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Lake Ee Khaw Günter A. B?hm Su Metcalfe James Staunton Peter F. Leadlay 《Journal of bacteriology》1998,180(4):809-814
The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by ΦC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this rapL mutant on media containing added l-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that rapL encodes a specific l-lysine cyclodeaminase important for the production of the l-pipecolate precursor. In the presence of added proline derivatives, rapL mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.Rapamycin is a 31-member macrocyclic polyketide produced by Streptomyces hygroscopicus NRRL 5491 which, like the structurally related compounds FK506 and immunomycin (Fig. (Fig.1),1), has potent immunosuppressive properties (24). Such compounds are potentially valuable in the treatment of autoimmune diseases and in preventing the rejection of transplanted tissues (16). The biosynthesis of rapamycin requires a modular polyketide synthase, which uses a shikimate-derived starter unit (11, 20) and which carries out a total of fourteen successive cycles of polyketide chain elongation that resemble the steps in fatty acid biosynthesis (2, 27). l-Pipecolic acid is then incorporated (21) into the chain, followed by closure of the macrocyclic ring, and both these steps are believed to be catalyzed by a pipecolate-incorporating enzyme (PIE) (18), the product of the rapP gene (8, 15). Further site-specific oxidations and O-methylation steps (15) are then required to produce rapamycin. Open in a separate windowFIG. 1Structures of rapamycin, FK506, and immunomycin.The origin of the pipecolic acid inserted into rapamycin has been previously established (21) to be free l-pipecolic acid derived from l-lysine (although the possible role of d-lysine as a precursor must also be borne in mind) (9). Previous work with other systems has suggested several alternative pathways for pipecolate formation from lysine (22), but the results of the incorporation of labelled lysine into the pipecolate moiety of immunomycin (Fig. (Fig.1)1) clearly indicate loss of the α-nitrogen atom (3). More recently, the sequencing of the rap gene cluster revealed the presence of the rapL gene (Fig. (Fig.2),2), whose deduced gene product bears striking sequence similarity to two isoenzymes of ornithine deaminase from Agrobacterium tumefaciens (25, 26). Ornithine deaminase catalyzes the deaminative cyclization of ornithine to proline, and we have proposed (15) that the rapL gene product catalyzes the analogous conversion of l-lysine to l-pipecolate (Fig. (Fig.3).3). Open in a separate windowFIG. 2A portion of the rapamycin biosynthetic gene cluster which contains ancillary (non-polyketide synthase) genes (15, 27). PKS, polyketide synthase.Open in a separate windowFIG. 3(A) The conversion of l-ornithine to l-proline by ornithine cyclodeaminase (17). (B) Proposed conversion of l-lysine to l-pipecolic acid by the rapL gene product.Here, we report the use of ΦC31 phage-mediated gene replacement (10) to introduce a frameshift mutation into rapL and the ability of the mutant to synthesize rapamycins in the absence or presence of added pipecolate or pipecolate analogs. 相似文献
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Visualization of Protein-RNA Interactions in Cytoplasmic Polyhedrosis Virus 总被引:16,自引:1,他引:16
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H. Zhang J. Zhang X. Yu X. Lu Q. Zhang J. Jakana D. H. Chen X. Zhang Z. H. Zhou 《Journal of virology》1999,73(2):1624-1629
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Apoptotic caspases, such as caspase-7, are stored as inactive protease zymogens, and when activated, lead to a fate-determining switch to induce cell death. We previously discovered small molecule thiol-containing inhibitors that when tethered revealed an allosteric site and trapped a conformation similar to the zymogen form of the enzyme. We noted three structural transitions that the compounds induced: (i) breaking of an interaction between Tyr-223 and Arg-187 in the allosteric site, which prevents proper ordering of the catalytic cysteine; (ii) pinning the L2′ loop over the allosteric site, which blocks critical interactions for proper ordering of the substrate-binding groove; and (iii) a hinge-like rotation at Gly-188 positioned after the catalytic Cys-186 and Arg-187. Here we report a systematic mutational analysis of these regions to dissect their functional importance to mediate the allosteric transition induced by these compounds. Mutating the hinge Gly-188 to the restrictive proline causes a massive ∼6000-fold reduction in catalytic efficiency. Mutations in the Arg-187–Tyr-223 couple have a far less dramatic effect (3–20-fold reductions). Interestingly, although the allosteric couple mutants still allow binding and allosteric inhibition, they partially relieve the mutual exclusivity of binding between inhibitors at the active and allosteric sites. These data highlight a small set of residues critical for mediating the transition from active to inactive zymogen-like states.Caspases are a family of dimeric cysteine proteases whose members control the ultimate steps for apoptosis (programmed cell death) or innate inflammation among others (for reviews, see Refs. 1 and 2). During apoptosis, the upstream initiator caspases (caspase-8 and -9) activate the downstream executioner caspases (caspase-3, -6, and-7) via zymogen maturation (3). The activated executioner caspases then cleave upwards of 500 key proteins (4–6) and DNA, leading to cell death. Due to their pivotal role in apoptosis, the caspases are involved both in embryonic development and in dysfunction in diseases including cancer and stroke (7). The 11 human caspases share a common active site cysteine-histidine dyad (8), and derive their name, cysteine aspartate proteases, from their exquisite specificity for cleaving substrate proteins after specific aspartate residues (9–13). Thus, it has been difficult to develop active site-directed inhibitors with significant specificity for one caspase over the others (14). Despite difficulties in obtaining specificity, there has been a long-standing correlation between efficacy of caspase inhibitors in vitro and their ability to inhibit caspases and apoptosis in vivo (for review, see Ref. 31). Thus, a clear understanding of in vitro inhibitor function is central to the ability control caspase function in vivo.Caspase-7 has been a paradigm for understanding the structure and dynamics of the executioner caspases (15–21). The substrate-binding site is composed of four loops; L2, L3, and L4 are contributed from one-half of the caspase dimer, and L2′ is contributed from the other half of the caspase dimer (Fig. 1). These loops appear highly dynamic as they are only observed in x-ray structures when bound to substrate or substrate analogs in the catalytically competent conformation (17–19, 22) (Fig. 1B).Open in a separate windowFIGURE 1.Allosteric site and dimeric structure in caspase-7. A, the surface of active site-bound caspase-7 shows a large open allosteric (yellow) site at the dimer interface. This cavity is distinct from the active sites, which are bound with the active site inhibitor DEVD (green sticks). B, large subunits of caspase-7 dimers (dark green and dark purple) contain the active site cysteine-histidine dyad. The small subunits (light green and light purple) contain the allosteric site cysteine 290. The conformation of the substrate-binding loops (L2, L2′, L3, and L4) in active caspase-7 (Protein Data Bank (PDB) number 1f1j) is depicted. The L2′ loop (spheres) from one-half of the dimer interacts with the L2 loop from the other half of the dimer. C, binding of allosteric inhibitors influences the conformation of the L2′ loop (spheres), which folds over the allosteric cavity (PDB number 1shj). Subunit rendering is as in panel A. Panels A, B, and C are in the same orientation.A potential alternative to active site inhibitors are allosteric inhibitors that have been seeded by the discovery of selective cysteine-tethered allosteric inhibitors for either apoptotic executioner caspase-3 or apoptotic executioner caspase-7 (23) as well as the inflammatory caspase-1 (24). These thiol-containing compounds bind to a putative allosteric site through disulfide bond formation with a thiol in the cavity at the dimer interface (Fig. 1A) (23, 24). X-ray structures of caspase-7 bound to allosteric inhibitors FICA3 and DICA (Fig. 2) show that these compounds trigger conformational rearrangements that stabilize the inactive zymogen-like conformation over the substrate-bound, active conformation. The ability of small molecules to hold mature caspase-7 in a conformation that mimics the naturally occurring, inactive zymogen state underscores the utility and biological relevance of the allosteric mechanism of inhibition. Several structural changes are evident between these allosterically inhibited and active states. (i) The allosteric inhibitors directly disrupt an interaction between Arg-187 (next to the catalytic Cys-186) and Tyr-223 that springs the Arg-187 into the active site (Fig. 3), (ii) this conformational change appears to be facilitated by a hinge-like movement about Gly-188, and (iii) the L2′ loop folds down to cover the allosteric inhibitor and assumes a zymogen-like conformation (Fig. 1C) (23).Open in a separate windowFIGURE 2.Structure of allosteric inhibitors DICA and FICA. DICA and FICA are hydrophobic small molecules that bind to an allosteric site at the dimer interface of caspase-7. Binding of DICA/FICA is mediated by a disulfide between the compound thiol and Cys-290 in caspase-7.Open in a separate windowFIGURE 3.Movement of L2′ blocking arm. The region of caspase-7 encompassing the allosteric couple Arg-187 and Tyr-223 is boxed. The inset shows the down orientation of Arg-187 and Tyr-223 in the active conformation with DEVD substrate mimic (orange spheres) in the active site. In the allosteric/zymogen conformation, Arg-187 and Tyr-223 are pushed up by DICA (blue spheres).Here, using mutational analysis and small molecule inhibitors, we assess the importance of these three structural units to modulate both the inhibition of the enzyme and the coupling between allosteric and active site labeling. Our data suggest that the hinge movement and pinning of the L2-L2′ are most critical for transitioning between the active and inactive forms of the enzyme. 相似文献
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Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12
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Yong Yang Ho-Ching Tiffany Tsui Tsz-Kwong Man Malcolm E. Winkler 《Journal of bacteriology》1998,180(7):1814-1821
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Identification of the Leader-Body Junctions for the Viral Subgenomic mRNAs and Organization of the Simian Hemorrhagic Fever Virus Genome: Evidence for Gene Duplication during Arterivirus Evolution 总被引:2,自引:1,他引:1
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E. K. Godeny A. A. F. de Vries X. C. Wang S. L. Smith R. J. de Groot 《Journal of virology》1998,72(1):862-867
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Cristina A. Viegas Paulo F. Almeida Miguel Cavaco Isabel Sá-Correia 《Applied and environmental microbiology》1998,64(2):779-783
Saccharomyces cerevisiae plasma membrane H+-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth. The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pHi). The cell population used as inoculum exhibited a significant heterogeneity of pHi, and the fall of pHi correlated with the loss of cell viability as determined by plate counts. When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.The biological target sites of octanoic acid in Saccharomyces cerevisiae may be related to processes of transport across membranes, particularly the plasma membrane (21). Like other weak acids at low pH, octanoic acid, a highly toxic by-product of yeast alcoholic fermentation (23) and an antimicrobial food additive (6), leads to the reduction of cytosolic pH (pHi) due to its dissociation in the approximately neutral cytoplasm following the entrance of the undissociated toxic form into the cell by passive diffusion (5, 20, 23). It is likely that this highly liposoluble weak acid significantly affects the spatial organization of the plasma membrane, affecting its function as a matrix for enzymes and as a selective barrier, thereby leading to the dissipation of the proton motive force across the plasma membrane and to intracellular acidification (16, 18). Significantly, the H+-ATPase in the plasma membrane in yeast, which creates the electrochemical proton gradient that drives the secondary transport of solutes and is implicated in the maintenance of pHi around neutrality, has been pointed out as a critical component of yeast adaptation to weak acids (8, 19, 24). Indeed, yeast plasma membrane H+-ATPase is activated during exponential growth with octanoic acid (19, 24), and the duration of lag phase before yeast cells enter exponential growth in the presence of sorbic acid is significantly extended in a mutant with reduced levels of plasma membrane ATPase activity (8). The activation of the H+-ATPase in the plasma membrane in yeast cells exposed to other stresses that also lead to the dissipation of the H+ gradient and intracellular acidification (such as subcritical inhibitory concentrations of ethanol [12, 14, 15], supraoptimal temperatures below 40°C [25], presence of other organic acids at low pH [1, 5, 8], and deprivation of nitrogen source [2]) have also been observed. Several lines of evidence indicate that ATPase activation is due to posttranslational modifications of the PMA1 ATPase (2, 12, 24, 25). Considerable information has been obtained on the variation of plasma membrane ATPase activity during exponential growth and early stationary phase of yeast cells cultivated in media, at low pH, supplemented or not with octanoic acid (24). However, this is not the case during the period of latency preceding exponential growth at concentrations of octanoic acid close to the maximal concentration allowing growth. The main objective of the present work was to obtain information about the pattern of ATPase activity and the changes in pHi and cell viability during the lag phase necessary for yeast adaptation to the physiological effects of octanoic acid before exponential growth.
Duration of yeast growth latency in octanoic acid-supplemented media.
When cells of S. cerevisiae IGC3507III grown, at 30°C, in medium that had not been supplemented with octanoic acid were used to inoculate buffered YG media (30 g of glucose liter−1, 6.7 g of Yeast Nitrogen Base [Difco] liter−1) (pH 4.0) supplemented with increasing concentrations of this toxic acid up to around 0.35 mM total acid (19, 23), exponential growth was initiated without significant delay (Fig. (Fig.1a),1a), although a dose-dependent decrease in specific growth rate was observed (Fig. (Fig.1b).1b). However, for higher concentrations up to the maximal that allowed growth (0.42 mM), a lag phase was observed and its duration strongly increased with the severity of octanoic acid stress (Fig. (Fig.1a).1a). The duration of latency was drastically reduced when exponential cells used as inoculum were grown in medium with an identical concentration of octanoic acid (Fig. (Fig.1a),1a), but the specific growth rate was not modified (Fig. (Fig.1b).1b). At a concentration of total octanoic acid of 0.39 mM, a lag phase of around 55 h was necessary for yeast cells, which had been cultivated under nonstressing conditions, to adapt to the deleterious effects of octanoic acid and to initiate inhibited exponential growth (Fig. (Fig.2).2). Open in a separate windowFIG. 1Effect of the addition to the growth medium of increasing concentrations of octanoic acid on the duration of lag phase (a) and the specific growth rate of S. cerevisiae IGC 3507III (b) for exponentially growing cells (used as inoculum) cultivated at 30°C at pH 4.0 in the absence (○) or presence (•) of concentrations of toxic lipophilic acid identical to those present in the growth medium. Results are representative of the many growth experiments carried out.Open in a separate windowFIG. 2Specific activity of plasma membrane H+-ATPase (filled symbols) and growth curve (open symbols) of S. cerevisiae IGC 3507III during cultivation in the presence (a) or absence (b) of 0.39 mM total octanoic acid (at pH 4.0, 30°C). The data are averages with standard deviations for at least three enzyme assays using cells from at least two independent growth experiments. OD, optical density.Activation of plasma membrane ATPase during octanoic acid-induced latency.
The specific activity of plasma membrane ATPase assayed in crude membrane suspensions prepared from nonadapted cells, as previously reported (19, 25), during cultivation in buffered medium (at pH 4.0) supplemented with 0.39 mM octanoic acid, increased during the 55 h of latency (Fig. (Fig.2a).2a). A peak of activity was reached during the early stages of exponential growth and values of ATPase activity were consistently higher (twofold) in cells grown under octanoic acid stress (Fig. (Fig.2),2), as described by Viegas et al. (24). Yeast cells must adapt to the physiological effects of octanoic acid during an extended lag period, the length of which depended on the severity of acid stress (Fig. (Fig.1a),1a), before eventually recovering and entering exponential growth; the activation of plasma membrane H+-ATPase observed during this period of latency reinforces the idea that this proton pump is an important component of this adaptative response (5, 8, 19, 24). In fact, the ability of yeast cells to grow in the presence of lipophilic acids at a low pH reflects their capacity to maintain control over their internal pH by excluding protons. This adaptative phenomenon, reported for the first time in the present work, complements the observation of Holyoak et al. (8) that a strain with reduced plasma membrane H+-ATPase activity displayed increased lag phase in the presence of the weak-acid preservative sorbic acid. Significantly, plasma membrane H+-ATPase activity was also pointed out to play a critical role in yeast tolerance of ethanol (15) or supraoptimal temperatures (13, 25). The mechanism underlying plasma membrane ATPase activation during octanoic acid-induced latency remains obscure at the present time, but it is likely that this is due to a posttranslational modification of ATPase, as proposed for ATPase activation during octanoic acid-stressed exponential growth (24). It is likely that during lag phase the amount of H+-ATPase in the plasma membrane slightly decreases, as found by Benito et al. (2) in yeast cells deprived of nitrogen source where ATPase activation also occurred (2), as the estimated half-life of the enzyme is about 11 h (2). ATPase activation during latency can hardly be attributed to the adaptative modification of the ATPase lipid environment in cells grown under lipophilic acid stress, as suggested by Alexandre et al. (1).Changes in yeast pHi and viability during octanoic acid-stressed cultivation.
The change in pHi during cultivation of nonadapted cells with 0.39 mM octanoic acid was monitored by using an adaptation of the fluorescence microscopic image processing technique developed by Imai and Ohno (9); 5- (and 6)-carboxyfluorescein (cF) was used as the internal pH-dependent fluoroprobe. Cells washed and resuspended in cold CF buffer (citrate-phosphate buffer [at pH 4.0] with 50 mM glycine [Sigma], 110 mM NaCl, 5 mM KCl, and 1 mM MgCl2) to a cellular density of 2 × 108 ml−1 were loaded with cF by adding 20 μM of 5 (and 6)-carboxyfluorescein-diacetate (Sigma) and vortexing in two bursts of 1 min each, interspersed with 15 min on ice (9). After being washed twice with cold CF buffer, cF-loaded cells were immediately examined with a Zeiss Axioplan microscope equipped with adequate epifluorescence interference filters (Zeiss BP450-490 and Zeiss LP520) and connected to a video camera and to a computer with an image- analysis program (gel documentation system SW2000; UVP, San Gabriel, Calif.). Following a cell-by-cell analysis, the value of fluorescence intensity (fI) emitted by each cell, measured by direct densitometry, corresponded to the arithmetical mean value of fI measured in two or three different regions in the cytoplasm of the same cell, with the less fluorescent vacuole excluded. To estimate average pHi, an in vivo calibration curve was prepared (Fig. (Fig.3)3) by using cell suspensions grown in the absence of toxics which were loaded with cF as described above and incubated, at 30°C, with 0.5 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP) to dissipate the plasma membrane pH gradient (4), before adjustment of external pH (in the range 3.5 to 7.5) by the addition of HCl or NaOH at 2 M. Fluorescence images were fixed 15 s after the occurrence of the excitation radiation in order to minimize interferences due to leakage of cF as well as fluorescence quenching (3, 7). Cells were kept on ice throughout the procedure, and CF buffer lacked glucose; therefore, the active efflux of cF (3) was minimized as confirmed by measuring the fluorescence in the medium surrounding the cells, which was negligible. Under the experimental conditions used and for the purpose of the study, this technique proved to be highly useful and suitable despite the limitations that might be raised (3, 7). It allowed a clear-cut picture of the pHi of individual cells, giving information about the distribution of pHi values of a yeast population (Fig. (Fig.44 and and5a5a to c), instead of solely an estimation of the average value of the whole population, as is the case with techniques based on the distribution of radioactive propionic acid (20) or on the in vivo 31P nuclear magnetic resonance (5). Moreover, values calculated for the average pHi of the whole yeast population during latency and exponential growth in medium with octanoic acid (Fig. (Fig.5d)5d) were close to, although slightly lower than, the values previously obtained based on the distribution of [14C]propionic acid (20, 22). Results revealed that the cell population used to inoculate octanoic acid-supplemented medium exhibited a significant heterogeneity (Fig. (Fig.4);4); around 31% showed a pHi in the optimal range (above 6.5) (Fig. (Fig.4),4), with the average pHi value of the whole population estimated to be approximately 6.0. This low pHi value results from cell cultivation in a rich medium with high production of organic acids (11) (external pH, 3.6), followed by washing of the cells with YG medium buffered at pH 4.0 (17). The introduction of the inoculum in octanoic acid-supplemented medium led to the very rapid (5-min) increase in the percentage of the cell population with pHi below 5.5, consistent with the rapid kinetics of cytosol acidification when yeast cells are exposed to weak acids (5). During extended incubation with octanoic acid and until the end of latency, the percentage of the population with a very low pHi (below 5.5) continued to increase, reaching 80% of the cell population, while the percentage of cell population with a pHi above 6.0 suffered a corresponding decrease (Fig. (Fig.5).5). During exponential growth, the opposite pHi modification was observed, consistent with a recovery of pHi to physiological levels (Fig. (Fig.5).5). The time-dependent pattern of internal acidification during lag phase correlated with plasma membrane ATPase activation (Fig. (Fig.2a2a and and5),5), suggesting that this activation was triggered by intracellular acidification, as proposed for acetic acid (5)- or nitrogen starvation (2)-induced activation. Immediately before yeast cells entered exponential growth, 80% of the initial viable population had lost viability, as assessed by the number of CFU (21) (Fig. (Fig.6),6), suggesting that octanoic acid-induced death during latency is related to internal acidification down to critical values (Fig. (Fig.55 and and6),6), in agreement with the relationship established by Imai and Ohno (10) between yeast viability and intracellular pH. Only about 20% of the initial population was able to start cell division in octanoic acid-supplemented medium, presumably those cells that in the inoculum exhibited pHi values around neutrality (Fig. (Fig.55 and and6).6). These results suggest that despite plasma membrane H+- ATPase activation, this system of pH homeostasis may not be able to fully counteract the physiological effects of increasing octanoic acid concentrations and eventually fails at very severe acid stress. Open in a separate windowFIG. 3In vivo calibration curve, showing the pH dependence of the fI of cF-loaded-cells of S. cerevisiae IGC 3507III. Intracellular and extracellular pHs were equilibrated by incubation of cF-loaded cells, for 10 min at 30°C, with 0.5 mM CCCP. At each pH, values of fI correspond to the average fI of about 20 cells. The data are averages with standard deviations for three independent experiments.Open in a separate windowFIG. 4Distribution of cells with different pHi values present in the inoculum of S. cerevisiae IGC 3507III prepared in growth medium without octanoic acid supplementation.Open in a separate windowFIG. 5Percentage of yeast cells with pHi below 5.5 (a), between 5.5 and 6.0 (b), or above 6.0 (c); average pHi of the whole cell population (▴) during S. cerevisiae IGC3507III cultivation in medium supplemented with 0.39 mM total octanoic acid (pH 4.0, 30°C); and the optical density (OD) of the culture at 600 nm (▪). The average pHi values estimated for the whole cell population are the arithmetical mean values of the various average pHi values calculated for individual cells. The percentage of cells present in the inoculum with pHi values within the three ranges (○) and the average pHi of the inoculum cell population (▵) are indicated.Open in a separate windowFIG. 6Concentration of viable cells (▴) and culture optical density (O.D.) at 600 nm (□) during lag and exponential phases of S. cerevisiae IGC 3507III growth in medium supplemented with 0.39 mM octanoic acid, at pH 4.0 and 30°C.Adaptative response to octanoic acid.
The adaptation of yeast cells to octanoic acid at a low pH appears to depend on their H+-exporting ability, but this requires not only a highly active H+-ATPase in the plasma membrane but the provision of sufficient ATP to drive this energy-demanding process as indicated by the results of Holyoak et al. (8). It is likely that increased ATPase activity under octanoic acid stress may reduce cellular ATP levels and that ATP depletion contributes to the failure of the maintenance of pHi homeostasis, particularly among the subpopulation that in the inoculum exhibited the lowest pHi values. The loss of viability might occur for those cells where pHi decreased down to nonphysiological values. The eventual recovery of growth therefore depends on the remaining viable population, in agreement with the well-known critical role played by the physiology and number of viable cells in the inoculum in the duration of latency under acid stress. The observation that octanoic acid-adapted cells reinoculated into the same fresh medium can resume growth after a much shorter latency (Fig. (Fig.1a)1a) is a good example of the importance of the physiology of the inoculum cells. Besides the increased plasma membrane H+-ATPase activity of octanoic acid-adapted cells, other mechanisms may underlie the adaptation to acid stress, such as the increased cellular buffering capacity of octanoic acid-grown cells due to their lower intracellular volume (20), the more favorable plasma membrane lipid composition (1), and the possible induction of the active efflux of the anion (26). 相似文献17.
Daniel Gonzalez-Dunia Beatrice Cubitt Juan Carlos de la Torre 《Journal of virology》1998,72(1):783-788
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