Effect
on Glucose Transport and Anion Exchange in Human Erythrocytes by Mechanical
Force Factors
LI
Yue-Zhou, ZHOU Han-Qing, PENG Feng, ZHANG
Zhi-Hong*
( Liren Laboratory, Department of Physiology
and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433,
China )
Abstract The
mechanical forces play an important role in both the normal physiology and the
pathology of the cardiovascular system. It was observed that an increase in
speed of rotary flow of the intact erythrocyte suspension resulted in increase
in the rate of glucose entry across the erythrocyte membranes. The effects of
osmotic stress and a membrane curvature-altering drug, chlorpromazine, on the
glucose and anion transport were also shown. The decrease in the activation
energy for glucose and anion transport according to Arrhenius plot for
temperature dependence of membrane transport meant that thermodynamic barrier
for transporter reorientation in the membranes was reduced. The data from the
measurement of intrinsic fluorescence quench in ghosts indicated that the
conformations of both glucose transport protein (GLUT1) and anion transport
protein (band 3) in the erythrocyte membranes were affected by the mechanical
force factors. After inhibition of anion transport, a modification of the
response of glucose transport by the mechanical force was observed, which also
indicates that there is an information linkage between GLUT1 and band 3 in the
erythrocyte membranes.
Key
words mechanical force;
erythrocytes; glucose transport; anion exchange
Cells
in body are subjected to various forms of mechanical forces, including the
tangential shear stress that is due to fluid flow, the volume stress that
results from osmotic pressure, and other mechanical stimuli such as tensile
stress exerted by neighboring cells, the morphologic stress by drug and the
internal tension of the cytoskeleton[1,2]. It is well known that the
cardiovascular system generates an appropriate pressure gradient for the
delivery of blood flow to serve metabolic and other functional needs to
tissues. The rheological functions of the system result from the coordinated
activities of its components, especially the erythrocytes. The molecular events
are beneficial for the body to cope with altered hemodynamic demands, but they
may be inappropriate and detrimental under some disease states.
It
has been reported that physiological shear stresses enhanced the Ca2＋
permeability in human erythrocyte membranes[3]. Health erythrocytes
deformed by shear respond monovalent and divalent cations with increased
permeability[4]. Similarly, the erythrocytes of sickle cell anemia,
which acquire distinctive elongated spicules upon deoxygenation, exhibit
increased permeability to both monovalent and divalent cations[5].
Volume-sensitive ion transport in the erythrocytes was also observed[6].
Mechanosensitive ion channels are well known recently, especially because the
three-dimensional structure of KcsA potassium channel has been determined[7].
But, relatively little is known about the role of mechanical force in the
function performance of the membrane transport proteins.
Because of the simplicity of the
structure of the human erythrocyte, this physically induced response is
particularly amenable to study in detail, and may afford insight into the
responses of more complex mammalian cells. In this paper, we focused on the
effect of mechanical force, including osmotic stress and drug-induced cell
deformation, on the properties of membrane transporters, glucose transporter 1
(GLUT1) and band 3, in human erythrocyte membranes. GLUT1 is a 55 kD integral
membrane glycoprotein that facilitates the passive diffusion of D-glucose
across the cell membrane and contains 12 membrane-spanning helices[8].
Band 3 is a 911-amino-acid glycoprotein that exchanges Cl–
for HCO3– as part of the process of CO2
elimination. The protein comprises two domains: the 52 kD carboxyl-terminal
domain embedded in the membrane which contains the anion-transporting site, and
the 43 kD amino-terminal domain which contains the binding sites for the
cytoskeletal proteins, hemoglobin, and glycolytic enzymes[9]. We
attempt to determine the response of both the GLUT1 and band 3 to the
mechanical signal, and to examine is there any linkage between GLUT1 and band 3
during the process of signal transduction.
1 Materials and Methods
1.1
Materials
Human
blood samples were obtained from the Shanghai Red Cross Blood Center and used
within 2 weeks. D-glucose, phloretin, N-acetyltryptophanamide(NATA),
4,4′-diisothiocyno-stilbene-2,2′-disulfonate(DIDS), Con A and chlorpromazine
were purchased from Sigma. “GOD-PAP” kit for determination of glucose was from
Shanghai Kexin Biotech Institute. All other reagents were of analytical grade.
1.2
Preparation of erythrocytes and ghosts
The
erythrocytes were washed three times with PBS(155 mmol/L NaCl, 5 mmol/L sodium
phosphate, pH 7.4). For preparation of white-stripped ghosts, the erythrocytes
at 1:40 were hemolyzed in lysis medium (5 mmol/L sodium phosphate, pH 7.4) at 4
℃ for 30 min. When membrane fraction
became a pearly white appearance after several times washing and centrifuging
at 15 000 r/min, they were collected and then exposed to 10 volumes of ice-cold
alkaline lysis medium (5 mmol/L sodium phosphate, pH 12.0 adjusted with dilute
NaOH) for 20 s to deplete peripheral proteins. The ghosts were then washed
repeatedly with PBS until the pH of the supernatant was 7.4. Aliquots of the
ghosts were typically resuspended in PBS and incubated for 1 h at 37 ℃
to promote resealing, and used to measure the fluorescence under osmotic
stress. The membrane protein concentration was calibrated according to Lowry’s
method.
1.3
Fluorescence measurements
Fluorescence
measurements were performed at 20 ℃
with a Hitachi M850 spectrofluorometer equipped with a circulating water bath[10].
The wavelength of excitation was 295 nm with a bandwidth of 3 nm and the
emission was at 340 nm with a bandwidth of 9 nm to minimize the contribution of
tyrosine residues. A 310 nm filter was used. Samples containing 25 mg of
protein/L were constantly stirred in a 1 cm cuvette during measurements with a
magnetic stirrer bar at the bottom of the cuvette. The unspecific effects of
dilution, inner filter and ligands fluorescence reabsorption were corrected by
a standard, NATA.
Tryptophan
residues in GLUT1 can be divided into two distinct populations, i.e.,
“accessible” and “not accessible” to aqueous solvent, and for fluorescence
quenching studies modified Stern-Volmer equation was introduced[11],
F0/ΔF＝1/f＋1/fKq[Q] (1)
where ΔF(＝F0-F)
is fluorescence change as quencher [Q] binding, F0 and F
are the unquenched and quenched fluorescence intensity, respectively. f
is the fractional accessible protein fluorescence. Kq is
apparent quenching constant and can be obtained from a plot of F0/ΔF
vs. 1/[Q], which value equals intercept/slope of the straight line.
Dissociation constant (Kd) is deduced from Kq(Kd＝1/Kq).
1.4
Sugar transport measurements
Both
the zero-trans entry and infinite-cis exit were determined. For
measurement of zero-trans entry, washed erythrocytes were incubated at
37 ℃ in PBS for 40 min for depletion of
glucose inside the cells. 50 ml of the erythrocyte suspension (～3×108
cell/ml) was rapidly mixed with 2 ml PBS containing 200 mmol/L D-glucose in 1
cm cuvette which contained a stirring bar. The time course of the change in
absorbance of the cell suspension at 660 nm on a spectrometer (Model 722,
Shanghai No.3 Analytical Instrument Factory) was measured at 37 ℃
with the circular water. The output electrical signal was fed to a computer.
The sampling point for each measurement was 1000. The initial transport rate V0
was determined from the recorded curve of “absorbanre vs. time” and accumulated
over 5 times of measurement. For the erythrocytes treated with a series of
osmolalities, the infinite-cis exit of glucose was determined by use of
GOD-PAP method. The erythrocytes were incubated in the solution with a series
of osmolalities, which contained 80 mmol/L D-glucose, for 40 min at 37 ℃.
50 mg of packed erythrocytes was mixed rapidly with 1 ml the solution
containing a series of (0～10
mmol/L) D-glucose at 16 ℃.
Efflux was terminated at required interval (0～120
s) by the rapid addition of 3 ml ice-cold stopping buffer which contained 50
mmol/L phloretin and maintained the same osmolality as the reacting solution.
Then, the suspension was quickly centrifuged at 0 ℃,
and the D-glucose amount in the supernatant was determined. According to
the data of Solomon et al.[12] and our observation, the total
volume of soluble water inside the erythrocytes under different osmolalities
can be deduced. Thus, the glucose concentration inside of the erythrocytes at
any time interval was calculated from the exterior amount of glucose. The
initial rate of glucose exit was obtained by regression analysis of the
initially linear segment of the time course data. The maximal rate of glucose
infinite-cis exit (Vmax) and affinity constant (Km)
were deduced according to Harris[13].
1.5
Mechanical force factors
The
spectrometer was modified to set up a magnetic stirrer at the bottom of the
photometric cell holder. The solution in cuvette was driven to rotate by a
stirring bar and form a rotary flow field. The relationship between rotation
speed and mechanical force acted on the cells was studied when the erythrocytes
were rotated in the 1-cm cuvette. The mechanical force was increased with the
rotation speed of the stirring bar in this model[14]. The parameter
of the rotation speed is w(r/s, revolutions per second). In the experiments of
osmotic stress, PBS was required to diluted 1:1 with water, then added sucrose
to obtain the solutions with osmolalities from 240 to 480 mOsm. The
osmolalities of solution were measured by a vapor pressure osmometer, VAPRO
model 5520(Wescor Inc.). Cell deformation was induced by chlorpromazine. 50 ml
of the erythrocyte suspension (～3×108
cell/ml) was incubated with 2 ml PBS containing a series of chlorpromazine (0～200
mmol/L) at 37 ℃
for 15 min.
1.6
Anion transport measurements
50
ml of isotonic erythrocytes (～3×108
cell/ml) was rapidly mixed with 2 ml isotonic solution containing 15.5 mmol/L
NaNO2, 139.5 mmol/L NaCl, 5 mmol/L sodium phosphate, pH 7.4 in
cuvette. When NO2– ions reacted with hemoglobin after
penetrating into the erythrocytes, methemoglobin was formed, and hence the
optical properties of cell suspension were changed. According to the time
course of the change in absorbance of the cell suspension at 576 nm on
spectrometer, the rate constant, Kt, of NO2–
entry into the erythrocyte was obtained[15].
2 Results
Blood
flow and the associated shear stress have been shown to play an active role in
the regulation of the structure and function of vascular cells. We were
interested in examining the effect of mechanical force on the membrane
transport proteins in the human erythrocytes. Fig.1 shows the effect of
mechanical force induced by rotary flow on the properties of glucose transport
across the erythrocyte membranes. As rotation speed (w), i.e., mechanical
force, was increased the initial rate of glucose zero-trans entry, V0,
was increased significantly. V0 rose from 13.2 mmol・L-1/min
to 77.1 mmol・L-1/min
after rotation speed was changed to be 40 r/s from zero. Because the
concentration of glucose in the medium was 200 mmol/L, at which the binding
sites of GLUT1 for glucose were occupied completely, V0 can
be regarded as the maximum transport rate approximately[16]. Using
fluorescence quenching method and according to Eq.1 the value of dissociation
constant, Kd, was obtained. It was deduced from the
relationship between Kd and rotation speed [Fig.1(B)] that
the mechanical force would affect the conformational state of GLUT1. For
estimation of unstirred layer effect, we plotted 1/V vs. S/(Vmax–V)
during infinite-cis exit under rotary flow (w＝10
r/s)[10] according to Lieb and Stein, where V is the initial
efflux rate of glucose and S is glucose concentration in the medium[17].
Results from linear regression analysis showed that the P-1,
x-intercept of the extrapolated straight line, was 0.0022±0.0021.
The value of P-1 found to be not significantly different from
zero means that the unstirred layer on the outer side of the erythrocyte
membrane needs not be considered.
Fig.1 Effect of rotation speed, w, on
the initial rate of glucose zero-trans entry, V0, and
dissociation constant, Kd
(A) Relationship between the rotation
speed of the cell suspension, w, and V0. (B) Dissociation
constant, Kd, of glucose binding to GLUT1 at different
rotation speed at 20 ℃.
Mean values and standard deviation are indicated from three separate
experiments.
There
are several methods for studying glucose transport in the erythrocytes. One of
the rapid and simple means is optical method based on the osmotic swelling and
shrinking of the erythrocytes during the course of glucose transport. In the
experiment of osmotic stress, we must always keep the erythrocytes in a
condition of certain osmolality and avoid any possible volume change. So, an
enzymatic method, “GOD-PAP”, was used in this research instead. The maximal
rate of glucose infinite-cis exit (Vmax) and affinity
constant (Km) under a series of osmotic stresses were
determined at 16 ℃
(Fig.2). Both of them were increased with the enhancement of osmotic stress. Vmax
rose from 16.6 mmol・L-1/min
to 106.1 mmol・L-1/min
when the medium osmolality was increased from 240 mOsm to 480 mOsm. Meanwhile, Km
was increased from 4.0 mmol/L to 18.0 mmol/L. The anion transport mediated by
band 3 was also responded to the osmotic stress. The rate constant of NO2–
permeability, Kt, was increased from 7.0 mmol・L-1/min
to 81.6 mmol・L-1/min
with the osmolality enhancement from 240 mOsm to 480 mOsm [Fig.2(C)]. Fig.3
shows the relationship between medium osmolality and dissociation constant (Kd)
for glucose binding to GLUT1 or DIDS bound to band 3. Resealed white stripped
ghosts were treated with 20 mmol/L glucose or 10 mmol/L DIDS at 20 ℃,
pH 7.4. The intrinsic tryptophan fluorescence of the ghosts was measured.
According to the fluorescence quenching an apparent quenching constant was
obtained, and then dissociation constant, Kd, of glucose
binding to GLUT1 or DIDS binding to band 3 was deduced. It seems that the
environment of tryptophan residues in GLUT1 or band 3 was changed by the
osmotic stress.
Fig.2 Effect of osmolality on the glucose and
anion transport
Relationships between the maximal rate of
glucose infinite-cis exit, Vmax, (A), affinity
constant, Km, (B), rate constant, Kt, of NO2–
entry (C) and the medium osmolality. Mean values and standard deviation are
indicated from three separate experiments.
Fig.3 Relationship between osmolality and
dissociation constant (Kd) for glucose binding to GLUT1 (●) or DIDS binding to band 3 (○)
Knowledge
of the temperature dependence of membrane transport makes an appraisal of the
thermodynamic features of the transport system possible from Arrhenius plot of
the data. The Arrhenius plots of effect of mechanical forces on the temperature
dependence of glucose flux and anion transport are shown in Fig.4. The
activation energy, Ea, for glucose efflux was 95.8 kJ/mol for
osmolality of 240 mOsm , 78.4 kJ/mol for 300 mOsm, and 45.4 kJ/mol for 480 mOsm
[Fig.4(A)]. Mechanical force also affected the temperature dependency of
glucose influx. As shown in [Fig.4(B)], the slopes of the straight lines, Ea,
was decreased with increase in rotation speed, w. Fig.4C shows the temperature
dependence of anion transport with three different osmolalities. We found that
an increase in medium osmolality resulted in decrease in Ea for anion exchange.
When osmolality was increased from 240 mOsm to 300 mOsm , and finally reached
to 480 mOsm, the Ea was reduced from 47.4 kJ/mol to 42.7
kJ/mol, and then to 33.8 kJ/mol, respectively.
Fig.4 Temperature dependence of glucose
transport and anion exchange with various mechanical force factors
(A) For glucose efflux under three
different osmolalities: 240 mOsm (□),
300 mOsm (●)
and 480 mOsm (△).
(B) For glucose influx under various rotation speed of the cell suspension: w＝0
r/s (□), w＝10
r/s (●) and w＝40
r/s (△). (C) For anion exchange under three
different osmolalities: 240 mOsm (□),
300 mOsm (●)
and 480 mOsm (△).
A
wild range of amphipathic drugs can change membrane tension and curvature in
the erythrocytes according to the bilayer-couple hypothesis[18].
Chlorpromazine molecules preferentially partition into the inner leaflet of the
phospholipid bilayer to bend the erythrocytes inward and form cup shape. The
rates of both glucose and anion transport were affected by the shape change
induced by chlorpromazine (Fig.5). V0 of glucose influx was
increased from 10.3 mmol・L-1/min
to 82.4 mmol・L–1/min
when chlorpromazine concentration was risen from 0 to 200 mmol/L. In the
meanwhile, the rate constant of NO2– transport was
reduced from 9.8 mmol・L–1
/ min to 2.2 mmol・L–1/min.
Fig.5 Effect of chlorpromazine on the glucose
entry (A) and anion transport (B)
Mean
values and standard deviation are indicated from three separate experiments.
Janoshazi
et al suggested that a transport protein complex, centered on band 3 and
included GLUT1, was responsible for the entire transport process[19].
We used DIDS, a specific inhibitor for anion transport, as a probe to study the
interaction of GLUT1 and band 3. Treated with DIDS from 0 to 50 mol/L, the
response of V0 to the mechanical force (rotation speed, w was
lowered [Fig.6(A)]. It can be inferred that the conformational changes in band
3 after binding with DIDS would be somehow transferred to GLUT1. This would
alter the response behavior of GLUT1 to the mechanical force. Con A, a kind of
lectin that can agglutinate human erythrocytes, binds to band 3 through
oligosaccharide chain. In the experiments, Con A did not change the effect of
mechanical force on the transport property of GLUT1 [Fig.6(B)]. What interested
was that when we treated the erythrocytes with Con A and DIDS together, the
pattern of response of V0 to the mechanical stress was
different from that of only DIDS treated, i.e., V0 was
decreased with the enhancement of mechanical force. Meanwhile, Con A did not
affect the anion exchange in our experimental condition (data not shown).
Fig.6 Effect of DIDS and Con A on the rate of
glucose transport at various mechanical forces
(A) Relationship between the initial rate
of glucose zero-trans entry, V0 , and the rotation
speed of the cell suspension, w at a set of DIDS concentration: control(□),
2 mmol/L(■),
5 mmol/L(△),
10 mmol/L(�),
20 mmol/L(○),
50 mmol/L(●).
(B) Effect of Con A on the relationship between V0 and w:
control(■),
50 mmol/L DIDS(●),
4 mg/L Con A (△),
50 mmol/L DIDS＋4
mg/L Con A(○).
Mean values and standard deviation are indicated from at least three separate
experiments at 37 ℃.
3 Discussion
A
variety of cell types, such as endothelial cells, osteoblasts, kidney
epithelial cells, and fibroblasts have been shown to respond to fluid shear
stress in vitro. Results presented here demonstrate that the important
functions of erythrocyte membranes, i.e., glucose and anion transport across
the membranes, do have certain response to the mechanical force.
Unlike
endothelial cells in vascular system, in which the main stress to be endured is
tangential shear stress, the erythrocytes are subjected to complicated
hemodynamic force which was mimicked by a rotary flow field in our experiments.
The deformation of the erythrocyte is very important for performance of the
cell function in blood circulation. Deformation of the erythrocyte needs energy
that is provided completely by the ATP. The ATP inside the erythrocyte is
produced uniquely through glycolytic process, and its level closely
interrelates with the rate of glucose transport across the erythrocyte
membranes. We obtained that the rate of glucose influx (V0)
was increased with the enhancement of mechanical stress. In the meantime, the
rate of glucose exit was decreased[14]. This may play an important
role for the erythrocytes in the high mechanical force condition because in
such circumstances the cells need more ATP to make intensive cell deformation,
which can be satisfied by increase in glucose level inside the cell. The
temperature dependence of the glucose transport across the erythrocyte
membranes showed that the Arrehnius activation energy was responded to the
mechanical force. To make a comparison between Fig.1 or Fig.2 and Fig.4, it is
deduced that a decreased in the activation energy for glucose transport induced
by mechanical force results in an increase in the rate of glucose transport.
This result is obviously reasonable. Reduction of the activation energy means
the decrease in thermodynamic barrier for GLUT1 reorientation in the membrane
and then substrates are much easy to transport.
The
intrinsic protein fluorescence due to its endogenous tryptophan(s) is known to
be sensitive to local microenvironments and has served as useful probe in
studying protein tertiary structure and its dynamics. For GLUT1, quenching of
the intrinsic fluorescence by ligands has been studied to describe protein
dynamics induced by transporter substrates and inhibitors[20]. GLUT1
consists of 492 amino acid residues among them four tryptophan residues are
located in the hydrophobic environment and another two tryptophan residues are
in the hydrophilic environment. When binding with substrates or inhibitors the
fluorescence quantum yield of these exposed tryptophan residues will be
decreased for their moving from a hydrophilic to a hydrophobic environment.
Because the fluorescence quench came from binding of GLUT1 with their specific
substrates, glucose, so we could exclude the contribution of the tryptophan
residues of other membrane proteins from the value of fluorescence change (DF).
On account of the lack of a suitable method to eliminate the scattering effect
of the ghosts from the intrinsic fluorescence, the total amount of membrane
proteins for each sample was the same in the experiment. Under this condition
it was still possible to estimate the apparent quenching constant, Kq,
according to Eq. 1 from plot of “1/DF vs.1/[Q]” even if we did
not know the exact value of F0.
As
a consequence of the cell membrane permeability for water molecules, which
follows hydrostatic and osmotic pressure gradients, mammalian cell volume
constancy is challenged by any alteration in intracellular and/or extracellular
osmolality. A notable example is the kidney medulla, where extracellular
osmolality may range from isotonicity to more than 1 500 mOsm during water
deprivation. Many evidences show that osmotic stress is a ubiquitous stimulus.
An increase in osmolality leads to decrease in cell volume at constant surface
area, which would facilitate the passage of the erythrocytes through narrow
channels, and reduce the erythrocytes to deform in shear flow. A decrease in
osmolality causes the opposite actions. Erythrocyte osmotic regulatory
mechanisms are specifically important in limiting alterations of cell volume
during their passage through the hypertonic kidney medulla and during HCO3–
transport in the lung and the periphery. One disorder exacerbated by altered
erythrocytes cell volume regulatory mechanisms is sickle cell anemia. Our data
indicated that the change in membrane tension resulted from the osmotic stress
could regulate the structure and function of GLUT1.
It
was reported that the erythrocytes (normal or sickle cell anemia) deformed by
shear stress have an increased permeability to monovalent and divalent cations.
On the other hand, anion exchange is not affected by shear stress[21].
In spite of modifying membrane skeletons, changing ion concentration, altering
metabolism and aging condition, anion exchange did not respond to rotate speed
in our experiment (unpublished observation). It is very interesting that the
rate constant of NO2– permeability, Kt,
was increased with the enhancement of osmolality [Fig.2(C)]. Meanwhile, the
activation energy for NO2– binding to band 3 was
decreased [Fig.4(C)]. Shear stress and osmotic stress are two kinds of
mechanical force and different in action pattern. The force of former is
tangential to the membrane and that of latter is vertical. It is obvious that
responses of the membrane proteins to them are various, and this would be a
reason why band 3 answers to osmotic stress but not to shear stress. Band 3 is
the most abundant protein in the erythrocyte membranes and is responsible for
the exchange of HCO3– formed from tissue CO2
with Cl– in the erythrocyte cytoplasm. This anion exchange occurs
during the passage of the erythrocytes through the tissue capillaries, and then
later in the reverse direction, as the erythrocytes pass through the lung.
There was reported that conformational changes in band 3 mediated shape changes
of the human erythrocytes[22].
The
erythrocyte membranes can been directly manipulated by applying amphipathic
drugs known to change membrane tension and curvature[18]. This is
another way to mimic the effect of mechanical force. We applied chlorpromazine
to bend membranes inward and found the expected morphologic change in the
erythrocytes. It has long been known that anionic and cationic amphiphaths can
induce echinocytic shape and stomatocytic (cup) shape change in the
erythrocytes, respectively. However, neither of these shape changes was
associated with an increase in cation flux in the erythrocytes[23].
Our data shows that an increase in chlorpromazine concentration resulted in
enhancement of glucose influx and decrease of NO2– permeabilitiy
(Fig.6). It demonstrates that transport property of GLUT1 and band 3 was
sensitive to the membrane tension induced by chlorpromazine and different from
that of cations transport.
In
light of the above results that the three mechanical force factors do affect
the glucose transport and anion exchange in human erythrocytes, it is our
further task to establish more quantitative relationship and to unveil the
detailed molecular mechanism for inducing the change in conformation and
function of the membrane transports by the mechanical force.
Some
specific inhibitors of anion transport can modulate the transport function of
GLUT1 and the information can be transmitted from GLUT1 to band 3[15,19].
In the present study, we observed that DIDS lowered the response of the rate of
glucose transport to the mechanical force. This demonstrates once again that
there is signal linkage between GLUT1 and band 3 in the erythrocyte membrane.
To consider the effect of Con A [Fig.6(B)], it seems that oligosaccharide chain
may be involved in the transduction of mechanical signals between them.
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Received: March 20, 2001 Accepted: May 8, 2001
This work was supported by the grants
from the National Natural Science Foundation of China, No.39730150 and
No.30070205
*Corresponding author: Tel,
86-21-65643673; Fax, 86-21-65650149; e-mail, [email protected]