R E V I E W A R T I C L Einto the mechanisms of protein maturation, folding, and stability Yoshihiro Sambongi1, Susumu Uchiyama2,*, Yuji Kobayashi2, Yasuo Igarashi3and Jun Hasegawa4 1 Gr
Trang 1R E V I E W A R T I C L E
into the mechanisms of protein maturation, folding, and stability
Yoshihiro Sambongi1, Susumu Uchiyama2,*, Yuji Kobayashi2, Yasuo Igarashi3and Jun Hasegawa4
1
Graduate School of Biosphere Sciences, Hiroshima University, Japan;2Faculty of Pharmaceutical Science, Osaka University, Japan;
3 Department of Biotechnology, University of Tokyo, Japan; 4 Daiichi Pharmaceutical Co., Ltd, Tokyo, Japan
Cytochrome c is widely distributed in bacterial species,from
mesophiles to thermophiles,and is one of the
best-charac-terized redox proteins in terms of
biogenesis,folding,struc-ture,function,and evolution Experimental molecular
biology techniques (gene cloning and expression) have
become applicable to cytochrome c,enabling its engineering
and manipulation Heterologous expression systems for
cytochromes c in bacteria,for use in mutagenesis studies,
have been established by extensive investigation of the
bio-logical process by which the functional structure is formed
Mutagenesis and structure analyses based on comparative studies using a thermophile Hydrogenobacter thermophilus cytochrome c-552 and its mesophilic counterpart have pro-vided substantial clues to the mechanism underlying protein stability at the amino-acid level The molecular mechanisms underlying protein maturation,folding,and stability in bacterial cytochromes c are beginning to be understood Keywords: bacteria; biogenesis; cytochrome c; Hydrogeno-bacter thermophilus; mutations; protein stability
In mitochondria,electrons shuttle between the
membrane-bound cytochrome bc1complex and cytochrome oxidase via
a water-soluble protein cytochrome c This process is
required for generation of an electrochemical proton
gradient across the membrane (proton-motive force),which
drives the synthesis of ATP,a biological energy currency
Bacteria also have soluble monoheme Class I cytochromes c
functioning as similar electron carriers on the peripheral
surface of the cytoplasmic membrane This type of
cytochrome c is widely distributed in bacteria,from
meso-philes to thermomeso-philes,and is one of the best-characterized
proteins in terms of biogenesis,folding,structure,function,
and evolution
Cytochrome c is unique among heme proteins in having a
heme covalently attached to the polypeptide chain via two
thioether bonds,formed from the vinyl groups of the heme
and the two cysteine residues in the consensus
Cys-X-X-Cys-His motif [1–4] Site-directed mutagenesis studies of
cytochrome c are at a relatively early stage,because of the
difficulty of expressing the holoprotein heterologously
However,during the last decade,it has been found that
bacteria have specific cellular apparatus for covalent attachment of a heme to the cytochrome c polypeptide [1–4] Knowledge obtained from studies on the cytochrome c biogenesis pathway in bacteria has been used to produce heterologous cytochromes c in large quantities,which has facilitated mutagenesis studies and structure analysis
In this review,we will summarize how bacterial cyto-chromes c have been used in recent mutagenesis and structure studies to elucidate protein stability Through such investigations,we have gained insight into the molecular mechanisms underlying cytochrome c maturation and folding as well as stability Bacterial cytochrome c has contributed greatly to our understanding in these areas,and
is one of the most successful model proteins The basic ideas obtained with cytochromes c should be applicable to other proteins of industrial and/or medical interest
C Y T O C H R O M E c A S A M O D E L F O R
P R O T E I N S T A B I L I T Y S T U D I E S Proteins isolated from thermophilic bacteria are usually stable to heat and chemical denaturants,indicating that they must have most of the determinants of protein stability Initial clues to the relationship between protein structure and stability can be obtained by pairwise sequence com-parison of homologous proteins from thermophiles and mesophiles The cytochromes c,which play a central role in electron-transport chains in both thermophilic and mesophilic bacteria as well as in eukaryotes,are useful in investigations of the structural basis of protein stability at the amino-acid level
Highly homologous cytochromesc from thermophiles and mesophiles
For pairwise comparison to elucidate protein structure– stability relationships using cytochromes c,we must find
Correspondence to Y Sambongi,Graduate School of Biosphere
Sciences,Hiroshima University,1-4-4 Kagamiyama,
Higashi-Hiroshima,Hiroshima 739-8528,Japan.
Fax/Tel.: +81 824 24 7924,
E-mail: sambongi@hiroshima-u.ac.jp
Abbreviations: AA c-555, Aquifex aeolicus cytochrome c-555 s ;
HT c-552, Hydrogenobacter thermophilus cytochrome c-552;
PA c-551, Pseudomonas aeruginosa cytochrome c-551; TT c-552,
Thermus thermophilus cytochrome c-552.
*Present address: Department of Biotechnology,Faculty of
Engine-ering,Osaka University,2-1 Yamadaoka,Suita,Osaka 565-0871,
Japan.
(Received 24 January 2002,revised 5 June 2002,
accepted 10 June 2002)
Trang 2homologues in thermophiles and mesophiles that exhibit
high sequence identity Cytochrome c-552 from a
thermo-philic bacterium, Hydrogenobacter thermophilus (HT c-552),
is an 80-amino-acid protein with a heme covalently attached
to the polypeptide chain [5] The amino-acid sequence
(Fig 1) and main chain folding (Fig 2) of HT c-552 from
this thermophile closely resemble those of the 82-amino-acid
cytochrome c-551 from a mesophile, Pseudomonas
aerugi-nosa(PA c-551) Comparisons of the two proteins indicated
that the amino-acid residues are 56% identical [5],and that
the root mean square deviation for their main chain folding is
within 1 A˚ [6] However,as expected from the difference in
their optimal growth temperatures (H thermophilus,70C;
P aeruginosa,37C),HT c-552 is more stable to heat and chemical denaturants than PA c-551 [7–9] For instance,the former has a significantly higher mid-point denaturation temperature than the latter,as judged both spectrophoto-metrically and calorispectrophoto-metrically As described below,inves-tigation of the relationship between the 3D structures and thermodynamic properties accompanying protein denatu-ration of HT c-552 and PA c-551 (wild-types and mutants) has revealed the molecular mechanism underlying protein stability The difference in stability between them can be attributed to differences in side-chain interactions in a few select regions [9] (see below)
Other homologous cytochromesc
To determine the amino-acid residues responsible for protein stability,it would be better if sequence information was available from a larger number of homologous proteins
in a variety of bacteria that differ in optimal growth temperature Many homologous cytochromes c that exhibit sequence identity with HT c-552 and PA c-551 of more than 50% are also known in other mesophiles,e.g Pseudomonas, Azotobacter,and Nitrosomonas species (Fig 1) On direct sequence comparison of these proteins,
we can find amino-acid residues that exist in HT c-552,but not in most of the others at the corresponding positions It is likely that some of them are the residues responsible for stability,and in fact Ala7,Met13,and Tyr43 in HT c-552 have been shown,by mutagenesis and 3D structure analyses,to be determinants of the higher stability of
HT c-552 (see below) For clarity,the residue numbers used for HT c-552 are those of PA c-551
A hyperthermophile, Aquifex aeolicus,has an 86-amino-acid cytochrome c-555s(AA c-555),in its mature form [10] The sequence identity between HT c-552 and AA c-555 (33%) is not as high as that between HT c-552 and
PA c-551,but these three proteins still exhibit high sequence similarity (more than 50%,Fig 1),therefore their main-chain folding may be similar Furthermore,the optimal growth temperature greatly differs between H thermophilus and A aeolicus (70C and 95 C,respectively) Therefore,
AA c-555 should be included in the group of cytochromes c
to be examined for protein stability Thermodynamic analysis of AA c-555 will be of great interest
The homologous cytochromes c listed in Fig 1 are excellent models for determining the structural origin of protein stability Experimental data on the stability and 3D structure of these proteins in addition to their amino-acid sequences will provide valuable information on the mech-anism underlying protein stability
B I O G E N E S I S O F B A C T E R I A L
C Y T O C H R O M E S c
To confirm experimentally the amino-acid residues respon-sible for the stability indicated by sequence and 3D structure comparisons,site-directed mutagenesis needs to be per-formed on a series of homologous cytochromes c For this, the cytochrome c gene needs to be heterologously expressed
as a holoprotein that has a covalently attached heme and is
in a correctly folded form,like the authentic proteins isolated from the original bacteria Heme attachment,which must occur regardless of whether the cytochrome c gene is
Fig 1 Amino-acid sequence comparison of cytochromes c homologous
to HT c-552 Amino-acid sequences are aligned using residue numbers
(every 10) of PA c-551 HT, Hydrogenobacter thermophilus; PA,
Pseudomonas aeruginosa; PS, Pseudomonas stutzeri; PZ, Pseudomonas
stutzeri Zobell; PM, Pseudomonas mendocina; PD, Pseudomonas
denitrificans; PF, Pseudomonas fluorescens; AV, Azotobacter vinelandii;
NE, Nitrosomonas europaea; AA, Aquifex aeolicus.
Fig 2 3Dstructural comparison of HT c-552 and PA c-551 The
main-chain folding of HT c-552 (red) is overlaid with that of PA c-551
(green).
Trang 3endogenous or exogenous,is a unique step during
cyto-chrome c biogenesis To be able to attempt heterologous
expression of the cytochrome c gene,it is important to
determine how the heme attaches to the polypeptide in the
cell and how its functional structure is formed The
path-way of cytochrome c biogenesis has been extensively studied
[1–4] In this section,we briefly summarize recent advances
in our understanding of bacterial cytochrome c biogenesis
Genes required for cytochromec biogenesis in bacteria
Cytochrome c heme lyase has been identified as the enzyme
responsible for heme attachment to the mitochondrial
cytochrome c polypeptide [11] The apparatus for
cyto-chrome c biogenesis in bacteria is not analogous to that in
mitochondria,because no orthologue of the cytochrome c
heme lyase has been found in the genomes of bacteria that
can synthesize cytochrome c Instead,genetic evidence has
suggested that at least 12 genes (ccm,cytochrome c
maturation; dsb,disulphide bond formation) are required
for bacterial cytochrome c biogenesis [1–4,12] It would be
interesting to compare the biogenesis apparatus for
cytochromes c and nonheme iron-sulfur proteins (Nif
proteins involved in Fe/S cluster formation),as the latter
appears to be common in prokaryotes and eukaryotes to
some extent [13] Among the ccm gene products, Escherichia
coliCcmE was first biochemically characterized as a factor
transferring a heme to the cytochrome c apopolypeptide
[14] A subsequent biochemical study indicated that E coli
CcmC can interact with CcmE during heme transfer [15] It
was predicted that heme transfer occurs in the periplasm
in vivo A site-directed mutagenesis study on bacterial
cytochrome c polypeptides also supported the idea that
heme attachment takes place after the apoprotein has left
the cytoplasm [16]
Effect of thiol–disulfide redox conditions
A defect in an integral membrane protein,DsbD (also
known as DipZ),was first characterized as an E coli
mutation that prevented the synthesis of mature
cyto-chrome c in the periplasm [17] DsbD contains a domain
with potential disulfide isomerase activity facing the
periplasm [18–20] Other Dsb proteins,DsbA and DsbB,
which have been determined to oxidize cysteine thiols to
form the internal disulfide bonds of many proteins in the
E coli periplasm,are also required for cytochrome c
biogenesis [21,22]
Cytochrome c biogenesis in dsbD mutant cells can be
restored by adding low-molecular-mass thiol compounds to
the growth medium [17],and that in dsbA and dsbB mutants
by adding disulfide compounds [22] These
complementa-tion results are consistent with the general role of Dsb
proteins in the regulation of the thiol–disulfide redox
conditions during periplasmic protein folding Although
no biochemical evidence for the requirement of the Dsb
system during cytochrome c biogenesis has been obtained
yet,the genetic evidence suggests that thiol–disulfide redox
control is also essential for cytochrome c biogenesis in the
periplasm Importantly,these results indicated that the level
of cytochrome c production could easily be controlled by
the thiol–disulfide redox potential,and this is the case,as
described below
E X P R E S S I O N O F E X O G E N O U S
C Y T O C H R O M E c G E N E S I N B A C T E R I A For mutagenesis studies and structure analysis,it is necessary to obtain heterologously expressed,mature holo-cytochromes c in large quantities Knowledge obtained from studies on bacterial cytochrome c biogenesis needs to
be extended for the efficient production of mature holocyto-chromes c
Targeting to the periplasm The functional regions of gene products (Ccm and Dsb proteins) required for cytochrome c biogenesis are located
in the periplasm of bacteria Thus,cytochrome c apo-polypeptide targeting to the periplasm is a physiologically essential feature for its maturation This is also indicated
by the presence of a typical signal peptide in the precursor of bacterial soluble cytochrome c Requirement
of the signal peptide was experimentally verified by heterologous gene expression of Paracoccus denitrificans cytochrome c-550 in E coli; the holoprotein is produced
in the periplasm if the gene retains the coding region for
a native signal peptide,but the cytoplasmic apoprotein is produced if this signal is removed [23] Even a mitoch-ondrial soluble cytochrome c can be expressed as a holoprotein in the E coli periplasm when the eukaryotic gene product is targeted to the periplasm by fusing the signal peptide of bacterial cytochrome c at the N-terminus [16]
Periplasmically expressed exogenous cytochromes c in host cells so far have spectrophotometric characteristics identical with those of the authentic proteins produced in the original organisms [8,23–26] These findings indicate that the heme attachment mode and protein folding are correct during heterologous gene expression and protein maturation Thus,heterologously expressed cytochromes c, which are quality-controlled in the bacterial periplasm,can
be used for further biochemical analysis as if we are dealing with the native proteins
Control of production level The yields of heterologously expressed cytochromes c may depend on the copy numbers of the plasmids used In addition,coexpression with ccm genes is effective for higher levels of plasmid-borne cytochrome c gene expression [27] Not only the protein factors functioning in the periplasm, but also low-molecular-mass thiol/disulfide compounds, which can maintain the periplasmic redox balance [28], successfully control the cytochrome c production level For instance,the yields of exogenous and endogenous cytochromes c reach about 10% of the total periplasmic protein fraction in E coli with the addition of disulfide compounds to the medium [22] Furthermore,a certain
E coli strain,JCB7120,can produce exogenous holo-(PA c-551) up to 30% of the total periplasmic protein level [8],although the mechanism underlying this high expression
in this strain is not yet known Now,using bacterial expression systems,we can obtain large amounts of holocytochromes c,which in terms of visible absorption spectra and other properties are indistinguishable from the native proteins This progress has facilitated mutagenesis
Trang 4and structure analyses of bacterial cytochromes c,including
HT c-552 and PA c-551
M U T A G E N E S I S S T U D I E S F O R
S T A B I L I T Y
In general,site-directed mutagenesis is a powerful tool for
investigating the relationship between protein structure and
function Experimental techniques for mutagenesis are
applicable to bacterial cytochrome c,as discussed above
We are able to dissect the molecular mechanisms of
cytochrome c,in terms of biogenesis,protein folding,redox
properties,electron-transfer kinetics,and stability,at the
amino-acid residue level through mutagenesis studies In
this section,we describe successful mutagenesis using
PA c-551 variants modeled by the homologous and more
stable HT c-552
Amino-acid residues responsible for stability
As HT c-552 and PA c-551 have almost identical main-chain folding [6],subtle differences in the side-main-chain interactions must explain the remarkable difference in their stabilities By careful comparison of their 3D structures [6],
we found that aromatic amino-acid interactions uniquely occur between Arg37 and Tyr34 and/or Tyr43,the latter also having hydrophobic contacts with the side chains of Tyr34,Ala40,and Leu44 in HT c-552 These interactions are not found in PA c-551 Small hydrophobic cores formed
by the side chains of Ala7,Met13,and Ile78 in HT c-552 are more tightly packed than the corresponding regions formed
by Phe7,Val13,and Val78 in PA c-551 All these residues are distributed in three separate regions (Fig 3) We expected that these multiple residues spread over the separate regions of HT c-552 would cause overall protein
Fig 3 Side-chain packing in the regions responsible for the stability of HT c-552, and the corresponding regions in the wild-type PA c-551 and its quintuple mutant The mutated residues in the quintuple mutant,and the corresponding ones of the wild-type PA c-551 and HT c-552 are colored purple,green,and red,respectively (A) The region around A7/M13 in HT c-552 and the corresponding regions (B) The region around Y34/Y43 in
HT c-552 and the corresponding regions (C) The region around I78 and the heme (denoted as HEM) in HT c-552 and the corresponding regions.
Trang 5stability in an additive manner,and hoped that we would be
able to clearly determine the stabilizing factors by
muta-genesis studies
Engineering stable proteins
To characterize the factors that affect protein stability,we
attempted to achieve maximal enhancement of the stability
of PA c-551 by introducing minimal mutations into
spa-tially separate regions Five amino-acid residues in
PA c-551,which were selected on structure comparison,
were substituted with those found at the corresponding
positions in HT c-552,and the stabilities of the resulting
PA c-551 mutants were measured A single mutation [Val78
to Ile (V78I)] and double mutations [Phe7 to Ala/Val13 to
Met (F7A/V13M) and Phe34 to Tyr/Glu43 to Tyr (F34Y/
E43Y)] in the three regions of PA c-551 each individually
enhanced the overall protein stability [8] These studies,
together with structure analysis,provided substantial clues
to the mechanism of protein stability in HT c-552 Ala7/
Met13 and Ile78 fill small spaces present in the
correspond-ing regions of PA c-551,and Tyr34/Tyr43 cause a favorable
electrostatic interaction These side-chain interactions may
contribute to the enhanced stability of HT c-552 It would
be worth trying to mutate HT c-552 so that it has the
amino-acid residues found in PA c-551,and to examine whether
the resulting HT c-552 mutants have decreased stability
Surprisingly,a PA c-551 variant simultaneously carrying
the five mutations in the three separate regions (F7A/
V13M/F34Y/E43Y/V78I,quintuple mutant,Fig 3)
exhib-its almost the same stability as that of natural HT c-552
(Fig 4) [9] This demonstrates that it is possible to convert a
mesophilic protein into an artificial one with stability similar
to that of the natural thermophile by replacing a few
amino-acid residues Therefore,the thermophilic character of
HT c-552 may depend on a few strong noncovalent
interactions We further found that the increase in the
stability of the quintuple mutant is almost the same as the sum of the three individual stabilities [9,29] Thus, the mutation(s) in each of the three regions contribute to the overall stability in an additive manner The multiple mutations in the separate regions of PA c-551 provide experimental evidence on the mechanism underlying the enhanced stability of HT c-552,the relationship between local side-chain interactions,and overall protein stability The rational design resulting from careful structural comparison of HT c-552 and PA c-551 makes it possible to select a set of amino-acid residues that are completely responsible for the stability of a thermophilic protein Only
a small number of mutant proteins is required to experi-mentally confirm which amino acids are responsible for overall protein stability This is the advantage of structural comparison of highly homologous proteins If
we randomly selected five of the 35 amino-acid residues that differ between the two cytochromes c,we would have tested (35· 34 · 33 · 32 · 31)/(5 · 4 · 3 · 2 · 1) ¼
324 632 variants
What we can learn from thermophilic proteins The strategies used by thermophilic proteins to enhance stability are,for example,relatively increased polarity of the solvent-exposed surface area,increased packing density and core hydrophobicity,and generation of ion pairs or hydrogen bonds between polar residues [30–32] However, these interactions are often related to each other in a protein molecule,and subtle changes in them can affect the overall stability Thus,it is usually difficult to identify the exact factors that contribute to the enhanced stability of the proteins from thermophilic bacteria This is reasonable because proteins in the native state are stabilized by 10–
20 kJÆmol)1compared with those in the denatured state; the energy is equivalent to the formation of only a few hydrogen bonds Therefore,to understand protein stability,it is necessary to carry out precise comparative studies using homologues exhibiting high sequence identity with similar 3D structures,but differing in stability From such comparisons,we must carefully detect subtle differences in side-chain interactions,and examine their contributions to the overall protein stability by mutagenesis studies If the interactions are spatially separated,their contributions may
be additive,in which case we can clearly identify protein-stabilizing factors The combination of precise comparison
of the structures of thermophilic and mesophilic homolog-ous proteins and selection for multiple mutations in separate regions is a valuable approach to elucidating the relation-ship between structure and stability This approach has been successfully applied to cytochrome c (as discussed above), triose phosphate isomerase [33],ribonuclease HI [34],and cold shock protein [35]
Recent advances in genome projects have revealed the gene resources of thermophilic bacteria,providing further opportunity for systematic comparisons of homologous proteins A similar approach to that used for cytochromes c will be applicable to other proteins Not only sequence comparison to identify the determinants of protein stability, but also the thermophilic proteins themselves can be used to elucidate the basic mechanisms underlying protein functions because they are usually purified and handled more easily than their mesophilic counterparts Although thermophilic
Fig 4 Thermal stability of wild-type PA c-551, the quintuple mutant,
and HT c-552 The heat capacity curves obtained by differential
scanning calorimetry of wild-type PA c-551,the quintuple mutant,and
HT c-552 at pH 5.0 with 1.5 M guanidine hydrochloride are shown.
The peak temperatures represent respective denaturation
tempera-tures.
Trang 6proteins are isolated from diverse extreme environments,
they should reveal general features of protein structure,
function,and stability
M U T A G E N E S I S F O R M A T U R A T I O N
S T U D I E S
In addition to protein stability,mutagenesis studies with
cytochrome c have contributed to the understanding of
protein maturation As discussed above,physiological
attachment of heme to apo-(cytochrome c) takes place in
the periplasm An exception to periplasmic covalent heme
attachment was first found for HT c-552 a decade ago
through a mutagenesis study [36] This unique case has
unexpectedly shed light on the chemical aspects of heme
attachment and apoprotein folding through the follow-up
experiments
Heterologous holo-(HT c-552),which has the heme
attached covalently,is found in the cytoplasm when the
truncated gene coding for the mature protein without the
original signal sequence is transformed into E coli [23,36]
An apo-(HT c-552) variant carrying mutations at the heme
covalent binding site (C12A/C15A) has also been expressed
in the E coli cytoplasm This gene product was found to
have a compactly folded structure,which apparently differs
from that of the natural holoprotein with the heme attached
covalently [37] The folded apoprotein aggregates into
amyloid fibrillar structures over a long time period [38],but,
in the presence of excess heme,it retains the prosthetic
group noncovalently like a b-type cytochrome [39] In
contrast,mesophilic apocytochromes c seem to form a
random coil structure,and holoprotein formation does not
occur in the bacterial cytoplasm [23] These observations
suggest that apo-(HT c-552) has a sufficiently folded
structure to incorporate a heme at moderate temperature,
possibly because of its thermostable properties After
insertion of the heme,cytochrome c folding occurs
There-fore,heme is not only required for the redox properties of
cytochrome c,but is also essential for correct protein
folding during cytochrome c biogenesis
The unique case of cytoplasmic heme attachment also
leads to the hypothesis that covalent thioether bond
formation itself can proceed spontaneously without
enzy-matic assistance once the heme is inserted into the
apopro-tein [23,40] Recently, a thermophile, Thermus thermophilus,
cytochrome c-552 (TT c-552) was produced as a
holopro-tein in the E coli cytoplasm similar to the case of HT c-552
[26] Although the cytoplasmic holo-(TT c-552) has the
same function and spectra as the authentic one,the
cytoplasmic soluble protein fraction also contains a minor
product,which has a heme attached covalently but differs in
heme-binding mode [41] This heterogeneity found in the
cytoplasmic products also appears to indicate that the heme
attachment itself is not catalyzed enzymatically
C O N C L U S I O N S A N D P E R S P E C T I V E
Highly homologous monoheme Class I cytochromes c are
available from a wide range of bacteria,from mesophiles to
thermophiles Their small size and high sequence identity are
advantageous for determining the amino-acid residues
responsible for protein stability The heterologous expression
systems established for cytochrome c, together with the
results from rapidly increasing X-ray crystal and NMR analyses,have stimulated mutagenesis studies,which con-tribute to the understanding of the mechanisms underlying protein maturation,folding,and stability
A mutagenesis study has also been carried out on cytochrome c to investigate its redox properties [42] The redox function of stable HT c-552 will be promising for electrochemical applications,such as the creation of a useful molecular device A cytochrome c folding study will also reveal the basic features of protein conformational diseases,
as first demonstrated with a HT c-552 mutant [38] Various technologies,such as NMR relaxation analysis,temperature jump methods,high pressure NMR,and stopped flow and single–molecule analyses,have been established,and are applicable to the elucidation of the dynamic features of cytochromes c It is of interest to characterize cytochrome c with respect to protein maturation,folding,stability,and function using a variety of combined experimental tech-niques Cytochrome c molecules will become very well understood through such interdisciplinary methods
A C K N O W L E D G E M E N T S
We thank Ikuo Ueda for his support and encouragement,and Kazuaki Nishio and Yuko Iko for critical reading of the manuscript.
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