opportunities for
characterizing genes and for studying groups of related genes. We use
sequences drawn from the database to construct an evolutionary framework
for examining the important glycolytic enzyme phosphoglucomutase (PGM).
Phosphoglucomutase plays a pivotal role in the synthesis and utilization of
glycogen and is present in all organisms. In humans, there are three
well-described isozymes, PGMI, PGM2, and PGM3. PGM1 was cloned 5 years ago;
however, repeated attempts using both immunological approaches and
molecular probes designed from PGM1 have failed to isolate either PGM2 or
PGM3. Using a phylogenetic strategy, we first identified 47 highly
divergent prokaryotic and eukaryotic PGM-like sequences from the database.
Although overall amino acid identity often fell below 20%, the relative
order, position, and sequence of three structural motifs, the active site
and the magnesium-- and sugar-binding sites, were conserved in all 47
sequences. The phylogenetic history of these sequences was complex and
marked by duplications and translocations; two instances of transkingdom
horizontal gene transfer were identified. Nonetheless, the sequences fell
within six well-defined evolutionary lineages, three of which contained
only prokaryotes. Of the two prokaryotic/eukaryotic lineages, one contained
bacterial, yeast, slimemold, invertebrate, and vertebrate homologs to human
PGM1 and the second contained likely homologs to human PGM2. Indeed, an
amino acid sequence, derived from a partial human cDNA, that fell within
the second cross-kingdom lineage bears several characteristics expected for
PGM2. A third lineage may contain homologs to human PGM3. On a general
level, our phylogenetic-based approach shows promise for the further
utilization of the extensive molecular database.
相似文献
122.
Quantifying heterogeneity: flow cytometry of bacterial cultures 总被引:1,自引:0,他引:1
Douglas B. Kell Hazel M. Ryder Arseny S. Kaprelyants Hans V. Westerhoff 《Antonie van Leeuwenhoek》1991,60(3-4):145-158
Flow cytometry is a technique which permits the characterisation of individual cells in populations, in terms of distributions in their properties such as DNA content, protein content, viability, enzyme activities and so on. We review the technique, and some of its recent applications to microbiological problems. It is concluded that cellular heterogeneity, in both batch and continuous axenic cultures, is far greater than is normally assumed. This has important implications for the quantitative analysis of microbial processes. 相似文献
123.
On the extent of localization of the energized membrane state in chromatophores from Rhodopseudomonas capsulata N22. 总被引:5,自引:5,他引:0
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1. The principle of the double-inhibitor titration method for assessing competing models of electron transport phosphorylation is expounded. 2. This principle is applied to photophosphorylation by chromatophores from Rhodopseudomonas capsulata N22. 3. It is found that, in contrast to the predictions of the chemiosmotic coupling model, free energy transfer is confined to individual electron transport chain and ATP synthase complexes. 4. This conclusion is not weakened by arguments concerning, the degree of uncoupling in the native chromatophore preparation or the relative number of electron transport chain and ATP synthase complexes present. 5. Photophosphorylation is completely inhibited by the uncoupler SF 6847 at a concentration corresponding to 0.31 molecules per electron transport chain. 6. The apparent paradox is solved by the proposal, consistent with the available evidence on the mode of action of uncouplers, that uncoupler binding causes a co-operative conformation transition in the chromatophore membrane, which leads to uncoupling and which is not present in the absence of uncoupler. 相似文献
124.
Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration 总被引:14,自引:4,他引:10
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The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout. 相似文献
125.
Estimation with an ion-selective electrode of the membrane potential in cells of Paracoccus denitrificans from the uptake of the butyltriphenylphosphonium cation during aerobic and anaerobic respiration. 总被引:6,自引:0,他引:6
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1. Aerobic respiration by cells of Paracoccus dentrificans drives the uptake of the lipophilic cation butyltriphenylphosphonium. Anaerobiosis or addition of an uncoupler of oxidative phosphorylation (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) results in efflux of the cation. Changes in the concentration of butyltriphenylphosphonium in the suspension medium were measured by using an ion-selective electrode, the construction of which is described. 2. If the uptake of butyltriphenylphosphonium is used as an indicator of membrane potential, then at pH 7.3 an estimate of about 160 mV is obtained for cells of P. dentrificans respiring aerobically in 100 mM-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid/NaOH or 100mM-NaH2PO4/NaOH. This potential, however, is decreased by more than 20 mV in reaction media containing a high concentration of phosphate (100 mM) together with at least 1 mM-K+. 3. Anaerobic electron transport with NO3-, NO2- or N2O as terminal electron acceptor generates a membrane potential of about 150mV in described suspension media. The presence of these species under aerobic conditions, moreover, has negligible effect upon the extent of uptake of butyltriphenylphosphonium normally driven by aerobic respiration. These data indicate that none of these molecules exert a significant uncoupling effect on the protonmotive force. 4. No 204Tl+ uptake into respiring cells was detected. This adds to the evidence that 204Tl+ is not a freely permeable cation in bacterial cells and therefore not an indicator of membrane potential as has been proposed. The absence of respiration-driven 204Tl+ uptake indicates that P. denitrificans cells grown under the conditions specified in the present work do not possess K+-transport systems of either the Kdp or TrkA types that have been described in Escherichia coli. 相似文献
126.
We used parameter scanning to emulate changes to the limiting rate for steps in a fitted model of glucose-derepressed yeast glycolysis. Three flux-control regimes were observed, two of which were under the dominant control of hexose transport, in accordance with various experimental studies and other model predictions. A third control regime in which phosphofructokinase exerted dominant glycolytic flux control was also found, but it appeared to be physiologically unreachable by this model, and all realistically obtainable flux control regimes featured hexose transport as a step involving high flux control. 相似文献
127.
Quantification of microbial productivity via multi-angle light scattering and supervised learning 总被引:1,自引:0,他引:1
This article describes the use of chemometric methods for prediction of biological parameters of cell suspensions on the basis of their light scattering profiles. Laser light is directed into a vial or flow cell containing media from the suspension. The intensity of the scattered light is recorded at 18 angles. Supervised learning methods are then used to calibrate a model relating the parameter of interest to the intensity values. Using such models opens up the possibility of estimating the biological properties of fermentor broths extremely rapidly (typically every 4 sec), and, using the flow cell, without user interaction. Our work has demonstrated the usefulness of this approach for estimation of yeast cell counts over a wide range of values (10(5)-10(9) cells mL-1), although it was less successful in predicting cell viability in such suspensions. 相似文献
128.
G J Salter D B Kell L A Ash J M Adams A J Brown R James 《Enzyme and microbial technology》1990,12(6):419-430
A novel method of cell immobilization is described. The cell support consists of ceramic microspheres of approximately 50-75 microns diameter. The spheres are hollow, having a wall thickness of 10-15 microns and one entrance (ca. 20 microns diameter). The walls are porous with a mean pore size of approximately 90 nm. When a cell suspension (of S. cerevisiae) is passed through a column of such particles, cells are immobilized. Conditions are devised such that the overwhelming majority of cells are held in the central cavity of the support and not between the particles. Provided turbulence is avoided, the distribution of cells along the column length in the steady state is rather homogeneous. The facts that (a) essentially all particles, regardless of orientation, entrap cells, and (b) nonporous particles also entrap cells with high efficiency, indicate that filtration effects are irrelevant and that heretofore unrecognized hydrodynamic forces are alone responsible for the cell immobilization. Cells can be immobilized to high biomass densities, while the hydrodynamic properties of columns containing such immobilized cells are excellent. We describe an on-line electronic method for the real-time measurement of immobilized cellular biomass. Cell growth (so recorded) and metabolism continue to occur in such particles at high rates. Using the glycolytic production of ethanol by S. cerevisiae as a model reaction, volumetric productivities as great as any published are obtained. Thus the "lobster-pot effect" or "hydrodynamic deposition" represents a novel, promising, and generally applicable method of cell immobilization. 相似文献
129.
1. Two major problems are encountered when one wishes to fit audio- and radio-frequency dielectric spectra of biological cell suspensions (or other materials): (a) changes in the apparent frequency-dependent permittivity of the system due to the phenomena of electrode polarisation can dominate those due to the biological system, and (b) because of the overlap of different dispersions it may be very difficult to deconvolute the individual contributions of the underlying biophysical mechanisms. 2. The extent of electrode polarisation depends substantially upon the conductivity of the medium surrounding the cells, but only marginally on the nature of the ions of a given valency contribution to it. 3. This, and the fact that the apparent time constants of the phenomena contributing to electrode polarisation are much greater than those of biological dielectric dispersions, permits one to use a simple substitution method to extract the latter in the presence of the former. This is shown both by simulation and by experiments using suspensions of human erythrocytes. 4. A spreadsheet method is described for the display of dielectric data and their conformance to the double Cole-Cole equation. The method provides a rapid and convenient approach, based on interactive graphical outputs, for the fitting of dielectric data to this equation. 5. Estimates derived from the spreadsheet program may be used in a BASIC program to arrive at the optimal fit. 6. The method is applied to the strongly-overlapping - and -dispersions of erythrocytes, permitting their deconvolution and providing a high level of accuracy.
Offprint requests to: D. B. Kell 相似文献
130.
The physiology of Clostridium sporogenes was investigated in defined, minimal media. In batch culture, the major end products of glucose dissimilation were acetate, ethanol and formate. When L-proline was present as an electron acceptor, acetate production was strongly enhanced at the expense of ethanol. As judged by assay of the relevant enzymes, glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The growth energetics of Cl. sporogenes were investigated in glucose- or L-valine-limited chemostat cultures. In the former case, the addition of L-proline to the medium caused a significant increase in the molar growth yield (as calculated by extrapolation to infinite dilution rate). This finding adds weight to the view that the reduction of L-proline by Cl. sporogenes is coupled to the conservation of free energy. 相似文献