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1.
The 5-methyl-2-pyrrolylcarbonyl moiety of the aminocoumarin antibiotics clorobiocin and coumermycin A1 is the key pharmacophore for targeting the ATP-binding site of GyrB for inhibition of the bacterial type-II topoisomerase DNA gyrase. During the late stage of clorobiocin and coumermycin A1 biosynthesis, the pyrrolyl-2-carboxyl group is transferred from the peptidyl carrier proteins Clo/CouN1 to the 3'-hydroxyl of the 4-methoxy-L-noviosyl scaffold by the action of the acyltransferases Clo/CouN7. CouN1 and CouN7 have now been heterologously expressed and purified from Escherichia coli. The apo form of CouN1 is converted to the acyl-holo form by loading with pyrrolyl-2-carboxyl-S-pantetheinyl moieties from synthetic pyrrolyl- and 5-methylpyrrolyl-CoAs by the action of the phosphopantetheinyl transferase Sfp. CouN7 acts as an acyltransferase, moving the pyrrole acyl moieties from CouN1 to the noviose sugar of descarbamoylnovobiocin. When the 5-methylpyrrolyl-2-carboxyl-thioester of CouN1 is the cosubstrate, the in vitro product differs from clorobiocin only in a CH3 for Cl group change on the coumarin ring. Double transfer of this acyl moiety by CouN7 to the penultimate intermediate in coumermycin A1 assembly completes that antibiotic biosynthetic pathway. 相似文献
2.
The aminocoumarin antibiotics clorobiocin and coumermycin A(1) target the B subunit of DNA gyrase by presentation of the 5-methyl-pyrrolyl-2-carboxy ester moiety in the ATP-binding site of the enzyme. The pyrrolyl pharmacophore is derived by a four electron oxidation of a prolyl unit while tethered in phosphopantetheinyl thioester linkage to a peptidyl carrier protein (PCP) subunit. l-Proline is selected and activated as l-prolyl-AMP by adenylation domain enzymes (CloN4 and CouN4) and then installed as the thioester on the holo form of the PCP proteins CloN5 and CouN5. Enzymatic oxidation of the prolyl-S-PCP by the flavoprotein dehydrogenase CloN3 can be followed by rapid quench and subsequent electrospray ionization-Fourier transform mass spectrometry analysis of the acyl-S-protein substrate/product mixture to establish that a two-electron oxidized pyrrolinyl-S-enzyme transiently accumulates on the way to the four-electron oxidized, heteroaromatic pyrrolyl-2-carboxy-S-PCP acyl enzyme product. 相似文献
3.
Transposon Tn5 mutagenesis was used to generate random mutations in Shewanella baltica MAC1, a polyunsaturated fatty acid (PUFA)-producing bacterium. Three mutants produced 3–5 times more eicosapentaenoic acid (EPA 20:5 n−3) compared to the wild type at 10°C. One of the mutants produced 0.3 mg EPA g−1 when grown at high temperature (30°C). Moreover, 2 mg docosahexaenoic acid (DHA 22:6 n−3) g−1 was produced by S. baltica mutants at 4°C. Sequencing of insertion mutation(s) showed 96% homology to trimethylamine N-oxide (TMAO) reductase gene and 85% homology to rRNA operons of E. coli. Tn5 transposon mutagenesis therefore is a suitable technique to increase PUFA formation in bacteria. 相似文献
4.
The conversion of a cellulose-producing cell (Cel
+) fromGluconacetobacter hansenii PJK (KCTC 10505 BP) to a non-cellulose-producing cell (Cel
−) was investigated by measuring the colony forming unit (CFU). This was achieved in a shaking flask with three slanted baffles,
which exerted a strong shear stress. The addition of organic acid, such as glutamic acid and acetic acid, induced the conversion
of microbial cells from a wild type toCel
− mutants in a flask culture. The supplementation of 1% ethanol to the medium containing an organic acid depressed the conversion
of the microbial cells toCel
− mutants in a conventional flask without slanted baffles. The addition of ethanol to the medium containing an organic acid;
however, accelerated the conversion of microbial cells in the flask with slanted baffles. TheCel
+ cells from the agitated culture were not easily converted intoCel
−, mutants on the additions of organic acid and ethanol to a flask without slanted baffles, but some portion of theCel
+ cells were converted toCel
− mutants in a flask with slanted baffles. The conversion ratio ofCel
+ cells toCel
− mutants was strongly related to the production of bacterial cellulose independently from the cell growth. 相似文献
5.
The effects of UVB on the kinetics of stem elongation of wild type (WT) and photomorphogenic mutants of tomato were studied
by using linear voltage transducers connected to a computer. Twenty-one or twenty-six-day-old plants, grown in 12 h white
light (150 μmol m−2 s−1 PAR)/12 h dark cycles, were first transferred to 200 μmol m−2 s−1 monochromatic yellow light for 12 h, then irradiated with 0.1 or 4.5 μmol m−2 s−1 UVB for 12 h and finally kept in darkness for another 24 h. The measurements of the kinetics of stem elongation started after
4 h under yellow light. Significant differences in stem growth during the irradiation with yellow light, as well as during
the dark period, were found between the genotypes. In darkness, the magnitude of stem growth followed the order: tri > AC = fri > MMau > hp1. Two factors determined the large differences of growth in darkness: 1) the different stem elongation rate (SER) and 2) the
different duration of the growing phase among the genotypes. In darkness the stem growth of au and hp1 mutants lasted for about 18 h, whereas it continued for the whole experimental period (36 h) in the other genotypes. UVB
irradiation substantially reduced elongation growth of all genotypes (4.5 μmol m−2 s−1 being more effective than 0.1 μmol m−2 s−1). Both fluence rates of UVB induced a detectable reduction of SER already after 15 min of irradiation. Red light inhibited,
while far red light promoted stem growth of all the genotypes tested. fri (phyA null), tri (phyB1 null), hp1 (exhibiting exaggerated phytochrome responses) mutants and WT tomato showed similar levels of UVB–induced inhibition of growth,
while the aurea mutant showed the largest growth inhibition during the 12 h of irradiation. These results indicate that phytochrome is not
directly involved in UVB control of stem elongation. The results of dichromatic irradiations UVB + red or UVB + far red indicate
the presence of distinct and additive action of UVB photoreceptor and of the phytochrome system in the photoregulation of
stem growth.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
6.
Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica 总被引:1,自引:0,他引:1
Förster A Aurich A Mauersberger S Barth G 《Applied microbiology and biotechnology》2007,75(6):1409-1417
The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess.
Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production
bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l−1 CA with a yield Y
CA of 0.75–0.82 g g−1, whereas at pH 5.0, 87 g l −1 with a yield Y
CA of 0.51 gg−1 were produced. The CA-productivity Q
CA increased from 0.40 g l −1 h−1 at pH 5.0 up to 0.73 g l −1 h−1 at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA
production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression
resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production. 相似文献
7.
The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l−1 maltose, 66 g l−1 yeast extract, and 5 g l−1 K2HPO4 at 30°C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture,
when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l−1 ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l−1 h−1 and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst
in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion
reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l−1, and the productivity was 1.5 g l−1 h−1. The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately
17 g l−1 of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l−1 h−1 was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally
high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography
with a final yield of 75%. 相似文献
8.
In the wild type strain (stock no. 1227) of Thermoactinomyces vulgaris, as reported earlier [Sinha and Singh (1980) Biochem. J. 190, 457–460], all phosphatase isoenzymes (three alkaline — AlpI,
AlpII and AlpIII, and one acidic — Acp) are present. However, the auxotrophic mutants, the strains 1286 (thi
−), 1279 (nic
−, ura
−) and 1278 (thi
−, ura
−) exhibited two alkaline phosphatase isoenzymes (AlpII and AlpIII), but AlpI was lacking. In the strain 1261 (nic
−, thi
−), only AlpIII was expressed, and AlpI and AlpII isoenzymes were missing. The results suggest that the strains, which require
either thiamine (1286 and 1278) or nicotinamide (1279) for their growth, were AlpI
− mutants; and the strain (1261), which requires both thiamine and nicotinamide for its growth, was AlpI
−/AlpII
− double mutant. There was no direct correlation between uracil auxotrophy and the expression of phosphatases. The uniform
expression of AlpIII and Acp in all the strains, irrespective of their nutrient requirements, suggest that these constitutive
phosphatases are species-specific. The specific activities of the thermophilic acid and alkaline phosphatases were maximum
in the wild type strain (1227) of T. vulgaris. The next in phosphatase activity was the strain 1279 (an AlpI
− mutant), followed by their decrease, in order, in the strains 1286 and 1278 (which were also AlpI
− mutants); while least activity of these enzymes was observed in the obligate thermophile strain 1261 (AlpI
−/AlpII
− double mutant). 相似文献
9.
Ugarte G Delgado R O'Day PM Farjah F Cid LP Vergara C Bacigalupo J 《The Journal of membrane biology》2005,207(3):151-160
We report that Drosophila retinal photoreceptors express inwardly rectifying chloride channels that seem to be orthologous to mammalian ClC-2 inward
rectifier channels. We measured inwardly rectifying Cl− currents in photoreceptor plasma membranes: Hyperpolarization under whole-cell tight-seal voltage clamp induced inward Cl− currents; and hyperpolarization of voltage-clamped inside-out patches excised from plasma membrane induced Cl− currents that have a unitary channel conductance of ∼3.7 pS. The channel was inhibited by 1 mM Zn2+ and by 1 mM 9-anthracene, but was insensitive to DIDS. Its anion permeability sequence is Cl− = SCN−> Br−>> I−, characteristic of ClC-2 channels. Exogenous polyunsaturated fatty acid, linolenic acid, enhanced or activated the inward
rectifier Cl− currents in both whole-cell and excised patch-clamp recordings. Using RT-PCR, we found expression in Drosophila retina of a ClC-2 gene orthologous to mammalian ClC-2 channels. Antibodies to rat ClC-2 channels labeled Drosophila photoreceptor plasma membranes and synaptic regions. Our results provide evidence that the inward rectification in Drosophila retinal photoreceptors is mediated by ClC-2-like channels in the non-transducing (extra-rhabdomeral) plasma membrane, and
that this inward rectification can be modulated by polyunsaturated fatty acid.
G. Ugarte and R. Delgado contributed equally to this work. 相似文献
10.
Eiji Sakuradani Akinori Ando Jun Ogawa Sakayu Shimizu 《Applied microbiology and biotechnology》2009,84(1):1-10
Studies on the application of functional lipids such as polyunsaturated fatty acids (PUFAs) have proceeded in various fields
regarding health and dietary requirements in a search for novel and rich sources. Filamentous fungus Mortierella alpina 1S-4 produces triacylglycerols rich in arachidonic acid, ones reaching 20 g/L and containing 30–70% arachidonic acid as to
the total fatty acids. Mutants derived from M. alpina 1S-4, defective in Δ5 and Δ6 desaturases, accumulate triacylglycerols rich in unique PUFAs, i.e., dihomo-γ-linolenic acid
and Mead acid, respectively. Furthermore, various mutants derived from M. alpina 1S-4 have led to the production of oils containing n−1, n−3, n−4, n−6, n−7, and n−9 PUFAs. A variety of genes encoding fatty acid desaturases and elongases involved in PUFA biosynthesis in M. alpina 1S-4 has been isolated and characterized. Molecular breeding of M. alpina strains by means of manipulation of these genes facilitates improvement of PUFA productivity and elucidation of the functions
of enzymes involved in PUFA biosynthesis. 相似文献
11.
Jasmonic acid affects changes in the growth and some components content in alga Chlorella vulgaris 总被引:1,自引:0,他引:1
Romuald Czerpak Alicja Piotrowska Katarzyna Szulecka 《Acta Physiologiae Plantarum》2006,28(3):195-203
The present study was undertaken to test the influence of exogenous applied jasmonic acid upon the growth and changes in some
metabolites levels in the cells of green alga Chlorella vulgaris Beijerinck (Chlorophyceae). It was found, that JA in algal cells acted in a concentration-dependent manner. Treatment with
JA at high concentrations range of 10−5–10−4 M, resulted in the decrease in cell number and reduction of major photosynthetic pigments, monosaccharides, soluble cellular
and extracellular proteins levels as well as decrease in pH of the medium. In contrast to 10−5 and 10−4 M JA, this phytohormone applied at 10−8–10−6 M induced the increase in cell number, photosynthetic pigments and monosaccharides contents, significant accumulation and
extracellular secretion of soluble proteins over control and neutralization of the medium. Quantitative changes in polypeptide
pattern of total cellular proteins after treatment with the optimal concentration of 10−7 M JA on the 7th day of cultivation as analyzed by SDS-PAGE, was also observed. JA induced synthesis de novo of 15 specific polypeptides with molecular weight 334-16 kDa which were’t detected in the control. The data suggest that
JA plays a important role in algal growth and development. 相似文献
12.
Glutamate excretion due to amino acid starvation was investigated in “stringent” and “relaxed” strains ofEscherichia coli. The observed excretion process isrelA-dependent, carrier-mediated, and glutamate-specific. After induction, excretion was detected within less than 2 min and continued
for more than 5h with a rate of 7–10 nmol (mg dry weight)−1 min−1. Using carbonyl cyanidem-chlorophenylhydrazone or polymyxin B nonapeptide, together with valinomycin, it was shown that glutamate excretion is driven
by the membrane potential. 相似文献
13.
Lithospermum officinale callus produces shikalkin 总被引:1,自引:0,他引:1
Kamahldin Haghbeen Valiolah Mozaffarian Fatemeh Ghaffari Elahe Pourazeezi Mohammad Saraji Morteza Daliri Joupari 《Biologia》2006,61(4):463-467
To study biosynthetic abilities of Lithospermum officinale, callus formation from young leaves and stems of the plant was induced on Linsmaier-Skoog medium supplemented with 2,4-D
(10−6 M) and kinetin (10−5 M). Maintaining the calli on this medium resulted in polyphenolic compounds production. Their transfer onto White medium
containing IAA (10−7 M) and kinetin (10−5 M) resulted in the production of a red naphthoquinonic pigment named shikalkin. Shikalkin production from callus cultures
was suppressed on the White medium containing NAA instead of IAA. This observation indicates that both shikalkin and polyphenolic
acids biosynthetic pathways exist in the L. officinale callus cells and a regulatory system counterbalances the ratio of shikalkin to polyphenolic acids. 相似文献
14.
Production of clavulanic acid (CA) by Streptomyces clavuligerus in a shake-flask culture increased from 92 to 180 mg l−1 with an increased O2 transfer efficiency (0.039 → 0.058 s−1), which maintained the redox potential values above −250 mV. Compared with traditional measures, such as dissolved O2 concentration and respiratory activity, the redox potential can easily be determined and correlates closely with CA production.
It can therefore be used to monitor microbial activities during biosyntheses of secondary metabolites.
Revisions requested 5 April 2005 and 19 July 2005; Revisions received 19 July 2005 and 9 September 2005 相似文献
15.
Walleska De Jesús-Bonilla Anthony Cruz Ariel Lewis José Cerda Daniel E. Bacelo Carmen L. Cadilla Juan López-Garriga 《Journal of biological inorganic chemistry》2006,11(3):334-342
Ferryl compounds [Fe(IV)=O] in living organisms play an essential role in the radical catalytic cycle and degradation processes
of hemeproteins. We studied the reactions between H2O2 and hemoglobin II (HbII) (GlnE7, TyrB10, PheCD1, PheE11), recombinant hemoglobin I (HbI) (GlnE7, PheB10, PheCD1, PheE11),
and the HbI PheB10Tyr mutant of L. pectinata. We found that the tyrosine residue in the B10 position tailors, in two very distinct ways, the reactivity of the ferryl
species, compounds I and II. First, increasing the reaction pH from 4.86 to 7.50, and then to 11.2, caused the the second-order
rate constant for HbII to decrease from 141.60 to 77.78 M−1 s−1, and to 2.96 M−1 s−1, respectively. This pH dependence is associated with the disruption of the heme–tyrosine (603 nm) protein moiety, which controls
the access of the H2O2 to the hemeprotein active center, thus regulating the formation of the ferryl species. Second, the presence of compound I
was evident in the UV–vis spectra (648-nm band) in the reactions of HbI and recombinant HbI with H2O2, This band, however, is completely absent in the analogous reaction with HbII and the HbI PheB10Tyr mutant. Therefore, the
existence of a hydrogen-bonding network between the heme pocket amino acids (i.e., TyrB10) and the ferryl compound I created
a path much faster than 3.0×10−2 s−1 for the decay of compound I to compound II. Furthermore, the decay of the heme ferryl compound I to compound II was independent
of the proximal HisF8 trans-ligand strength. Thus, the pH dependence of the heme–tyrosine moiety complex determined the overall reaction rate of the
oxidative reaction limiting the interaction with H2O2 at neutral pH. The hydrogen-bonding strength between the TyrB10 and the heme ferryl species suggests the presence of a cycle
where the ferryl consumption by the ferric heme increases significantly the pseudoperoxidase activity of these hemeproteins. 相似文献
16.
Kamiel Spoelstra Serge Daan 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》2008,194(3):235-242
Mutations in each of the genes mPer1, mPer2, mCry1 and mCry2 separately cause deviations from the wild type circadian system. Differences between these mutant strains have inspired the
hypothesis that the duality of circadian genes (two mPer and two mCry genes involved) is related to the existence of two components in the circadian oscillator (Daan et al., J Biol Rhythms 16:105–116,
2001). We tested the predictions from this theory that the circadian period (τ) lengthens under constant illumination (LL)
in mCry1 and mPer1 mutant mice, while it shortens in mCry2 and mPer2 mutants. mCry1
−/−
and mCry2
−/−
knockout mice both consistently increased τ with increasing light intensity, as did wild type mice. With increasing illumination,
rhythmicity is reduced in mCry1, mCry2 and mPer1, but not in mPer2 deficient mice. Results for mPer mutant mice are in agreement with data reported on these strains earlier by Steinlechner et al. (J Biol Rhythms 17:202–209,
2002), and also with the predictions from the model. The increase in cycle length of the circadian system by light in the
mCry2 deficient mice violates the predictions. The model is thereby rejected: the mCry genes do not play a differential role, although the opposite responses of mPer mutants to light remain consistent with a functional Evening–Morning differentiation. 相似文献
17.
Victoria López-Rodas Antonio Flores-Moya Emilia Maneiro Nieves Perdigones Fernando Marva Marta E. García Eduardo Costas 《Evolutionary ecology》2007,21(4):535-547
Adaptation of Microcystis aeruginosa (Cyanobacteria) to resist the herbicide glyphosate was analysed by using an experimental model. Growth of wild-type, glyphosate-sensitive
(Gs) cells was inhibited when they were cultured with 120 ppm glyphosate, but after further incubation for several weeks, occasionally
the growth of rare cells resistant (Gr) to the herbicide was found. A fluctuation analysis was carried out to distinguish between resistant cells arising from rare
spontaneous mutations and resistant cells arising from other mechanisms of adaptation. Resistant cells arose by rare spontaneous
mutations prior to the addition of glyphosate, with a rate ranging from 3.1 × 10−7 to 3.6 × 10−7 mutants per cell per generation in two strains of M. aeruginosa; the frequency of the Gr allele ranged from 6.14 × 10−4 to 6.54 × 10−4. The Gr mutants are slightly elliptical in outline, whereas the Gs cells are spherical. Since Gr mutants have a diminished growth rate, they may be maintained in uncontaminated waters as the result of a balance between
new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, rare spontaneous pre-selective
mutations may allow the survival of M. aeruginosa in glyphosate-polluted waters via Gr clone selection. 相似文献
18.
Sharma Madhu Sood Anil Nagar P.K. Prakash Om Ahuja Paramvir Singh 《Plant Cell, Tissue and Organ Culture》1999,58(2):111-118
Micropropagation of tea (Camellia sinensis (L.) O. Kuntze) has been widely attempted but commercial exploitation of this method is limited by heavy losses during the
hardening procedures. In the present study, optimization of time of harvesting (spring and early summer) of microshoots, shoot
size, soil pH (4.0–6.4), plant growth regulator treatment (IBA; 500 mg l-1, 30 min) CO2 (9.09/10×10−5 mol l-1 to 10.22/10×10-5 mol l-1 and 20/11×10−5 mol l-1 to 80/13×10−7 mol l-1) enrichment and light (15 μ mol m-2 s-1) conditions in specially designed hardening chambers, made a significant impact on the percent of success for hardening.
Following the standardized procedure, up to 71.6% root induction and 73% survival could be achieved. Successful field transfer
was also accomplished.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
19.
Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm−3 of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic.
Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within
two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer
to medium with gibberellic acid (GA3, 1.0 mg dm− 3) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm−3 BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots.
Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm−3 BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages. 相似文献
20.
A system for rapid plant regeneration through somatic embryogenesis from shoot tip explants of sorghum [Sorghum bicolor (L.) Moench] is described. Somatic embryogenesis was observed after incubation of explants in dark for 6–7 weeks through
a friable embryogenic callus phase. Linsmaier and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2 mg l−1) and kinetin (0.1 mg l −1) was used for induction of friable embryogenic calli and somatic embryos. Germination of somatic embryos was achieved about
5 weeks after transfer onto Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (2 mg l−1) and indole-3-acetic acid (0.5 mg l −1) under light. Seeds from in vitro-regenerated plants produced a normal crop in a field trial, and were comparable to the crop grown with the seeds of the mother
plant used to initiate tissue culture. The simplicity of the protocol and possible advantages of the system for transformation
over other protocols using different explants are discussed.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献