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1.
Oka T Nishimoto Y Sasagawa T Kanouchi H Kawasaki Y Natori Y 《Cellular and molecular life sciences : CMLS》1999,55(1):131-134
An efficient Escherichia coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. Complementary DNA encoding
PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E. coli was expressed by isopropyl 1-thio-β-D-galactopyranoside (IPTG), the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The
purified product showed the expected NH2-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic
PSP isolated from rat liver.
Received 8 October 1998; received after revision 6 November 1998; accepted 6 November 1998 相似文献
2.
The role of hsp70 in protection and repair of luciferase activity in vivo; experimental data and mathematical modelling 总被引:1,自引:0,他引:1
J. E. M. Souren F. A. C. Wiegant R. Van Wijk 《Cellular and molecular life sciences : CMLS》1999,55(5):799-811
The stably transfected rat cell line HR24 expressing high levels of the inducible human hsp70 and its parental cell line
Rat-1 were used for in vivo studies to analyse the role of hsp70 during thermal protein denaturation and the subsequent renaturation.
In order to monitor denaturation and renaturation of a cellular protein in vivo, both cell lines were transiently transfected
with firefly luciferase (Luc). The continuous monitoring of Luc activity during and after heat stress allowed a detailed analysis
of the inactivation and reactivation kinetics in cells grown in monolayers. The aim of these studies was to distinguish a
protective effect of increased hsp70 levels during heat shock-induced protein inactivation from a stimulation of reactivation.
In this paper we show that in cells that are stably transfected with hsp70, thermal Luc inactivation decreased, and subsequent
reactivation yielded higher activity levels, compared with the parental cells. The difference in early inactivation kinetics
observed in the two cell lines suggests an immediate effect of the presence of an extra amount of hsp70 on enzyme inactivation.
Using different mathematical models, the heat-induced inactivation and reactivation kinetics was compared with simulations
of denaturation and renaturation. It is concluded that the model in which it is assumed that hsp70 is able to interact with
partially denatured proteins, which did not yet lose their enzymatic activity, most optimally explains the experimental observations.
Received 2 December 1998; received after revision 19 February 1999; accepted 18 March 1999 相似文献
3.
Signalling roles of mammalian phospholipase D1 and D2 总被引:11,自引:0,他引:11
S. Cockcroft 《Cellular and molecular life sciences : CMLS》2001,58(11):1674-1687
Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidate
(PA) and choline. PLD activity in mammalian cells is low and is transiently stimulated upon activation by G-protein-coupled
and receptor tyrosine kinase cell surface receptors. Two mammalian PLD enzymes (PLD1 and PLD2) have been cloned and their
intracellular regulators identified as ARF and Rho proteins, protein kinase Cα as well as the lipid, phosphatidylinositol
[4, 5] bisphosphate (PIP2). I discuss the regulation of these enzymes by cell surface receptors, their cellular localisation and the potential function
of PA as a second messenger. Evidence is presented for a role of PA in regulating the lipid kinase activity of PIP 5-kinase,
an enzyme that synthesises PIP2. A signalling role of phospholipase D via PA and indirectly via PIP2 in regulating membrane traffic and actin dynamics is indicated by the available data.
Received 25 April 2001; received after revision 15 June 2001; accepted 15 June 2001 相似文献
4.
Neurodegeneration changes in primary central nervous system neurons transfected with the Alzheimer-associated neuronal thread protein gene 总被引:6,自引:0,他引:6
The AD7c-NTP gene is over-expressed in brains with Alzheimer's disease (AD), and increased levels of the corresponding protein
are detectable in cortical neurons, brain tissue extracts, cerebrospinal fluid, and urine beginning early in the course of
AD neurodegeneration. In the present study, we utilized a novel method to transfect post-mitotic primary neuronal cell cultures,
and demonstrated that over-expression of the AD7c-NTP gene causes cell death and neuritic sprouting, two prominent abnormalities
associated with AD. These results provide further evidence that aberrantly increas-ed AD7c-NTP expression may have a role
in AD-type neurodegeneration. In addition, we demonstrate that primary post-mitotic neurons can be efficiently transfected
with conventional recombinant plasmid DNA to evaluate the effects of gene over-expression in relevant in vitro models.
Received 31 January 2001; received after revision 31 March 2001; accepted 4 April 2001 相似文献
5.
The intracellular signaling pathways mediating the nuclear exclusion of the androgen receptor (AR) by melatonin were evaluated
in PC3 cells stably transfected with the AR. The melatonin-induced nuclear exclusion of the AR by melatonin (100 nM, 3 h)
was blocked by LY 83583 (an inhibitor of guanylyl cyclases). 8-Bromo-cGMP (a cell-permeable cGMP analog), mimicked the effect
of melatonin, as did ionomycin (a calcium ionophore) and PMA [an activator of protein kinase C (PKC)], and their effects were
blocked by GF-109203X (a selective PKC inhibitor). BAPTA (an intracellular calcium chelator) blocked the effects of melatonin
and 8-bromo-cGMP but not of PMA. Inhibition or activation of the protein kinase A pathway did not affect basal or melatonin-mediated
AR localization. We conclude that the melatonin-mediated rise in cGMP elicits AR nuclear exclusion via a pathway involving
increased intracellular calcium and PKC activation. These results define a novel signaling pathway that regulates AR localization
and androgen responses in target cells.
Received 31 July 2001; received after revision 18 September 2001; accepted 30 October 2001 相似文献
6.
Protein farnesylation in mammalian cells: effects of farnesyltransferase inhibitors on cancer cells 总被引:3,自引:0,他引:3
F. Tamanoi C.-L. Gau C. Jiang H. Edamatsu J. Kato-Stankiewicz 《Cellular and molecular life sciences : CMLS》2001,58(11):1636-1649
Protein farnesylation, catalyzed by protein farnesyltransferase, plays important roles in the membrane association and protein-protein
interaction of a number of eukaryotic proteins. Recent development of farnesyltransferase inhibitors (FTIs) has led to further
insight into the biological significance of farnesylation in cancer cells. A number of reports point to the dramatic effects
FTIs exert on cancer cells. In addition to inhibiting anchorage-independent growth, FTIs cause changes in the cell cycle either
at the G1/S or at the G2/M phase. Furthermore, induction of apoptosis by FTIs has been reported. FTIs also affects the actin
cytoskeleton and cell morphology. This review summarizes these reports and discusses implications for farnesylated proteins
responsible for these FTI effects.
Received 17 April 2001; received after revision 28 May 2001; accepted 28 May 2001 相似文献
7.
Jaspamide (jasplakinolide) is a natural peptide isolated from marine sponges of Jaspis species and has fungicidal and growth-inhibiting activities. We characterized the jasplakinolide-induced loss of viability
by programmed cell death in the HL-60 human promyelocytic leukemia cell line and found that this process was accompanied by
neutral endopeptidase (NEP)/CD10 expression on the surface of the apoptotic cells. HL-60 cells do not normally express detectable
amounts of NEP/CD10 on their surface or intracytoplasmically, but upon jaspamide treatment, CD10 was synthesized de novo,
its expression being inhibited by cycloheximide pretreatment. Once synthesized, NEP/CD10 interfered with the jasplakinolide
signal delivered to HL-60 cells. Inhibition of NEP/CD10 by the NEP inhibitor phosphoramidon or by an anti-CD10 monoclonal
antibody significantly increased apoptosis induction. The appearance of CD10 on the cell surface was blocked by preincubation
of the cells with the monocytic/macrophage-differentiating agents vitamin D3 and phorbol 12-myristate 13-acetate, but not
by the granulocytic differentiating agents retinoic acid or dimethyl sulfoxide. Moreover, in the promonocytic U937 and mature
monocytic THP-1 cell lines, jaspamide induced apoptosis but not CD10 expression. In HL-60 cells, CD10 expression was partially
but not totally blocked by the broad-spectrum caspase inhibitor benzyloxacarbonyl-Val-Ala-Asp-fluoromethylketone, indicating
a connection between apoptosis induction and CD10 synthesis. Our findings suggest that the CD10 expression is related to the
programmed cell death induction by jaspamide, and also with the process of granulocytic differentiation in HL-60 cells.
Received 22 April 2002; received after revision 8 June 2002; accepted 10 June 2002 相似文献
8.
Elevated levels of butyrylcholinesterase activity occur under a number of hypertriglyceridemic conditions, including diabetes
and obesity. This study examines whether butyrylcholinesterase activity has a direct effect on triglyceride production, using
Caco-2 cells, a human intestinal adenocarcinoma cell line. Caco-2 cells were incubated with 500 μM oleate to stimulate triglyceride
production, and butyrylcholinesterase activity was measured in the cellular homogenate. Butyrylcholinesterase activity was
approximately 3 × 10-3 mmol/min per milligram protein. Although triglyceride production increased by almost five-fold after 18 h of stimulation
with oleate, butyrylcholinesterase activity was not increased. Furthermore, inhibition of butyrylcholinesterase activity using
1 mM tetraisopropylpyrophosphoramide did not significantly affect triglyceride production or secretion. Human insulin (100
μU/ml) increased the production of butyrylcholinesterase without increasing triglyceride production. This demonstrates that
stimulation of fatty acid production and butyrylcholinesterase activity occur by independent mechanisms and suggests that
their correlation in hyperlipidemic conditions is not due to a direct relationship in production in situ.
Received 23 April 2001; received after revision 25 May 2001; accepted 20 June 2001 相似文献
9.
Olfactory ensheathing cells have been used in several studies to promote repair in the injured spinal cord. However, cellular
interaction between olfactory ensheathing cells and glial cells induced to be reactive in the aftermath of injury site has
not been investigated. Using an in vitro model of astrogliosis, we show that reactive astrocytes expressed significantly less glial fibrillary acidic protein (GFAP)
when cultured both in direct contact with olfactory ensheathing cells and when the two cell types were separated by a porous
membrane. Immunofluorescence staining also suggested that reactive astrocytes showed decreased chondroitin sulfate proteoglycans
in the presence of olfactory ensheathing cells, although the reduction was not statistically significant. No down-regulation
of GFAP was observed when reactive astrocytes were similarly cultured with Schwann cells. Cell viability assay and bromodeoxyuridine
uptake showed that proliferation of reactive astrocytes was significantly increased in the presence of olfactory ensheathing
cells and Schwann cells.
Received 27 February 2007; received after revision 30 March 2007; accepted 3 April 2007 相似文献
10.
Levy-Favatier F Leroux A Antoine B Nedelec B Delpech M 《Cellular and molecular life sciences : CMLS》2004,61(22):2886-2892
In a previous study, we identified and purified a 99-amino-acid rat liver-kidney perchloric-acid-soluble 23-kDa protein (P23) which displays 30% identity with a highly conserved domain of heat shock proteins (HSPs), as well as an AT-rich 3 untranslated region, which has also been described to play a role in H70 mRNA life span and protein expression. An identical perchloric-acid-soluble protein inhibiting protein synthesis in a rabbit reticulocyte lysate system was also found 2 years later by another group. More recently, the novel, the YjgF, protein family has been described, comprising, 24 full-length homologues, including P23, highly conserved through evolution, and consisting of approximately 130 residues each and sharing a common ternary structure. Independent studies from different laboratories have provided various hypothetical functions for each of these proteins. The high degree of evolutionary conservation may suggest that these proteins play an important role in cellular regulation. Although the function of none of these proteins is known precisely, we present experimental evidence which, combined with the relationship to glucose-regulating protein revealed here, and the relationship to fatty-acid-binding protein revealed by others, allow us to propose a role for P23. In rat liver, P23 expression is developmentally regulated and modulated by dietary glucose, and its mRNA is induced by starvation, in the presence of fatty-acids and in 3-MeDAB-induced hepatomas. The mRNA encoding mouse liver P23 is also hormonally modulated in a mouse line AT1F8. These data indicate that P23 protein might be a key controller of intermediary metabolism during fasting.Received 7 June 2003; received after revision 8 September 2004; accepted 10 October 2004 相似文献
11.
Expansion of amino acid homo-sequences, such as polyglutamines or polyalanines, in proteins has been directly implicated in
various degenerative diseases through a mechanism of protein misfolding and aggregation. However, it is still unclear how
the nature of the expansion and the protein context influence the tendency of a protein to aggregate. Here, we have addressed
these questions using spinocerebellar ataxia type-3 (ATX3) protein, the best characterised of the polyglutamine proteins,
chosen as a model system. Using a transfected mammalian cell line, we demonstrate that ATX3 aggregation is noticeably reduced
by deletion or replacement of regions other than the polyglutamine tract. The nature of the amino acid homo-sequences also
has a strong influence on aggregation. From our studies, we draw general conclusions on the effect of the protein architecture
and of the amino acid homo-sequence on pathology.
Received 3 March 2006; received after revision 19 April 2006; accepted 22 May 2006 相似文献
12.
R. G. MacDonald R. H. McCusker D. J. Blackwood J. A. Vanderhoof J. H. Y. Park 《Cellular and molecular life sciences : CMLS》1998,54(2):158-166
To determine if intestinal stromal cells secrete diffusible factors such as insulin-like growth factors (IGFs) capable of
regulating epithelial cell growth in vitro, stromal cells were isolated by enzymatic digestion of rat intestine. Incorporation
of [3H]thymidine into DNA and [14C]leucine into protein of IEC-6 cells, a model intestinal epithelial cell line, was significantly increased (two- to threefold)
when the IEC-6 cells were co-cultured with stromal cells, relative to IEC-6 cells grown alone. Medium conditioned by stromal
cells stimulated DNA synthesis of IEC-6 cells in a dose-dependent manner. Analysis of the conditioned medium revealed that
intestinal stromal cells secreted IGF-I, but little IGF-II, in addition to an M
r 32,000 IGF-binding protein (IGFBP-2) and an IGFBP having M
r∼ 24,000. We conclude that rat intestinal stromal cells secrete one or more diffusible factors, which may include IGF-I and
IGFBPs, capable of stimulating proliferation of IEC-6 cells in vitro.
Received 25 August 1997; received after revision 7 November 1997; accepted 20 November 1997 相似文献
13.
Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria 总被引:3,自引:0,他引:3
Thomson M 《Cellular and molecular life sciences : CMLS》2002,59(2):213-219
The finding that mitochondria contain substrates for protein kinases lead to the discovery that protein kinases are located
in the mitochondria of certain tissues and species. These include pyruvate dyhydrogenase kinase, branched-chain α-ketoacid dehydrogenase kinase, protein kinase A, protein kinase Cδ, stress-activated kinase and A-Raf as well as unidentified kinases. Recent evidence suggests that mitochondrial protein kinases
may be involved in physiological processes such as apoptosis and steroidogenesis. Additionally, the novel finding of low-molecular-weight
GTP-binding proteins in mitochondria suggests the possibility that these may interact with mitochondrial protein kinases to
regulate the activity of mitochondrial effector proteins. The fact that there are components of cellular regulatory systems
in mitochondria indicates the exciting possibility of undiscovered systems regulating mitochondrial physiology.
Received 19 June 2001; received after revision 7 August 2001; accepted 8 August 2001 相似文献
14.
Brem R Oraszlan-Szovik K Foser S Bohrmann B Certa U 《Cellular and molecular life sciences : CMLS》2003,60(6):1235-1248
Interferon (IFN)-inducible proteins of the 1-8 gene family mediate homotypic adhesion and transduction of antiproliferative
signals. Their induction correlates with inhibition of cell growth while they are often repressed in the course of malignant
transformation and tumor development. Ras-mediated transformation of mouse mast cells is associated with downregulation of
1-8U expression and interferon-α (IFN-α) treatment reverts the proliferation rate to normal levels together with induction
of 1-8U. Conversely, the antiproliferative responses of IFN-α in sensitive human melanoma cells are accompanied by 1-8U induction.
Here we provide direct evidence that recombinant expression of 1-8U in human cell lines is sufficient to block cell proliferation.
Based on the abundant expression and subcellular localization to the plasma membrane and exosome-like structures, we propose
a model capable of explaining the pleiotropic functions of 1-8 family proteins in tumor cells and during normal development.
Received 15 January 2003; received after revision 21 March 2003; accepted 25 March 2003
RID="*"
ID="*"Corresponding author. 相似文献
15.
B. C. Yoo S-H. Hong J-L. Ku Y-H. Kim Y-K. Shin S-G. Jang I-J. Kim S-Y. Jeong J-G. Park 《Cellular and molecular life sciences : CMLS》2009,66(2):350-364
Comparative analysis of proteomes using 5-fluorouracil (5-FU)-resistant human colon cancer cell line revealed that decreased
galectin-3 expression was significantly associated with retarded proliferation. However, in the presence of 5-FU proliferation
rate of cells with suppressed galectin-3 expression did not differ from that of cells with normal galectin-3 expression, even
galectin-3 suppression augmented apoptosis. Mechanism by which galectin-3 regulates cancer cell proliferation has been identified
in immunoprecipitates of the anti-galectin-3 antibody. Heterogeneous nuclear ribonucleoprotein Q (hnRNP Q) was identified
as a protein interacting with galectin-3. Interestingly, while galectin-3 protein was not affected by the hnRNP Q level, its
suppression was accompanied by a decrease in hnRNP Q expression. The present study demonstrates that galectin-3 stabilizes
hnRNP Q via complex formation, and reduction in the hnRNP Q level leads to slow proliferation and less susceptibility to 5-FU.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
B.C.Yoo; S-H.Hong; These two authors contributed equally to this work.
Received 10 September 2008; received after revision 19 October 2008; accepted 07 November 2008 相似文献
16.
The processing and presentation of endogenous and exogenous antigen by Schwann cells in vitro 总被引:1,自引:0,他引:1
Lilje O 《Cellular and molecular life sciences : CMLS》2002,59(12):2191-2198
The expression of major histocomatibility complex class II in vitro and in vivo by Schwann cells indicates a potential facultative
role of Schwann cells in the presentation of antigen to neuritogenic T cells during inflammatory demyelinating neuropathies.
Using a T cell proliferation assay, this study demonstrated that processing and presentation of endogenous and exogenous antigen
by Schwann cells influences T cell proliferation. Statistical analysis of proliferation and its relation to processing and
presentation of antigen by Schwann cells had not been previously addressed. Different combinations of factors including treatment
of cultures (untreated, irradiated or fixed), concentration of exogenous antigen (0 or 40 μmg/ml), the presence of interferon-γ
and the timing of exogenous antigen addition influence the proliferation P2-specific, non-mammalian protein ovalbumin-specific T cell lines and naive T cells.
Received 25 July 2002; received after revision 9 September 2002; accepted 7 October 2002 相似文献
17.
Culture in low levels of oxygen enhances in vitro proliferation potential of satellite cells from old skeletal muscles 总被引:4,自引:0,他引:4
Chakravarthy MV Spangenburg EE Booth FW 《Cellular and molecular life sciences : CMLS》2001,58(8):1150-1158
The proliferation ability of satellite cells (considered the 'stem cells' of mature myofibers) declines with increasing age
when cultured under standard cell culture conditions of 21% oxygen. However, actual oxygen levels in the intact myofiber in
vivo are an order of magnitude lower. No studies to date have addressed the issue of whether culturing satellite cells from
old muscles under more 'physiologic' conditions would enhance their proliferation and/or differentiation ability. Therefore,
we analyzed satellite cells derived from 31-month-old rats in standard cultures with 21% O2 and in lowered (∼3%) O2. Under the lowered O2 conditions, we noted a remarkable increase in the percentage of large-sized colonies, activation of cell cycle progression
factors, phosphorylation of Akt, and downregulation of the cell cycle inhibitor p27Kip1. These data suggest that lower O2 levels provide a milieu that stimulates proliferation by allowing continued cell cycle progression, to result ultimately
in the enhanced in vitro replicative life span of the old satellite cells. Such a method therefore provides an improved means
for the ex vivo generation of progenitor satellite cell populations for potential therapeutic stem cell transplantation.
Received 20 April 2001; received after revision 28 May 2001; accepted 31 May 2001 相似文献
18.
Oxidative stress and hypoxia-like injury cause Alzheimer-type molecular abnormalities in central nervous system neurons 总被引:11,自引:0,他引:11
de la Monte SM Neely TR Cannon J Wands JR 《Cellular and molecular life sciences : CMLS》2000,57(10):1471-1481
Neuronal loss and neuritic/cytoskeletal lesions (synaptic disconnection and proliferation of dystrophic neurites) represent
major dementia-associated abnormalities in Alzheimer’s disease (AD). This study examined the role of oxidative stress as a
factor contributing to both the cell death and neuritic degeneration cascades in AD. Primary neuron cultures were treated
with H2O2 (9–90 μM) or desferrioxamine (2–25 μM) for 24 h and then analyzed for viability, mitochondrial mass, mitochondrial function,
and pro-apoptosis and sprouting gene expression. H2O2 treatment causes free-radical injury and desferrioxamine causes hypoxia-type injury without free radical generation. The
H2O2-treated cells exhibited sustained viability but neurite retraction, impaired mitochondrial function, increased levels of
the pro-apoptosis gene product CD95/Fas, reduced expression of N2J1-immunoreactive neuronal thread protein and synaptophysin,
and reduced distribution of mitochondria in neuritic processes. Desferrioxamine treatment resulted in dose-dependent neuronal
loss associated with impaired mitochondrial function, proliferation of neurites, and reduced expression of GAP-43, which has
a role in path-finding during neurite outgrowth. The results suggest that oxidative stress can cause neurodegeneration associated
with enhanced susceptibility to apoptosis due to activation of pro-apoptosis genes, neurite retraction (synaptic disconnection),
and impaired transport of mitochondria to cell processes where they are likely required for synaptic function. In contrast,
hypoxia-type injury causes neuronal loss with proliferation of neurites (sprouting), impaired mitochondrial function, and
reduced expression of molecules required to form and maintain synaptic connections. Since similar abnormalities occur in AD,
both oxidative stress and hypoxic injury can contribute to AD neurodegeneration.
Received 24 May 2000; received after revision 7 July 2000; accepted 27 July 2000 相似文献
19.
Trimeric guanine nucleotide-binding proteins (G proteins) function as the key regulatory elements in a number of transmembrane
signaling cascades where they convey information from agonist-activated receptors to effector molecules. The subcellular localization
of G proteins is directly related to their functional role, i.e., the dominant portion of the cellular pool of G proteins
resides in the plasma membrane. An intimate association of G protein subunits with the plasma membrane has been well known
for a long time. However, results of a number of independent studies published in the past decade have indicated clearly that
exposure of intact target cells to agonists results in subcellular redistribution of the cognate G proteins from plasma membranes
to the light-vesicular membrane fractions, in internalization from the cell surface into the cell interior and in transfer
from the membrane to the soluble cell fraction (high-speed supernatant), i.e., solubilization. Solubilization of G protein
α subunits as a consequence of stimulation of G protein-coupled receptors (GPCRs) with agonists has also been observed in
isolated membrane preparations. The membrane-cytosol shift of G proteins was detected even after direct activation of these
proteins by non-hydrolyzable analogues of GTP or by cholera toxin-induced ADP-ribosylation. In addition, prolonged stimulation
of GPCRs with agonists has been shown to lead to down-regulation of the relevant G proteins. Together, these data suggest
that G proteins might potentially participate in a highly complex set of events, which are generally termed desensitization
of the hormone response. Internalization, subcellular redistribution, solubilization, and down-regulation of trimeric G proteins
may thus provide an additional means (i.e., beside receptor-based mechanisms) to dampen the hormone or neurotransmitter response
after sustained (long-term) exposure.
Received 31 August 2001; received after revision 31 October 2001; accepted 7 November 2001 相似文献
20.
The correct repair of double-strand breaks (DSBs) is essential for the genomic integrity of a cell, as inappropriate repair
can lead to chromosomal rearrangements such as translocations. In many hematologic cancers and sarcomas, translocations are
the etiological factor in tumorigenesis, resulting in either the deregulation of a proto-oncogene or the expression of a fusion
protein with transforming properties. Mammalian cells are able to repair DSBs by pathways involving homologous recombination
and nonhomologous end-joining. The analysis of translocation breakpoints in a number of cancers and the development of model
translocation systems are beginning to shed light on specific DSB repair pathway(s) responsible for the improper repair of
broken chromosomes.
Received 19 June 2001; received after revision 6 September 2001; accepted 11 September 2001 相似文献