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1.
The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic α-helical peptide that exhibits a broad range of antimicrobial activity. To explore
a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid.
The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni2+-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity
chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K12D31, Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but
only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein.
The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting
milligram quantities of ABP-CM4 that is biologically active. 相似文献
2.
Sung-Hee Lee Seo-Jin Kim Yoo-Sup Lee Min-Dong Song Ick-Hee Kim Hyung-Sik Won 《Regulatory peptides》2011,166(1-3):36-41
As potential therapeutic agents, antimicrobial peptides with shorter length and simpler amino acid composition can be better candidates for clinical and commercial development. Here, we attempted de novo design of short (5- to 11-residue) antimicrobial peptides with three kinds of amino acids. Amphipathic helical properties were conferred by using leucines and lysines and two tryptophan residues were positioned at the critical amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. According to this specified rule, 12 model peptides were generated and their helical propensity was confirmed by circular dichroism spectroscopy. Antimicrobial and hemolytic activities were compared with those of the known 12-residue peptide agent, omiganan, which is currently under therapeutic and commercial development. Antimicrobial activities against Gram-negative and Gram-positive bacteria, including a multi-drug resistant strain, were observed for certain 7- to 11-residue models. Among them, the most potent activity was found for a 9-residue peptide (L5K2W2), although it also had severe hemolytic activity. Alternatively, an 11-residue peptide (L4K5W2) with little hemolytic activity was potentially the most useful agent, as it showed higher antibacterial activity than omiganan. These results not only suggest useful candidates for novel antibiotic development, but also provide an efficient strategy to design such peptides. 相似文献
3.
Liangfan Zhou Qingping Lin Baocun Li Nannan Li Shuangquan Zhang 《Biotechnology letters》2009,31(3):437-441
The antimicrobial peptide CM4 is a 35-residue cationic peptide. To explore a new approach for the expression and purification
of CM4 in Escherichia coli, the CM4 gene was cloned into the vector pET32a to construct an expression vector pET32a-CM4. The fusion protein Trx-CM4,
purified by Ni2+-chelating chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant CM4. Purification of recombinant
CM4 was achieved by reverse HPLC chromatography, and about 1.4 mg/l active recombinant CM4 with the purity more than 98% was
obtained. The recombinant CM4 showed antimicrobial activities that were similar to synthetic one.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
An erratum to this article can be found at 相似文献
4.
Dimitra Triantafillidou Maria Simitsopoulou Francois Franceschi Theodora Choli-Papadopoulou 《Journal of Protein Chemistry》1999,18(2):215-223
The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol. The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli. Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus. The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms. Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion. The similar structural and biochemical properties as well as the significant homology between L11 from E. coli and B. stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function. 相似文献
5.
Four different strains ofLactobacillus delbrueckii subsp.bulgaricus (Ss1 and Yop12) andStreptococcus salivarius subsp.thermophilus (Ss2 and Yop9) were isolated from two different yogurt sources in Argentina. In medium containing different carbon sources: lactose, fructose, sucrose or glucose plus fructose, the growth of a mixed culture (Yop12+Ss2) shows stimulation ofS. thermophilus and inhibition ofL. bulgaricus with respect to pure cultures. Both microorganisms in mixed culture grew less well on glucose plus galactose. However, in medium with glucose or galactose, both microorganisms were stimulated. 相似文献
6.
Toshio Takahashi Osamu Koizumi Eisuke Hayakawa Sumiko Minobe Rinako Suetsugu Yoshitaka Kobayakawa Thomas C. G. Bosch Charles N. David Toshitaka Fujisawa 《Development genes and evolution》2009,219(3):119-129
From an evolutionary point of view, Hydra has one of the most primitive nervous systems among metazoans. Two different groups of peptides that affect neuron differentiation
were identified in a systematic screening of peptide signaling molecules in Hydra. Within the first group of peptides, a neuropeptide, Hym-355, was previously shown to positively regulate neuron differentiation.
The second group of peptides encompasses the PW family of peptides that negatively regulate neuron differentiation. In this
study, we identified the gene encoding PW peptide preprohormone. Moreover, we made the antibody that specifically recognizes
LPW. In situ hybridization and immunohistochemical analyses showed that the PW peptides and the gene encoding them were expressed
in ectodermal epithelial cells throughout the body except for the basal disk. The PW peptides are produced by epithelial cells
and are therefore termed “epitheliopeptides.” Together with Hym-355, the PW family peptides mediate communication between
neurons and epithelial cells and thereby maintain a specific density of neurons in Hydra.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Toshio Takahashi, Osamu Koizumi equally contributed to this study. 相似文献
7.
Liu B Hong Y Wu L Li Z Ni J Sheng D Shen Y 《Extremophiles : life under extreme conditions》2007,11(5):733-739
A glycerate kinase (GK) gene (PH0495) from the hyperthermophilic archaeon Pyrococcus horikoshii, was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and ion exchange chromatography. The enzyme
was likely a homodimer based on SDS-PAGE (47 kDa) and gel filtration chromatography (100 kDa) analysis. A radioisotope-labeling
examination method was initially used for the enzymatic activity detection, and the enzyme (GKph) was found to catalyze the formation of 2-phosphoglycerate using d-glycerate as the substrate. The enzyme exhibited unique phosphoryl donor specificity with maximal activity towards pyrophosphate.
The temperature and pH optima of the enzyme were 45°C and 7.0, respectively, and about half of the maximal activity remained
at 100°C. The enzyme was highly thermostable with almost no loss of activity at 90°C for 12 h. Based on sequence alignment
and structural comparison it was assigned to group I of the trichotomy of GKs.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
8.
Xu-De Wang Xian-En Zhang Yong-Chao Guo Zhi-Ping Zhang Zhu-An Cao Ya-Feng Zhou 《Biotechnology letters》2009,31(5):711-717
The gdh and gdhr genes, encoding B12-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the β-subunit was lost during GDH purification when co-expressing α, β and γ subunit. This was overcome by fusing
the β-subunit to α- or γ-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties
of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein
GDHALB/C, constructed by linking the N-terminal of β-subunit to the C-terminal of α subunit through a (Gly4Ser)4 linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based
inactivation by glycerol during catalysis and could be reactivated by GDHR.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
9.
Lee JH Moon YH Kim N Kim YM Kang HK Jung JY Abada E Kang SS Kim D 《Biotechnology letters》2008,30(4):749-754
The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K
m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl
moiety of sucrose to cytosine monophosphate (CMP).
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
10.
Z. L. Urshev K. N. Pashova-Baltova Z. P. Dimitrov 《World journal of microbiology & biotechnology》2006,22(11):1223-1228
Three-component starters for yogurt were obtained on the base of starter LBB.BY 5-12 for traditional Bulgarian yogurt, containing strains Lactobacillus delbrueckii ssp. bulgaricus B5 and Streptococcus thermophilus A with the addition of either an exopolysaccharide-producing S. thermophilus strain 6V or the fast acidifying S. thermophilus strain N1. To differentiate between the three strains in the starter cultures, randomly amplified polymorphic DNA (RAPD) technique was applied to develop strain-specific probes. Southern hybridization against dot-blots of chromosomal DNA from the three S. thermophilus strains confirmed that two probes, derived from a 770 bp RAPD product obtained with primer RAPD-4 and a 290 bp sequence obtained with primer OPP-7 were specific for S. thermophilus 6V and S. thermophilus A, respectively, while no hybridization to S. thermophilus N1 DNA was observed. The selected probes were used to differentiate between S. thermophilus colonies on a solid agar medium by colony hybridization. The evaluation of the viable cell counts revealed that the populations of S. thermophilus A and the added S. thermophilus strains 6V or N1 in the three-component starters and in yogurt had nearly equal proportion allowing each strain to contribute to the enriched properties of starter and product. 相似文献
11.
Joanna Will Andreas Kyas William S. Sheldrick Dirk Wolters 《Journal of biological inorganic chemistry》2007,12(6):883-894
An automated multidimensional protein identification technology, which combines biphasic liquid chromatography with electrospray
ionisation tandem mass spectrometry (MS/MS), was employed to analyse tryptic peptides from Escherichia coli cells treated with the antiproliferation agent [(η6-p-cymene)RuCl2(DMSO)], where DMSO is dimethyl sulfoxide. MS/MS spectra were recorded for molecular ions generated by neutral loss of p-cymene from intensive peptide ions coordinated by the (η6-p-cymene)RuII fragment. Matching of the MS/MS spectra of the ruthenated peptides to spectra of proteins in the E. coli database enabled the identification of five protein targets for [(η6-p-cymene)RuCl2(DMSO)]. One of these is the constitutive cold-shock protein cspC, which regulates the expression of genes encoding stress-response
proteins, and three of the other targets, ppiD, osmY and sucC, are proteins of the latter type. The DNA damage-inducible helicase
dinG was likewise established as a protein target. Aspartate carboxylate functions were identified as the probable Ru binding
sites in cspC, ppiD and dinG, and threonine and lysine side chains in osmY and sucC, respectively.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
12.
Khan SA Hamayun M Kim HY Yoon HJ Seo JC Choo YS Lee IJ Kim SD Rhee IK Kim JG 《Biotechnology letters》2009,31(2):283-287
Plant growth-promoting endophytic fungi with gibberellin-producing ability were isolated from the roots of Carex kobomugi Ohwi, a common sand-dune plant, and bioassayed for plant growth-promotion. A new strain, Arthrinium phaeospermum KACC43901, promoted growth of waito-c rice and Atriplex gemelinii. Analysis of its culture filtrate showed the presence of bioactive GA1 (0.5 ng/ml), GA3 (8.8 ng/ml), GA4 (4.7 ng/ml) and GA7 (2.2 ng/ml) along with physiologically inactive GA5 (0.4 ng/ml), GA9 (0.6 ng/ml), GA12 (0.4 ng/ml), GA15 (0.4 ng/ml), GA19 (0.9 ng/ml) and GA24 (1.8 ng/ml). The fungal isolate was identified through sequence homology and phylogenetic analysis of 18S rDNA (internal
transcribed region).
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
13.
Ole Petter Thangstad Per Winge Harald Husebye Atle Bones 《Plant molecular biology》1993,23(3):511-524
The glucosinolate hydrolyzing enzymes myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) are encoded by a multigene family consisting of two subgroups. The first two nuclear genes representing each of these two subgroups of the new gene family, Myr1.Bn1 and Myr2.Bn1, from Brassica napus have been cloned and sequenced. Based on conserved regions in cDNA of three species, PCR (polymerase chain reaction) primers were made, and used to amplify and characterize the structure of the myrosinase genes in seven species of Brassiceae. Southern hybridization analysis of PCR products and genomic DNA indicates that myrosinase is encoded by at least 14 genes in B. napus, with similar numbers in the other species of Brassicaceae investigated. The Myr1 gene cloned from B. napus has a 19 amino acid signal peptide and consists of 11 exons of sizes ranging from 54 to 256 bp and 10 introns of sizes from 75 to 229 bp. The Myr2 gene has a 20 amino acid signal peptide and consists of 12 exons ranging in size from 35 to 262 bp and 11 introns of sizes from 81 to 131 bp. The exons from the two genes have 83% homology at the amino acid level. The intron-exon splice sites are of GT..AG consensus type. The signal peptides and presence of sites for N-linked glycosylation, suggest transport and glycosylation through the ER-Golgi complex. The differences between the two genes are discussed on the basis of their predicted expression at different developmental stages in the plant. Both genes show homology to a conserved motif representing the glycosyl hydrolase family of enzymes. 相似文献
14.
Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking
machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons
and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous
to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed
that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression
of OsNPSNs was significantly activated in rice seedlings treated with H2O2, but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs
were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing
various concentrations of H2O2, but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H2O2 and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in
different aspects of the signal transduction in plant and yeast responses to abiotic stresses.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
15.
Tsuji S Ueda K Nishiyama T Hasebe M Yoshikawa S Konagaya A Nishiuchi T Yamaguchi K 《Journal of plant research》2007,120(2):281-290
We determined the complete nucleotide sequence of the chloroplast genome of Selaginella uncinata, a lycophyte belonging to the basal lineage of the vascular plants. The circular double-stranded DNA is 144,170 bp, with
an inverted repeat of 25,578 bp separated by a large single copy region (LSC) of 77,706 bp and a small single copy region
(SSC) of 40,886 bp. We assigned 81 protein-coding genes including four pseudogenes, four rRNA genes and only 12 tRNA genes.
Four genes, rps15, rps16, rpl32 and ycf10, found in most chloroplast genomes in land plants were not present in S. uncinata. While gene order and arrangement of the chloroplast genome of another lycophyte, Hupertzia lucidula, are almost the same as those of bryophytes, those of S. uncinata differ considerably from the typical structure of bryophytes with respect to the presence of a unique 20 kb inversion within
the LSC, transposition of two segments from the LSC to the SSC and many gene losses. Thus, the organization of the S. uncinata chloroplast genome provides a new insight into the evolution of lycophytes, which were separated from euphyllophytes approximately
400 million years ago.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
16.
Monosaccharide transporter (MST) genes of Lupinus polyphyllus and L. albus were cloned, expressed and characterised. The isolation and functional characterisation of a cDNA clone and its corresponding
genomic clone of a sugar transporter from L. polyphyllus (LpSTP1) is reported. Phylogenetic comparison of the nucleic and amino acid sequences showed the highest similarity to the
AtSTP1 gene from Arabidopsis thaliana, which encodes a high affinity sugar transporter. The similar topology as well as the substrate specificity and expression
pattern of LpSTP1 encoded protein additionally support the high similarity to the AtSTP1 gene product. The 1,590 bp LpSTP1 cDNA clone was heterologously expressed in yeast resulting in a fully functional specific sugar transporter. This transformation
restored the viability of a yeast deletion mutant, which is devoid of all intrinsic MSTs and thus unable to take up and grow
on hexose-containing media. The LpSTP1 protein is postulated to be a high-affinity MST since it supported growth best on media
containing 0.2% hexose. Tissue-specific expression of LaSTP1 in L. albus was assayed by real-time PCR, which revealed that the lupin STP1 is mainly expressed in flower buds, flowers and young leaves. The results suggest that the main role of LaSTP1 is to catalyse
monosaccharide import in sink tissues to meet increased carbohydrate demand during plant development.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
17.
Nonnative heme coordination structures emerging upon guanidine hydrochloric acid (GdnHCl) induced unfolding of Hydrogenobacter thermophilus ferricytochrome c
552 were characterized by means of paramagnetic NMR. The heme coordination structure possessing the N-terminal amino group of
the peptide chain in place of axial Met (His–Nterm form) was determined in the presence of GdnHCl concentrations in excess of 1.5 M at neutral pH. The stability of the His–Nterm form at pH 7.0 was found to be comparable with that of the bis-His form which has been recognized as a major nonnative heme
coordination structure in cytochrome c folding/unfolding. Consequently, in addition to the bis-His form, the His–Nterm form is a substantial intermediate which affects the pathway and kinetics of the folding/unfolding of cytochromes c, of which the N-terminal amino groups are not acetylated.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
18.
Paavo Ahvenniemi Matthias Wolf Mari J. Lehtonen Paula Wilson Malgorzata German-Kinnari Jari P. T. Valkonen 《Journal of molecular evolution》2009,69(2):150-163
The rRNA cistron (18S–ITS1–5.8S–ITS2–28S) is used widely for phylogenetic analyses. Recent studies show that compensatory
base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between
hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG
determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact
during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared
to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3–21 nuclei per cell), 1–3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish
them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described
as R. solani are a species distinct from other AGs. The ITS1–5.8S–ITS2 sequences were analyzed by direct sequencing of PCR products from
497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability
in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing
cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly,
the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
19.
We studied the short-term impact of sediment load on the photosynthetic performance of Saccharina latissima sporophytes exposed to ultraviolet radiation (UVR). The algae were collected from different sediment-influenced environments
in Svalbard in August 2007. Initial optimum quantum yield (F
v/F
m) of sediment-covered sporophytes was significantly higher compared to sediment-free sporophytes. Experimental sediment coating
on blade discs had a photoprotective function by screening out 92% of the weighted UV-B (UVery) treatment. No UVR-induced photoinhibition was observed in sediment-coated blade discs while sediment removal caused a reduction
in F
v/F
m not only after 12-h UVR exposure but also after 6-h recovery in low white light compared to the initial value. Thus, sediment
coating has a short-term functional significance in mitigating the negative effect of UVR on photosynthesis of an important
kelp species and set a baseline for further studies.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
20.
Narasimha Rao Nizampatnam Harinath Doodhi Yamini Kalinati Narasimhan Sujatha Mulpuri Dinesh Kumar Viswanathaswamy 《Planta》2009,229(4):987-1001
Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking.
To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The
cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control
vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent
transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male
sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile
plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis.
RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed
cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait
was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by
introducing orfH522 gene that could be useful for genetic engineering of male sterility.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献