Mutagenesis, Vol. 15, No. 6, 507-515,
November 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Changes in microtubule organization after exposure to a benzimidazole derivative in Chinese hamster cells
Research and Development, Sigma-Tau, Pomezia, Rome, 1 Department of Genetics and Molecular Biology, University `La Sapienza', Rome, 2 CNR Centre of Evolutionary Genetics, c/o Department of Genetics and Molecular Biology, University `La Sapienza', Rome and 3 University `Roma Tre', V.le Marconi 146, 00146 Rome, Italy
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Abstract |
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Many aneugenic compounds are known to affect one or several components of the mitotic apparatus. The mechanisms and targets of the aneuploidy-inducing activity of the benzimidazole derivative thiabendazole remain uninvestigated. In our experiments we found that thiabendazole-treated Chinese hamster cells (Cl-1) exhibited low levels of newly synthesized tubulin, indicating microtubule poisoning. In addition, microtubule growth and organization were substantially affected at mitosis. This was revealed by the reduced length of both interpolar and astral microtubules. Furthermore, thiabendazole strongly induced multipolar and asymmetric
-tubulin-positive metaphase spindles, characterized, however, by the absence of fragmentation of centrosome material as evaluated by anti-
-tubulin antibody staining. Interestingly, we found that microtubule poisoning induced by thiabendazole was qualitatively different from that of colchicine, the best known microtubule depolymerizing agent. In fact, in interphase cells colchicine was comparatively more effective than thiabendazole in promoting depolymerization of cytoplasmic microtubules. However, colchicine could not depolymerize a sub-population of stable, acetylated microtubules, which were however significantly reduced after thiabendazole exposure. In conclusion, the capability of thiabendazole to promote chromosomal malsegregation could be related to an effect on microtubule polymerization that specifically promotes formation of aberrant spindles. |
Introduction |
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TopAbstractIntroductionMaterials and methodsCell stainingResultsDiscussionReferences |
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The correct segregation of sister chromatids to daughter cells during mitosis depends on the formation of a bipolar spindle composed primarily of microtubules (MTs). The centrosomes, which constitute the major microtubule organizing centres (MTOCs), regulate the temporal and spatial organization of different arrays of MTs. Centrosomes not only promote nucleation but also anchor the resulting MT array and establish MT length, number and polarity. Proper arrangement of MT arrays is crucial for spindle function at mitosis and essentially relies on an extremely precise balance between assembly and disassembly of tubulin molecules. Indeed, MTs are highly dynamic elements, as they can spontaneously switch between phases of growth and shrinkage, through a continuous exchange of tubulin subunits between the monomer pools and MTs (Mitchinson and Kirshner, 1984
; Cassimeris et al., 1988; Sammak and Borisy, 1988). Moreover, fluorescence recovery after photobleaching studies of fluorescently labeled tubulin in mammalian cultured cells have shown that MT dynamics are increased during mitosis, as single fibers undergo more frequent transitions from growth to shrinkage (Saxton et al., 1984). This makes the mitotic spindle the main target of injury by physical and chemical agents that interfer with MT behavior, thus invariably disturbing the correct segregation of chromosomes during cell division. For this reason, MT poisons can be thought of and have extensively been used as useful tools for the investigation of MT-mediated events in mitosis.
Thiabendazole (TB) is a benzimidazole derivative, fungicide and antihelmintic chemical. In a recent review, TB was classified as a positive compound in an in vitro test for aneuploidy, whereas inconclusive results were found in in vivo studies (Aardema et al., 1998). Among the end-points investigated, TB was shown to be a powerful inducer of CREST-positive micronuclei (MN) in Cl-1 cells in the in vitro CREST MN assay (Antoccia et al., 1991).
TB is known to affect tubulin assembly in a purified MT assembly assay (Wallin et al., 1988; Albertini, 1990). Such an effect is related to the capability of TB to selectively bind ß-tubulin and prevent MT formation (Martin et al., 1997), similarly to the effects observed for colchicine (Luduena, 1979).
In this work we intended to assess in detail the mechanisms responsible for chromosomal malsegregation at mitosis and induction of MN containing entire chromosomes after TB treatment. Particular attention was focused on the effect of the drug on cellular structures containing tubulins. To this end, we analyzed: (i) inhibition of tubulin synthesis; (ii) defects in the spatial organization of cellular MTs in mitosis; (iii) alterations of the MT network in interphase cells, comparing the damage induced by TB with that occurring after colchicine exposure; (iiii) the integrity of centrosomal material by simultaneous staining for - and -tubulin.
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Materials and methods |
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Cell line, culture conditions and chemicals
The hamster cell line used in this study (Cl-1) is a stabilized clonal derivative of male embryonic lung cells with a modal number of 22 chromosomes (85% of the cells) and a relative frequency of 1% polyploid cells. Cell culture conditions were as previously described (Degrassi and Tanzarella, 1988
). All chemicals were provided by J.M.Parry (Swansea, UK). Colchicine was dissolved in bidistilled water. TB was dissolved in dimethylsulfoxide (DMSO), freshly prepared before use. Control cultures received 1% DMSO.
Tubulin analysis by 35S in vivo protein labeling
Aliquots of 2x105 cells/ml were seeded in 16 mm miniwells (Falcon; Becton Dickinson, Plymouth, UK) and treated for 24 h with 1–25 µg/ml TB (5x10–6–1.25x10–4 M). For production of [35S]methionine-labeled proteins, cells were labeled in methionine-free Dulbecco's modified Eagle's medium (DMEM) (BioWhitaker, Belgium) containing no serum. During the last hour of TB treatment cells were incubated with 150 µl of medium containing ~20 µCi/ml [35S]methionine (Amersham, Little Chalfont, UK). At the end of the treatment the medium was aspirated and replaced with 500 µl lysis buffer (25 mM Tris–HCl, pH 7.4, 0.4 M NaCl, 0.1% deoxycholate, 1% NP-40 and 0.5% SDS). After 30 min at room temperature, the cell lysate was transferred to a 2 ml Eppendorf tube. The samples were then boiled for 3 min and centrifuged at 11 000 g for 5 min. The pellets were removed and the remaining supernatant was brought to a concentration of 0.1% 2-mercaptoethanol and boiled again for 3 min. Proteins were precipitated at –20°C with 3 vol acetone for 30 min. The pellet obtained after centrifugation at 11 000 g for 5 min was dried and then resuspended in 300 µl of phosphate-buffered saline (PBS). Aliquots (150 µl) from each sample were taken for quantitative estimation of total protein content and for immunoprecipitation experiments.
Tubulin immunoprecipitation assay.
Immunoprecipitation of 35S-labeled tubulin was carried out by the technique previously described by Cleveland et al. (1981) with some modifications. Aliquots of 2.5 mg IgG (Miles) anti-- and anti-ß-tubulin antibodies were conjugated to 3 g CNBr-activated Sepharose (Amersham). Aliquots of initial protein extracts were incubated for 1 h with equal volumes of Sepharose-bound antibodies at 4°C. The pellets obtained after centrifugation at 8000 g for 5 min were washed three times with 1 ml of PBS. Then, the precipitated tubulin was solubilized by adding PBS containing 1 M acetic acid and centrifuging at 11 000 g for 5 min. Finally, the supernatants containing the solubilized tubulin were analyzed in a scintillation counter. The c.p.m. obtained were normalized to the relative protein content of each sample.
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Cell staining |
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TopAbstractIntroductionMaterials and methodsCell stainingResultsDiscussionReferences |
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Giemsa staining
One day before the treatment, 2x105 cells were seeded onto coverslips in 30 mm Petri dishes and treated for 24 h with 25 µg/ml TB (1.25x10–4 M), fixed in methanol:acetic acid (3:1) and stained for 5 min in 10% Giemsa. A total of 200 Giemsa stained mitotic cells (100 metaphases and 100 anaphases) were scored in replicate cultures from two repeated experiments.
Double indirect immunofluorescence
Growing cells were treated with 100 µg/ml TB (5x10–4 M) for 2 h. Prior to fixation cells were washed with 2 ml of PBS and then were fixed by immersion in methanol at –20°C for 5 min, then in acetone at –20°C for 1–2 min and, finally, in PBS containing 1% Triton X-100 and 0.5% acetic acid for 10 min at room temperature. The coverslips were then washed three times in PBS (5 min each) and incubated overnight with the anti-acetylated--tubulin antibody, diluted 1:10 in PBS, washed three times in PBS and incubated for 5 h with secondary Texas red-labeled sheep anti-mouse IgGs (Amersham), diluted 1:50 in PBS. Coverslips were washed three times in PBS (10 min each), then blocked for 60 min with goat serum to allow complete saturation of non-specific binding sites. Then, coverslips were immunostained for -tubulin with primary anti--tubulin antibody (Amersham) for 1 h, diluted 1:10 in PBS, and secondary antibody FITC-labeled goat anti-mouse IgGs (Vector, Burlingame, CA) for 45 min, diluted 1:50 in PBS. Finally, slides were washed thoroughly with PBS and mounted in 50% glycerol in PBS. Similar conditions of incubation and detection were also followed for the double immunostaining procedure with -tubulin and TU-30 mouse monoclonal antibody directed against -tubulin (Novakova et al., 1996). Secondary antibodies were Texas red and FITC conjugated for - and -tubulin, respectively. Experiments were repeated at least twice on replicate cultures. Slides were analyzed with a Zeiss III photomicroscope with epifluorescence equipment, using the 09 combination of filters (BP 450-490, FT 510 and LP 420 or BP 546, FT 580 and LP 590). Micrographs were taken with Kodak T-Max 100 film. Photographic images of two color immunofluorescence were taken by a cooled CCD camera (Photometrics) and processed using Adobe Photoshop on an Apple Quadra Computer.
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Results |
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TopAbstractIntroductionMaterials and methodsCell stainingResultsDiscussionReferences |
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Analysis of chromosome segregation
A treatment effective in inducing kinetochore-containing MN (Antoccia et al., 1991
) was adopted to analyze the effects of TB on chromosome segregation. Several alterations in chromosome segregation were observed (Figure 1). Only 29% of metaphases showed a normal chromosome arrangement in TB-treated samples, as against 92% of controls; metaphases with an alterated arrangement of chromosomes, such as tripolar and tetrapolar, were classified as metaphases with non-congressed chromosomes (54%). In addition, 58% of abnormalities at anaphase were scored, including multipolar anaphases (27%) and lagging chromosomes (13%).
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Effect of TB on tubulin synthesis
Defects in chromosome segregation, together with the notion that TB affects tubulin assembly in a purified microtubule assembly assay, suggested that tubulin might be the target of TB in mammalian cells. In order to exactly evaluate the decrease in cellular tubulin occurring after TB treatment, we performed an immunoprecipitation assay for tubulin with which we could quickly and unambiguously determine the rate of synthesis of the protein. In these experiments equal numbers of Cl-1 cells were seeded into microtiter wells and incubated with different concentrations of TB for 24 h at 37°C. Cells were plated at low density so that at the end of treatment they were sub-confluent. 35S-labeled extracts were then prepared and aliquots were taken for quantitative estimation of total protein content. The newly synthesized 35S-labeled tubulin fraction was immunoprecipitated and finally normalized to the total protein content. Thus, we noticed that TB produced a dose-dependent inhibition of tubulin synthesis: 50% inhibition occurred at 10 µg/ml (5x10–5 M), whereas ~80% inhibition was observed at 25 µg/ml (Figure 2
). These results led us to assume that TB may act as an inhibitor of cellular MT assembly. In fact, it is believed that increased levels of unpolymerized tubulin subunits following treatment with MT drugs depress the synthesis of new tubulin monomers.
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Analysis of spindle morphology
As a next step, we attempted to identify the type of spindle disturbances occurring upon TB treatment. A high dose of drug was chosen in order to analyze the effects of TB on MT organization restricted to cells in mitosis, with particular attention to the mechanistic aspects. Cells were therefore processed for immunofluorescence staining with anti-tubulin antibodies in order to visualize spindle morphology. Both an anti-
-tubulin and anti-acetylated -tubulin antibodies were used. In control cells, astral MTs grow throughout prophase and reach their maximum length in prometaphase. In contrast, we observed that TB produced a marked inhibition of growth of astral MTs in prophase, such that hardly any fibers were detectable around each centrosome (Figure 3g, h, j and k).
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In TB-treated cultures at metaphase, abnormalities were observed in the number of poles (multipolar spindles) and in their respective dimensions (asymmetric spindles) (Figure 4g and h
), sometimes appearing as small dots of apparently non-nucleating centrosomal material (Figure 4j–l). Apparently, these findings are suggestive of strong centrosome disorganization occurring after incubation with TB.
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From our observations it was also apparent that TB affects the assembly of another class of MTs, the non-kinetochore interzonal fibers. These, growing with opposite polarity from each pole, increase considerably in length throughout anaphase of control cells, thus contributing to elongation of the central spindle. Staining of treated anaphase cells with the anti-acetylated
-tubulin antibody revealed that these fibers, as well as the central spindle, are considerably shortened (Figure 5d and e).
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At telophase the residual interzonal fibers of the spindle persist in bundles which closely assemble into a rod-like structure, the midbody, which persists in order to separate the two daughter cells at the end of mitosis. Staining with the anti-acetylated
-tubulin antibody enabled us to determine that in controls the midbody is thick and long (Figure 6a and b), whereas in TB-treated cells it is much thinner and shorter (Figure 6d and e). Therefore, both the number and growth of interzonal fibers appear to be affected by TB. Finally, we observed a significant percentage (9%) of binucleated cells among the interphase cell population which may well have arisen from reduced growth of the central spindle.
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Analysis of the interphase microtubule array
To analyze tubulin organization in interphase MTs, cells were treated with a high concentration of TB for 2 h (100 µg/ml, 5x10–4 M). Interestingly, when we examined the acetylated MT array in interphase cells, we found that the damage induced by TB was strikingly different from that produced by 4x10–2 µg/ml (10–7 M) colchicine. The latter led to almost complete disruption of cytoplasmic MTs, as shown by the background staining of depolymerized tubulin subunits (Figure 7e
). Acetylated fibers, however, appeared to be comparatively more resistant to the depolymerizing effect of colchicine; in fact, immunostaining with the anti-acetylated -tubulin antibody remained intense and enabled us to visualize an aster-like arrangement of these fibers around the centrosome (Figure. 7f). This result is consistent with the observation that cytoplasmic MTs resistant to depolymerizing drugs, such as colchicine and nocodazole, are modified by acetylation of the -tubulin molecule (Piperno et al., 1987).
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On the other hand, TB proved to be clearly less effective than colchicine in promoting microtubule depolymerization, as a much higher concentration was needed to produce only partial disassembly of cytoplasmic MTs (Figure 7c
). Yet, interestingly, staining of these cells with the anti-acetylated -tubulin antibody revealed that acetylated fibers were less abundant than in control or colchicine-treated cultures and occasionally completely absent or virtually undetectable (Figure 7d).
Analysis of spindle poles
Abnormalities in the number of poles or in their respective dimensions (Figure 4j–l) prompted us to investigate whether the centrosomal structure was damaged by TB treatment. A more detailed analysis of centrosomal material fragmentation was achieved by simultaneous staining of metaphase spindles untreated and TB-treated cells for - and -tubulin. Examples off cells with multiple and asymmetric asters are shown in Figure 8b and c, respectively. -Tubulin staining revealed that 52% of metaphases displayed three or more MT asters, 40% asymmetric asters and only 5% monopolar asters. Interestingly, antibodies raised against -tubulin stained only and always two spots in 100% of each of the above-mentioned categories, indicating that the number of -tubulin-positive MT asters in multipolar spindles was higher than those reactive to anti--tubulin. In contrast, in untreated cultures the number of multiple asters detected constantly matched the number of -tubulin spots.
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Discussion |
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TopAbstractIntroductionMaterials and methodsCell stainingResultsDiscussionReferences |
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Drugs affecting the dynamics of MTs are expected to perturb one or more aspects of mitosis, since this is the time during the cell cycle when MT dynamics are particularly increased (Saxton et al., 1984
; Mitchinson, 1989). Since TB proved to inhibit MT assembly in an in vitro tubulin assembly assay (Wallin et al., 1988; Albertini, 1990), we first intended to assess whether TB produced similar effects in living cells. To accomplish this, we took advantage of the autoregulatory control of tubulin synthesis. This predicts that depression of tubulin synthesis levels stems from a rise in the free tubulin pool after MT depolymerization or block of MT assembly (Cleveland et al., 1981). The autoregulatory mechanism of tubulin synthesis provides that tubulin subunits may modulate tubulin mRNA stability either by protein–RNA or protein–protein interactions (Gay et al., 1987). We consistently found that TB treatment produced a depression of tubulin synthesis, thus strengthening the assumption that TB is an inhibitor of MT growth in cultured mammalian cells.
Consequently, we found that TB disturbs the normal arrangement of MT arrays. The defects observed at mitosis were decreased number and length of both astral and interpolar fibers and abnormal asters. During prometaphase, the astral MTs repeatedly grow and shorten, apparently probing the cytoplasm until kinetochore attachment is achieved (Rieder and Alexander, 1990). Therefore, it can be envisaged that interfering with astral MT growth might result in an inability of MTs to reach the kinetochore of a chromosome. This observation is in good agreement with the high proportion of metaphases containing imperfectly congressed chromosomes. In addition, it is plausible that a cell which eventually passed through metaphase during incubation with TB would not undergo accurate chromosome segregation. The scoring of 13% of anaphase cells as having lagging chromosomes is consistent with this hypothesis, as well as our previous finding that TB is a powerful inducer of CREST-positive MN (Antoccia et al., 1991). Together these findings provide further support for the idea that alterations in MT dynamics are among the main causes of aneuploidy.
The observed decrease in pole-to-pole distance and the short and thin midbody of telophase cells may well arise from TB inhibition of interpolar fiber and central spindle growth. In these cells inhibition of cytokinesis probably results from such alterations, thus providing a possible explanation for the 9% of binucleated cells we scored in the TB-treated cell population. In fact, it could well be that a specific intracellular signal required for completion of cytokinesis is abolished when central spindle assembly is perturbed by treatment with spindle poisons or agents interfering with the formation of central spindles (Fishkind et al., 1996; Cimini et al., 1999).
Furthermore, we examined the spatial arrangement of cytoplasmic acetylated MTs obtained after exposure to either colchicine or TB, by immunofluorescence staining with an anti-acetylated tubulin antibody. Acetylation is a post- translational modification of -tubulin that occurs in MT frameworks such as axonemes, basal bodies (Piperno and Fuller, 1985; Le Dizet and Piperno, 1986), primary cilia, midbodies, centrioles and in a sub-population of single cytoplasmic MTs (Piperno et al., 1987). Acetylated MTs are more stable under conditions promoting MT disassembly (i.e. exposure to colchicine or nocodazole) (Piperno and Fuller, 1985). In interphase cells TB proved to be clearly less effective than colchicine in promoting microtubule depolymerization, as a much higher concentration was needed to produce only partial disassembly of cytoplasmic MTs. Yet, interestingly, staining of these cells with the anti-acetylated -tubulin antibody revealed that the acetylated fibers were less abundant than in control or in colchicine-treated cultures and occasionally completely absent or virtually undetectable, thus suggesting that different molecular mechanisms underlie the damage produced by the two drugs as evaluated in interphase cells. In contrast, acetylated -tubulin associated with asters appeared only slightly diminished in mitosis-arrested cells after TB treatment. Similarly, the acetylated -tubulin fraction of MTs was shown to be resistant to the benzimidazole derivative carbendazim (Can and Albertini, 1997). The capability of benzimidazole derivatives to promote MT depolymerization is highly variable. In contrast to the effect observed after TB treatment of interphase cells, carbendazim was shown to exhibit selective disruption of astral and interpolar spindle MTs in M phase cells, whereas no effect was detected in interphase (Can and Albertini, 1997).
Besides interfering with tubulin polymerization, chemical agents may also affect centrosome integrity, leading to chromosomal missegregation at mitosis. As a probe to analyse the centrosome we used an antibody directed against -tubulin, a member of the tubulin superfamily known to be a universal component of MTOCs and probably responsible for their nucleating activity (Oakley and Oakley, 1989; Stearns et al., 1991; Oakley, 1992). In our immunofluorescence experiments TB seemed to affect centrosome integrity only marginally, if at all. TB induces a significant disorganization of the mitotic apparatus. In fact, an unusual number of -tubulin-positive asters and/or asters characterized by different amounts of tubulin were detected at a high rate: such an effect could arise as a result of either spindle or centrosome fragmentation. It should be noted, however, that independently of the number and dimensions of asters, two spots of -tubulin of equal dimensions were always detected.
The ability of TB to induce centrosome fragmentation seems to be ruled out by the observation that metaphase cells do not display multiple and/or diminished -tubulin staining, as was observed in cells treated with nocodazole (Novakova et al., 1996) or diazepam (Izzo et al., 1998). Yet there is no evidence as to the mechanisms by which MT drugs affect centrosome organization. Indeed, a full understanding of these phenomena will have to await a better analysis of the molecular and structural nature of the functional components of this organelle.
Finally, it should be noted that we detected ~30% of cells showing multipolar segregation of chromosomes, even though the number of -tubulin-reactive poles scored at metaphase never exceeded two. This may be indicative of a certain proportion of -tubulin-free poles maintaining a functional MT nucleating activity after TB treatment and promoting chromosomal malsegregation. The presence of -tubulin- positive poles lacking staining for stable markers for centrosomes (anti--tubulin or 5051 antiserum) was also recorded after treatment of mammalian cells with taxol (Novakova et al., 1996) and carbendazim (Can and Albertini, 1997). Interestingly, poles not labeled by stable marker antibodies appeared positive when probed with antibodies directed against transient markers of centrosomes, such as NuMA and centrophilin (Can and Albertini, 1997).
In conclusion, TB seems to affect MT polymerization and specifically promotes formation of aberrant spindles which are able to interact with chromosomes leading to aneuploid daughter cells.
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Acknowledgments |
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M.Izzo and L.Mattace are greatly acknowledged for their contribution to the experiments and editorial assistance, respectively. Acetylated-
-tubulin and TU-30 anti--tubulin antibodies were kindly provided by Dr G.Piperno (Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, NY) and Dr P. Draber (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic), respectively. |
Notes |
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4 To whom correspondence should be addressed. Tel: +39 06 5517 6336; Fax: +39 06 5517 6321; Email: tanzarel{at}bio.uniroma3.it
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Received on April 19, 2000; accepted on June 20, 2000.
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