Unidad de Endocrinología Molecular, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, 28029 Madrid, Spain
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ABSTRACT |
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Uncoupling protein (UCP), the mitochondrial protein specific to brown adipose tissue, is activated transcriptionally in response to cold and adrenergic agents. We studied the role of triiodothyronine (T3) on the adrenergic stimulation of UCP mRNA expression by use of primary cultures of rat brown adipocytes. Basal UCP mRNA levels are undetectable. Norepinephrine (NE) increases UCP mRNA during differentiation, not during proliferation. In hypothyroid conditions, UCP mRNA response to NE is almost absent. The presence of T3 (0.2-20 nM) greatly increases the adrenergic response (30-fold). The sensitivity of UCP mRNA responses to NE is potentiated ~100-fold by the presence of T3. The effect is proportional to the dose and time of preexposure to T3. The increases obtained with NE and T3 are prevented by actinomycin and cycloheximide. T3 greatly stabilizes UCP mRNA transcripts. The effects of thyroxine and retinoic acid are weaker than those of T3. In conclusion, in cultured rat brown adipocytes, T3 is required and both synergizes with NE to increase UCP mRNA and stabilizes its mRNA transcripts.
thyroid hormones; brown adipose tissue
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INTRODUCTION |
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BROWN ADIPOSE TISSUE (BAT) is a thermogenic tissue that plays an important role in hibernating animals, newborns, and cold-exposed mammals. BAT also seems important in maintaining energy balance, as it is activated in response to diet (diet-induced thermogenesis). The main function of BAT is to produce heat under adrenergic stimulation (facultative thermogenesis). This function is accomplished by the uncoupling protein (UCP), nowadays called UCP-1 (UCP in this paper), a BAT-specific protein present only in the inner mitochondrial membrane of BAT (37). When activated adrenergically, UCP provides heat by uncoupling the oxidative phosphorylation, dissipating as heat the energy otherwise stored as ATP (25).
BAT thermogenesis is activated in response to the norepinephrine (NE)
released by sympathetic nerve endings. The adrenergic stimulation
induces UCP synthesis, a process that has been studied in vivo in
several species and different situations, measuring increases in the
activity, protein, or UCP mRNA expression (4, 6, 16, 24, 27, 38, 46,
47). The presence of UCP has also been characterized in isolated
floating rat brown adipocytes (2) or in primary cultures of brown
adipocytes obtained from mice or hamsters as donor animals (9, 17, 36).
In the latter culture systems, UCP mRNA is stimulated in response to
NE, adrenergic analogs, or cAMP, and the stimulation has been
characterized as -adrenergic, acting through the
1-
and
3-adrenergic receptors (9, 17, 30, 36). Other
studies have been done using murine cell lines from hibernomas in which
different sensitivity to NE was observed (18, 21, 39, 40).
Thyroid hormones are also important in thermogenesis. Hypothyroid animals are intolerant to cold and even die when exposed to cold for several hours (4). This is due to a defective basal and facultative thermogenesis in the hypothyroid animals, because during cold exposure the increases in UCP expression are lower than in control animals. This deficiency can be restored by adequate replacement therapy with thyroxine (T4) (4, 46). Basal and stimulated UCP mRNA is also low in hypothyroid fetuses and newborns, and either triiodothyronine (T3) or T4 restores UCP levels in fetuses (26, 28). Therefore, thyroid hormones seem necessary for the full expression of UCP in vivo (4, 46), and T3 amplifies the adrenergic stimulation of UCP mRNA expression and transcription in cold-exposed rats (3). In addition, there is a high correlation between the occupancy of nuclear T3 receptors and UCP increases (5). Recently, certain regions of the rat UCP promoter have been identified as thyroid hormone and retinoic acid response elements (TRE and RARE, respectively) (1, 8, 33, 34).
The production of T3 in BAT is catalyzed by the type II 5'-iodothyronine deiodinase (DII), an enzyme stimulated mainly by cold and adrenergic agents (44); its role is to produce T3 locally. DII seems necessary for an optimal thermogenic response of BAT to cold (4).
Although the role of thyroid hormones for the full expression of UCP has been well documented in rats in vivo (3), studies using cultured rat brown adipocytes are scarce (9), and the role and need of thyroid hormones for the adrenergic stimulation of UCP are not always recognized (9, 36) because the results depend on the culture model used. Several groups include T3 as a so-called "differentiation medium," a beneficial factor for the differentiation of cultured brown preadipocytes (1, 18). Nevertheless, the precise role of T3 has not been fully clarified in cultures of brown adipocytes.
The aim of the present study is to study the effect of thyroid hormones on the adrenergic stimulation of UCP mRNA expression by use of primary cultures of rat brown adipocytes, a system in which the stimulation of UCP by NE is not easily achieved. Precursor cells were isolated from rat BAT pads and, after a period of active proliferation, were used during the differentiation period. We analyze the role of T3 that is required and that potentiates the adrenergic activation of UCP mRNA in cultured rat brown preadipocytes. The present model remarks on the importance of T3, which seems more important than T3 as previously reported in other culture systems.
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MATERIALS AND METHODS |
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Materials.
DMEM was obtained from Life Technologies (Uxbridge, UK). Newborn calf
serum (NCS) was purchased from Flow (Paisley, Scotland). Antibiotics
were obtained from the local pharmacy. BSA (in solution at 22%, pH = 7.2) was obtained from Ortho Diagnostic Systems, Johnson & Johnson
(Raritan, NJ). Collagenase, bovine insulin, ascorbic acid, guanidinium
HCl, MOPS, T3, T4, NE, and retinoic acid (RA)
were obtained from Sigma (St. Louis, MO). Cycloheximide (CHX),
actinomycin D (Act D), and restriction enzymes were obtained from
Boehringer Mannheim (Mannheim, Germany). Ion exchange resin AG1-X8 was
from Bio-Rad (Richmond, CA). All other chemicals were reagent or
molecular biology grade. Radiolabeled
deoxy-[-32P]CTP (3,000 Ci/mmol) was
purchased from Amersham International (Aylesbury, UK), and the
oligolabeling system was obtained from Pharmacia (Uppsala, Sweden).
Nytran membranes were purchased from Schleicher & Schuell (Dassel, Germany).
Cultures of brown adipocytes. Precursor cells were obtained from the interscapular brown adipose tissue of 20-day-old rats (Sprague-Dawley), isolated according to the method described by Néchad et al. (23), with modifications (13) by use of collagenase digestion (0.2%) in DMEM+1.5% BSA at 37°C and filtration through 250-µm pore size silk filters. Mature cells were allowed to float, and the infranatant was filtrated through 25-µm pore size silk filters and centrifuged. Precursor cells were seeded in 25-cm2 culture flasks to get 1,500-2,000 cells/cm2 on day 1 and were grown in DMEM supplemented with 10% NCS, 3 nM insulin, 10 mM HEPES, 50 IU penicillin, 50 µg streptomycin/ml, and 15 µM ascorbic acid. Culture medium was changed on day 1 and every 2nd day thereafter. Precursor cells proliferated actively under these conditions, reached confluence on the 4th or 5th day after seeding (40,000-80,000 cells/cm2), and by day 8 were fully differentiated into mature brown adipocytes. Studies were performed during the period of differentiation (5th-8th culture day) using NCS or hypothyroid serum in the presence of thyroid hormones or other treatments, as specified.
Both NCS and hypothyroid serum (HS) were used for culture. The latter was obtained by depleting NCS of thyroid hormones with the anion exchange resin AG1X8, as previously described (43). HS containedRNA preparation and Northern blot analysis.
Total cellular RNA was extracted in guanidinium-HCl as described (13),
with use of ethanol precipitation. The recovery was 50-90 µg
total RNA/25-cm2 flasks, ~5 × 106 cells
(~25 µg RNA in the 4th-5th days). For Northern analysis, total
RNA (15-20 µg) was denatured and electrophoresed on a 2.2 M
formaldehyde/1% agarose gel in 1× MOPS buffer and transferred to
nylon membranes. A 1,200-bp fragment of the rat UCP cDNA clone [kindly provided by Dr. D. Ricquier (7)] was used as a
probe by labeling with deoxy-[-32P]CTP by
use of random primers (>108 cpm/µg DNA). Filters were
hybridized for 20 h at 50°C [40% formamide, 5× sodium
saline citrate (SSC), 2× Denhardt's solution, and 0.1% SDS] and washed 4 times in 2× SSC-0.2% SDS at room
temperature for 15 min and then twice in 0.1× SSC-0.2% SDS at
65°C for 20 min. Autoradiograms were obtained from the filters and
quantified by laser computer-assisted densitometry (Molecular Dymanics,
Sunnyvale, CA). The membranes were routinely dyed using methylene blue
to visualize the ribosomal RNAs (rRNA), and differences between lanes were used to correct the results obtained from UCP mRNA. Usually rRNA
is presented in the figures. Some filters were also hybridized with the
cDNA for cyclophilin or vimentin as a loading control between
lanes. All experiments were repeated 2-3 times using
duplicates. The more complete and representative experiments are shown
in the figures, and the graphs show the means of 2-3 experiments.
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RESULTS |
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Adrenergic stimulation of UCP mRNA during differentiation.
The expression of UCP mRNA during the differentiation of rat
preadipocytes into adipocytes was examined. The basal expression of UCP
is undetectable under standard culture conditions, namely 10% NCS
(Fig. 1, odd lanes). The presence of NE (10 µM) significantly increased UCP mRNA (Fig. 1, even lanes, P
< 0.05) on any day after the cells reached confluence, being
maximal around confluence and decreasing thereafter (P < 0.05). NE does not increase UCP mRNA during proliferation (day
3). The two bands correspond to the two molecular sizes of the UCP
mRNA, 1.6 and 1.9 kb. In Fig. 1 we also show, as control, the rRNA
pattern and the expression of vimentin, a constitutive protein of the
intermediate filaments in brown adipocytes. Therefore, rat confluent
cells are true brown adipocytes, as they express UCP mRNA.
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Effect of T3 on the adrenergic response of UCP.
Because the responses to NE in the presence of NCS were frequently very
low, we analyzed the effect of T3 by using several T3 concentrations, the effect of the differentiation state
of the cells, and the kinetics of induction by NE and T3.
The effect of T3 on the adrenergic response of UCP mRNA
expression was studied at days 6 and 8 (Fig.
2) by using 2 or 40 nM T3 for
20 h and 10 µM NE during the last 4 h (day 7 was also
studied). In the absence of NE, T3 per se
does not increase UCP mRNA levels. The adrenergic response when NE
is used in hypothyroid medium is barely detectable at day 6 and
absent at day 8. But when T3 is added together with NE, there is a marked potentiation of the adrenergic response [5-30 times, depending on the T3 concentration
used (P < 0.05), as well as on the time of exposure to
T3 (lower at 4 h, data not shown)]. This effect of
T3 is observed on any day after confluence.
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Kinetics of induction of UCP mRNA with NE and T3. We also examined the minimal time required for both NE and T3 to increase UCP mRNA levels. The kinetics of induction by NE are shown in Fig. 5A in cells preexposed for 24 h to 2 or 20 nM T3. NE requires <2 h to increase UCP mRNA, and the effect is quicker when 20 nM T3 is used, because at 2 h a strong signal is already observed. The effects are prolonged throughout 24 h and amplified at the larger T3 dose.
The kinetics of induction for T3 were also studied using two T3 doses (2 and 20 nM) during 4, 8, 16, or 28 h, when NE (10 µM) was added during the last 4 h (Fig. 5B). The effect of T3 on the adrenergic response is quick, as it is obtained in <4 h, and it is proportional to the dose of T3 used (P < 0.05).Effect of CHX and Act D on UCP gene expression.
The effect of T3 on the adrenergic stimulation is inhibited
by CHX, so it requires de novo protein synthesis. Figure
6A shows that CHX inhibits the
induction observed by use of NE, as well as NE+T3, at two
different doses, after either short (4 h) or long incubations
(16-24 h). The experiment was repeated several times and in
different conditions, and usually the inhibition was not complete
(50-70%).
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Stabilization of UCP mRNA by T3.
Figure 7 shows the effect of T3
on the stabilization of the UCP mRNA transcripts. The adrenergic
stimulation of UCP mRNA was tested by using two doses of
T3, 10 nM and 0.5 nM T3, in HS in the presence
of NE. Act D was added after 12-h exposure to NE+T3, and
cells were collected at different times from 2 to 9 h. Results show
that T3 stabilizes the UCP mRNA transcripts, increasing UCP mRNA half-life from 5 to >24 h.
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Effects of RA and T4.
The effects of RA and T4 on the adrenergic response were
also examined and, in general, were found to be less strong than when
T3 was used. Figure 8 shows a
dose-response curve for T4+NE. The adrenergic stimulation
is higher at either low or high T4 concentrations, and
lower at the intermediate T4 doses. This experiment was
repeated 3 times, with doses ranging from 0.1 to 20 nM T4. The effect of T4+NE (at low doses) is much lower than that
of T3+NE (5 times lower, results not shown). In Fig.
9 we show the effect of RA alone or with NE
or NE and T3, compared with T3. A clear effect
of RA+NE is observed, although it is lower than that of
T3+NE. RA does not synergize with T3 or
T3+NE.
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DISCUSSION |
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BAT thermogenesis is stimulated in vivo by the NE released in the
sympathetic nerve endings. The adrenergic stimuli induce the expression
of UCP at the transcriptional level, as demonstrated in vivo in rats
(3, 6, 16, 35, 38) as well as in vitro, by use of cultures of mouse
brown adipocytes or cell lines (9, 21, 36). Most of these increases are
mediated through -adrenergic receptors, mainly
3-adrenergic receptors (9, 17-19, 21, 30, 36, 39),
although the participation of an
1-adrenergic component has also been suggested (36). Most of the studies published on the
regulation of UCP mRNA expression have been performed using cultures of
brown adipocytes from mice or hamsters, species in which the adrenergic
stimulation is more easily demonstrated (36), whereas studies using rat
brown adipocytes are scarce (9, 11). The reasons for this lack of
successful studies have not been clarified.
In the present paper, we show that UCP mRNA can be stimulated
adrenergically in primary cultures of rat brown adipocytes obtained from precursor cells, demonstrating that the cells obtained after proliferation and differentiation in culture are true brown adipocytes, because UCP expression is the specific marker of BAT. The adrenergic stimulation of UCP, although weak, is obtained at any day after the
cells reach confluence, indicating that, even when the accumulation of
lipids and lipogenic markers begins on later days (10, 29), the cells
are equipped for thermogenic functions and behave earlier as brown
adipocytes. In the absence of NE, the basal expression of UCP is absent
when standard culture conditions are used. Maximal adrenergic
stimulation of UCP is obtained around confluence, and no increases are
observed during active proliferation. In this aspect, the results
obtained in rat primary cultures are similar to the ones described when
primary cultures of mouse preadipocytes are used (36). The stimulation
by NE is quick (2 h), suggesting, as described in vivo, an activation
at the transcriptional level (3, 35). We observed that the stimulation
is via -adrenergic receptors, in agreement with data obtained by
other authors (9, 19, 21, 36), and it is fully mimicked by
3-adrenergic agonists (unpublished observations).
Nevertheless, the adrenergic stimulation of UCP mRNA in primary cultures of rat adipocytes is generally weak or absent (even when different sources of serum are used), and the optimal conditions for such studies have not been described, except for one brief study that compares UCP mRNA expression in mouse and rat primary cultures (9). Thus studies on the regulation of UCP mRNA in cultured rat brown adipocytes are scarce (11). The low levels of UCP mRNA observed in rat cultures after adrenergic stimulation can be attributed to 1) growth factors or hormones present in the serum used in the cultures that might inhibit the expression of UCP or 2) the low amount of T3 present in the culture medium, which we demonstrated in this study to be essential for the NE effect. In fact, although cellular T4 concentrations are normal, T4 concentrations measured in cultures using 10% NCS were ~0.07 nM T3 in the medium and 0.7 nM T3 in the cells, resulting in a cellular T3 concentration 5-6 times lower than that found in BAT in vivo (3-4 nM, which increases to 8 nM T3 after cold exposure; our unpublished results). Thus we came to the conclusion that our cells are really in hypothyroid conditions. This seems to be due to the presence of active pathways for T3 degradation, namely, inner ring deiodinase activity (DIII), activated in response to the serum used for culture (13, 15). We have also demonstrated that, in the same cultures of brown adipocytes (14), a minimal amount of T3 is required for the adrenergic activation of DII, the enzyme responsible for T3 production in BAT.
The main finding of the present work is that T3 is absolutely necessary for the adrenergic stimulation of UCP expression, reinforcing the results obtained in rats in vivo and in floating adipocytes (2, 3, 46). In the absence of thyroid hormones (hypothyroid medium), the adrenergic stimulation of UCP is almost undetectable, and T3 potentiates the effect of a given NE dose. We demonstrate that the effect of T3 takes place at any day after confluence and is proportional to the dose and time exposure to T3. T3 amplifies the adrenergic stimulation of UCP by ~30-fold, depending on the T3 dose used, and a 10-fold increase in T3 concentrations multiplies the NE effect by 100-fold. This gives an idea of the magnitude and importance of the T3 effect. This T3 effect is already observed at very low T3 concentrations (0.2 nM) in the lower physiological range. These facts are easier to demonstrate in culture systems, where those small changes can be handled, whereas in vivo, potent mechanisms are present to maintain stable and high T3 concentrations in BAT (increased T3 uptake and high 5' deiodination), so that the diminution of T3 concentrations and therefore of UCP expression would be more difficult to show (26).
The effect of T3 is detected in <4 h, suggesting an effect on transcription, as already described (3, 35, 12), although a maximal effect requires longer times with the use of physiological T3 concentrations. This fact suggests that the effect of T3 requires the synthesis of an intermediate product and possibly de novo protein synthesis. The experiments using CHX demonstrate that this is the case. It is difficult to discern whether the inhibitory effect of CHX is exerted on the action of T3 or of NE, as separately each does not have an effect. The inhibition seems to be exerted on the combined use of NE+T3, possibly on a protein required for its combined action. Nevertheless, the effect of CHX is more potent when added before T3 (18 h) than when added after T3 and just before NE (6 h), suggesting that T3 per se is able to induce the synthesis of a protein or transcription factor required for the adrenergic stimulation of UCP. These data are contrary to those reported in vivo, where the T3 effect did not require de novo protein synthesis (3).
Our data using Act D and the prompt responses observed suggest that the effect is clearly at the transcriptional level, confirming previous results (12, 35, 36). However, the full T3 effect is not only exerted at the transcriptional level, because the effect on the stabilization of the mRNA transcripts seems even more important, contributing to a marked elongation of UCP mRNA half-life. Previous in vivo studies (35) described a brisk increase in transcription during the first hour of exposure of rats to cold, whereas the stabilization effect takes place at longer times (8 h). In our cultured cells, the stabilization effect is important even at low doses of T3, making difficult a discrimination between low and high T3 doses.
Important studies have been conducted to study the effect of
T3 on UCP at the promoter level, and an enhancer region has
been defined in the 2490/
2280 bp of the rat UCP gene
promoter (8, 34). Two TREs have been defined at
2391/
2376
(upTRE) and at
2348/
2334 (downTRE) (34), both of which
seem needed for the stimulation of UCP and the downTRE for the
potentiation of the cAMP effect by T3. Another group of
investigators (1) has identified three RAREs, in the same region as the
downTRE, at
2357/
2330 (1), whereas the former authors
(33) define the RAREs further upstream of the upTRE region
(
2490/
2399). It is very much a possibility that this
enhancer region might act promiscuously with both RA and T3
to enhance UCP mRNA expression, and the results depend on the specific
proteins present in the transfection cell system used or the origin of
the cells used (32). In contrast to the mouse UCP gene promoter in
which cAMP response elements (CREs) were described in an enhancer
element located in an enhancer region similar to that of the rat
promoter (20), CREs were not defined in the rat UCP promoter until
recently (48), one near the proximal promoter region and a second one
within the enhancer element described above (32). It seems that the
synergism between NE and T3 involves interaction with a
protein or coactivator, but this area demands further investigation.
T3 has also been described to induce per se the transcription of UCP mRNA without the concurrence of NE (12) and to stabilize its mRNA in primary cultures of fetal brown adipocytes. It is not clear whether this fact is linked to the fetal state of the donor animals or to the culture conditions used (serum-free medium), pointing to a possible inhibitory effect of serum on UCP mRNA expression.
In our cultures, T4 also has an effect on the adrenergic stimulation of UCP mRNA expression. This effect is much lower than that of T3 and follows a biphasic pattern, suggesting that at low T4 doses (0.2 nM), the effect is mediated through the generation of T3 from T4 through DII activity, when DII is not inhibited by T4 (45), whereas at higher T4 doses that inhibit DII, it might be mediated through the occupancy of nuclear T3 receptors by T4. We find striking similarities between the adrenergic stimulation of DII activity and UCP mRNA expression with respect to the T3 requirements (14). Both genes require the combined presence of T3 and NE to get adrenergic increases. In both, the potentiating effect of T3 is large, increases with time, and requires protein synthesis, and in both UCP and DII, NE or T3 alone does not exert per se a significant effect.
We have also found an effect of RA on the adrenergic stimulation of UCP, somewhat lower than that of T3. This is contrary to other reports using cultured murine adipocytes (1, 31), where RA exerts a higher effect than T3. Nevertheless, it has to be taken into account that, in the last report, T3 was added to the medium to induce differentiation, and the effect of RA should only be considered as "pure" when depleted serum + RA were used. The effects we observed are not synergic or additive. This suggests that RA and T3 use the same pathway to increase the adrenergic stimulation of UCP, competing for the same sites or for the retinoid X receptor present, because no synergism is found between T3 and RA. These results are also different from the only report using floating rat brown adipocytes stimulated with NE (33).
In summary, we have studied the conditions required for the adrenergic
stimulation of UCP in primary cultures of rat brown adipocytes,
especially with respect to the need for T3. We conclude that there is an absolute need of T3 for the adrenergic
stimulation of UCP mRNA, because in hypothyroid conditions, basal and
adrenergically induced UCP expression is absent. The role of
T3 in the stabilization of the mRNA transcripts is also of
major importance. However, the T3 effects are exerted at
multiple levels: at the UCP promoter, contributing to the stabilization
of the transcripts, and at other levels, such as the modulation of the
-adrenergic receptor population (41, 42).
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ACKNOWLEDGEMENTS |
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We thank Dr. D. Ricquier for the gift of the rat UCP cDNA.
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FOOTNOTES |
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This work was supported by Grants PB 92/0061 and PB 95/0097 from Dirección General de Investigación Científica y Técnica and Fondo de Investigaciones Sanitarias de la Seguridad Social Grant 94/0274 from Fondo de Investigaciones Sanitarias of Spain.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. J. Obregón, Instituto Investigaciones Biomédicas (C.S.I.C.), Arturo Duperier 4, 28029 Madrid, Spain (E-mail: mjobregon{at}iib.uam.es).
Received 24 June 1999; accepted in final form 15 November 1999.
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