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Brazilian Journal of Medical and Biological Research

On-line version ISSN 1414-431X

Braz J Med Biol Res vol.38 no.9 Ribeirão Preto Sept. 2005

http://dx.doi.org/10.1590/S0100-879X2005000900002 

Braz J Med Biol Res, September 2005, Volume 38(09) 1293-1302 (Review)

CaMKII d overexpression in hypertrophy and heart failure: cellular consequences for excitation-contraction coupling

L.S. Maier

Abteilung Kardiologie and Pneumologie, Herzzentrum, Georg-August-Universität Göttingen, Göttingen, Germany

Abstract
Text
References
Acknowledgments
Correspondence and Footnotes


Abstract

Ca/calmodulin-dependent protein kinase II d (CaMKII d) is the predominant isoform in the heart. During excitation-contraction coupling (ECC) CaMKII phosphorylates several Ca-handling proteins including ryanodine receptors (RyR), phospholamban, and L-type Ca channels. CaMKII expression and activity have been shown to correlate positively with impaired ejection fraction in the myocardium of patients with heart failure and CaMKII has been proposed to be a possible compensatory mechanism to keep hearts from complete failure. However, in addition to these acute effects on ECC, CaMKII was shown to be involved in hypertrophic signaling, termed excitation-transcription coupling (ETC). Thus, animal models have shown that overexpression of nuclear isoform CaMKII dB can induce myocyte hypertrophy. Recent study from our laboratory has suggested that transgenic overexpression of the cytosolic isoform CaMKII dC in mice causes severe heart failure with altered intracellular Ca handling and protein expression leading to reduced sarcoplasmic reticulum (SR) Ca content. Interestingly, the frequency of diastolic spontaneous SR Ca release events (or opening of RyR) was greatly enhanced, demonstrating increased diastolic SR Ca leak. This was attributed to increased CaMKII-dependent RyR phosphorylation, resulting in increased and prolonged openings of RyR since Ca spark frequency could be reduced back to normal levels by CaMKII inhibition. This review focuses on acute and chronic effects of CaMKII in ECC and ETC. In summary, CaMKII overexpression can lead to heart failure and CaMKII-dependent RyR hyperphosphorylation seems to be a novel and important mechanism in ECC due to SR Ca leak which may be important in the pathogenesis of heart failure.

Key words: Calcium, Calmodulin, CaM kinase, Excitation-contraction coupling, Heart


Structure of Ca/calmodulin protein kinase II

Ca/calmodulin protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase mainly found in the heart (1,2) which can phosphorylate several proteins in response to increasing intracellular Ca concentrations, [Ca]i (3). There are four different CaMKII genes ( a, ß, g, d) of which CaMKII d protein is the most abundant isoform in the heart (1,4). Subcellular localizations of CaMKII d were found, with dB being specifically compartmentalized to the nucleus by means of an 11-amino acid long nuclear localization sequence and with dC being the cytosolic isoform without nuclear localization sequence (4). Each CaMKII isoform contains a catalytic domain, a central regulatory domain (containing partially overlapping autoinhibitory and CaM binding regions) and an association domain responsible for oligomerization (1,2). When Ca/CaM binds to the regulatory region (amino acid 296-311) the active site on the catalytic domain gains access to target substrates.

The CaMKII holoenzyme consists of homo- or heteromultimers of 6-12 kinase subunits forming a wheel-like structure (1,3). During CaMKII activation, Ca/CaM binds to the regulatory domain and displaces the autoinhibitory domain (amino acid 273-309) on CaMKII thereby activating the enzyme with a half maximal activation at [Ca]i of 500-1000 nM (i.e., systolic [Ca]i). Subsequently, the kinase locks itself into an activated state by autophosphorylation of Thr-287 on the autoinhibitory segment (4). Autophosphorylation is critical in maintaining the enzyme active even after [Ca]i has declined and Ca/CaM has dissociated from its binding region (autonomous state). It is important to know that autophosphorylation itself is not essential for initial CaMKII activity, but does have important consequences, i.e., by increasing the affinity of the kinase-CaM complex (5). This effect traps Ca/CaM in the autophosphorylated subunit. At high [Ca]i, the affinity of Ca/CaM for CaMKII increases ~700-fold from 45 nM to 60 pM (1,5). Even when [Ca]i declines to resting levels during diastole (i.e., ~100 nM), CaM is still trapped for several seconds. As a result, the kinase retains 100% activity as long as CaM is trapped, regardless of the [Ca]i level (5). Interestingly, autophosphorylation is sufficient to disrupt the autoinhibitory domain, with the enzymes remaining partially active even after CaM has dissociated from this autonomous state (20-80%) (6-9). For complete inactivation to occur, CaMKII can be dephosphorylated by protein phosphatases including PP1, PP2A, and PP2C (4).

Several CaMKII inhibitors have been widely used in cardiac myocytes, including the organic inhibitors KN62 and KN93 (2) which competitively inhibit CaM binding at the regulatory domain to CaMKII (Ki ~0.37 µM) and are quite selective. Unfortunately, some of these agents appear to have direct ion channel effects which may be independent of CaMKII action (10,11). In contrast, peptide inhibitors are not known to affect ion channels. Two useful peptides are autocamtide-2-related inhibitory peptide (AIP) with 13 amino acids (12), and autocamtide-2 inhibitory peptide (AC3-I; Ref. 13).

CaMKII and excitation-contraction coupling

During excitation-contraction coupling (ECC) Ca enters the cell mainly via voltage-dependent L-type Ca channels (ICa), triggering Ca release from the sarcoplasmic reticulum (SR) via SR Ca channels (ryanodine-receptors, RyR), a process termed Ca-induced Ca release (14). These processes increase intracellular [Ca]i, causing Ca binding to troponin C which activates the myofilaments leading to contraction (systole). For diastolic relaxation to occur, Ca must be removed from the cytoplasm. The SR Ca-ATPase (SERCA) and the Na/Ca-exchanger (NCX) are the main mechanisms for Ca removal (14). CaMKII can modulate ECC by phosphorylating several important Ca regulatory proteins in the heart in response to Ca signals, including RyR (15,16) and phospholamban (PLB; Refs. 17,18), and possibly L-type Ca channels (2), with multiple functional consequences.

Facilitation of ICa

CaMKII modulates L-type Ca channels and thereby ICa, and this is most clearly seen functionally as a positive staircase of ICa with repeated depolarization from -90 to 0 mV, a process termed facilitation (19,20). ICa amplitude increases and inactivation is slowed progressively over 2-5 pulses at 1 Hz. Several groups independently demonstrated that Ca-dependent ICa facilitation is mediated by CaMKII-dependent phosphorylation (21-23). At the single channel level this CaMKII-dependent ICa facilitation is manifest as longer single channel openings (24). This positive ICa staircase is Ca-dependent, very local at the mouth of the L-type Ca channels, and still occurs in the absence of SR Ca release, since it is still observed when cells are heavily Ca buffered with 10 mM EGTA. The physiological role of ICa facilitation is not entirely clear, but it may partly offset reduced L-type Ca channel availability at high heart rates (caused by direct Ca-dependent inactivation).

Sarcoplasmic reticulum Ca release

CaMKII also affects RyR activity. Based on sequence analysis, there are up to four serine and two threonine residues at the RyR that are consensus phosphorylation sites. Witcher et al. (15) first reported direct phosphorylation of cardiac RyR at Ser-2809 by CaMKII activating the SR Ca release channel. Additional studies also showed that RyR are substrates of CaMKII (16,25), but the specific effects of phosphorylation reported in these studies remain controversial. That is, CaMKII either increases (15,16) or decreases RyR open probability (25). Most of the work on the effects of CaMKII on RyR was conducted using RyR in lipid bilayers or by measuring Ca release from SR vesicles, but few data have been obtained for more physiological environments. Li et al. (10) reported that in intact voltage-clamped cardiac myocytes endogenous CaMKII increased the amount of SR Ca release for a given SR Ca content and ICa trigger. In that study, the effect of CaMKII on RyR was evaluated when both L-type Ca current and SR Ca load were constant under control conditions and in the presence of the CaMKII inhibitor KN-93. This conclusion is also consistent with observations that protein phosphates (PP1 and PP2A) can reduce RyR activity for a given ICa and SR Ca load, and that, conversely, phosphatase inhibitors enhance it (26). However, Wu et al. (27) obtained opposite results suggesting that constitutively active CaMKII inhibited SR Ca release, while CaMKII inhibition (by AC3-I) enhanced SR Ca release. Since they did not measure SR Ca content in the same protocols, the dichotomy raised is still unresolved.

Exciting reports from our laboratory and others over the past two years have provided new evidence in isolated cardiac myocytes that CaMKII indeed is directly associated with RyR (28) and overexpression of CaMKII increases SR Ca release as shown by increased spontaneous SR Ca release events termed Ca spark frequency (see in detail below: CaMKII and hypertrophy and heart failure). In contrast, when blocking CaMKII (using KN-93) Ca spark frequency decreases dramatically (29). These results in myocytes from CaMKII transgenic mouse hearts were confirmed recently by Currie et al. (30) showing that the CaMKII peptide inhibitor AIP (1 µM) depresses Ca spark frequency in rabbit hearts due to decreased endogenous CaMKII binding to RyR. Interestingly, Wehrens et al. (31), using site-directed mutagenesis, most recently showed that CaMKII binding at RyR might not be at Ser-2809 (protein kinase A, PKA, site with subsequent FKBP12.6 dissociation) but at Ser-2815. In addition, they showed that CaMKII-dependent RyR activation resulted in even more active RyR compared to PKA-dependent RyR phosphorylation. They clearly showed that CaMKII-dependent RyR phosphorylation increased RyR open probability using single channel measurements in lipid bilayers (31). Interestingly, the exact mechanism of RyR activation remained unclear since FKBP12.6 dissociation (as seen by PKA-dependent phosphorylation) does not seem to occur.

Frequency-dependent acceleration of relaxation

PLB is an endogenous inhibitor of SERCA in its unphosphorylated state (32). Upon PLB phosphorylation SERCA activity and SR Ca uptake are enhanced. PLB can be phosphorylated by PKA at Ser-16 and by CaMKII at Thr-17 (18,32) with a reduction of Km(Ca) of SERCA similar to Ser-16 phosphorylation by PKA. Although less generally accepted, there are also reports that suggest an increase of Vmax due to CaMKII phosphorylation of PLB with less effect on Km vs PKA. Bassani et al. (33) showed that CaMKII phosphorylation of PLB might be responsible for the frequency-dependent acceleration of relaxation (FDAR) of twitches and SR Ca uptake typically seen in intact myocytes. Hagemann et al. (34) recently showed a frequency-dependent increase in PLB Thr-17 phosphorylation in rat myocytes even in the absence of Ser-16 phosphorylation, and that the level of CaMKII-dependent Thr-17 phosphorylation correlated with the rate of relaxation.

Physiologically, FDAR may be an important intrinsic mechanism to allow faster relaxation (and diastolic filling) when heart rate is increased. FDAR is also manifest as slowing of twitch relaxation as time between beats is prolonged (i.e., at post-rest contractions; Ref. 33). As mentioned above, FDAR is also reflected on the rate of [Ca]i decline and is attributable to altered SR Ca uptake (33). Schouten (35) proposed that FDAR might be due to enhanced SR Ca uptake via PLB phosphorylation by CaMKII, activated by the cyclic increase in [Ca]i during ECC. Conversely, rest would allow deactivation of CaMKII and dephosphorylation of PLB, reversing the stimulation of SR-dependent [Ca]i decline. In intact rat myocytes, the CaMKII inhibitor KN-62 prevents the acceleration of [Ca]i decline in steady-state vs post-rest twitches, whereas phosphatase inhibitors prevent the slowing of [Ca]i decline at the post-rest twitch (33). While PLB is a logical CaMKII target explaining FDAR, we found that FDAR is still quite prominent in PLB-deficient mice and still sensitive to CaMKII inhibition by KN-93 (36). Thus, while PLB might contribute to FDAR, it cannot be the sole mechanism. There have been several reports of direct CaMKII-dependent phosphorylation of cardiac SERCA, and increased Vmax for Ca transport; Refs. 2,37). While this might fit with FDAR results above, critical studies have shown that CaMKII does not directly phosphorylate SERCA2 (2,37). Thus, the identity of the CaMKII target involved in accelerating SR Ca transport during FDAR has not yet been clearly determined.

Acidosis

Acidosis, a substantial decrease in intracellular pH (pHi), depresses myocardial contractility mainly due to decreased myofilament Ca sensitivity (37). During long periods of acidosis, there is a progressive increase in D[Ca]i causing a partial recovery of contractions (37). Increased [H]i during acidosis displaces Ca ions from proteins (e.g., troponin C), thereby raising resting (diastolic) [Ca]i (37). The combination of increased D[Ca]i and diastolic [Ca]i may contribute to the recovery of contractility. Also, an increased SR Ca content contributes to this recovery which is probably due to a combination of higher diastolic [Ca]i, inhibition of SR Ca release, elevated [Na]i, and [Ca]i (37). The increase in SR Ca load may gradually increase [Ca]i transients and offsets a reduced SR Ca fractional release at low pH (37).

Inhibition of SR Ca-ATPase during acidosis is manifest by the slowed decline of Ca transients and contractions, which progressively recover during late acidosis and are accelerated concomitant with an increase in the amplitudes of [Ca]i transients and contractions (38). Interestingly, the recovery of Ca transients can be prevented by the CaMKII inhibitor KN-93 and it was proposed that CaMKII-dependent PLB phosphorylation may be responsible for the faster [Ca]i decline and recovery of contractions that partially overcome the direct inhibitory effect of acidosis (38,39). This is consistent with an inhibition of phosphatases during acidosis and increased PLB phosphorylation (40). We recently confirmed the importance of PLB phosphorylation by CaMKII in the absence of PKA phosphorylation in mouse myocytes (41). We showed also that PLB is required for the recovery of D[Ca]i during acidosis using isolated myocytes from PLB knockout compared to wild-type mice. In addition, KN-93 inhibited CaMKII-dependent protein phosphorylation (e.g., PLB Thr-17). Moreover, preliminary results from our laboratory have shown that acute overexpression of CaMKII in rabbit cardiac myocytes improves recovery during late acidosis leading to increased twitch shortening, [Ca]i, as well as accelerated relaxation parameters (42).

Arrhythmias

Early afterdepolarizations (EAD) are thought to initiate long QT-arrhythmias. CaMKII was first thought to be involved in arrhythmias in a study by Anderson et al. (11). The authors found that KN-93 decreases the amount of EAD which they induced in isolated rabbit hearts and they showed that ICa was the mechanism responsible. This was convincingly done by single cell studies showing that ICa and RyR are needed to initiate EADs (13). In a follow-up study, the same group described increased arrhythmias in a mouse model of transgenic CaMKIV overexpression leading to hypertrophy (43). Interestingly, in this mouse model increased CaMKII activity was found as a side effect. Electrophysiologically, this model is associated with increased AP duration, as also shown by specific overexpression of CaMKII (see below; Ref. 29) and QT prolongation leading to EAD. Arrhythmias could be increased by isoproterenol and decreased by KN-93 and a specific inhibiting peptide for CaMKII.

Early reports show that, in addition to the Ca channels, also cardiac Na channels might be regulated by CaMKII possibly contributing to cardiac arrhythmias (44). Also, in human atrial myocytes, transient outward K current can be accelerated using KN-93 (45). In summary, the role of CaMKII in arrhythmias needs to be further investigated and the subcellular mechanisms need to be explored in detail.

CaMKII and hypertrophy and heart failure

In addition to the acute effects of CaMKII on ECC in the heart discussed in the previous sections, CaMKII is also involved in longer-term regulation of gene expression, termed excitation-transcription coupling, especially in hypertrophy and heart failure (2).

It has been known for several years that Ca can bind to CaM in different cell types which then translocate into the nucleus activating CaMK, resulting in phosphorylation of the transcription factor cAMP response element binding protein (CREB) which promotes transcription of c-fos (3). CREB residue Ser-133 was the major site of phosphorylation by the CaMK in vitro and of phosphorylation after membrane depolarization in vivo (2). Interestingly, phosphorylation of Ser-142 seems to negatively regulate CREB-dependent transcription. CaM might also be associated with the nuclear envelope. In the heart, overexpression of CaM in transgenic mice causes severe cardiac hypertrophy (46) and results in higher CaMKII phosphorylation (and activity), and the expression of the hypertrophic marker atrial natriuretic factor (Ref. 47). Interestingly, the CaM antagonist W-7 and the CaMKII antagonist KN-62 (48) could prevent hypertrophy in cultured myocytes, further implicating Ca-CaM as a mediator of the hypertrophic response. Activated CaMKIV (and I) can also induce hypertrophic responses in cultured cardiomyocytes and transgenic mice (49). CaMKIV is expressed at very low levels in the heart vs CaMKII, but like CaMKII dB it can enter the nucleus (1).

Recently, it was shown that CaMKII might also be involved in the transduction of hypertrophic stimuli through class II histone deacetylases (HDAC) which contain two CaMK phosphorylation sites and usually are bound to myocyte elongation factor 2 (MEF2), thereby inhibiting transcription (50). When HDAC are phosphorylated, dissociation from MEF2 and nuclear export of HDAC results in activation of MEF2 (51) which interacts with nuclear factor of activated T cells and GATA transcription factors leading to hypertrophic gene expression. Interestingly, HDAC translocation from the nucleus might be a very local process due to perinuclear Ca release (as a consequence of IP3 increase) independent of cytosolic Ca transients during ECC leading to local CaMKII activation, i.e., nuclear isoform CaMKII dB (52). In agreement with this are preliminary data showing InsP3 receptors concentrated in the nuclear envelope of cardiac myocytes which are associated with CaMKII d (53).

New and exciting reports have also indicated that chronic ß-adrenergic stimulation, in contrast to short-term stimulation which typically activates the cAMP/PKA pathway, activates CaMKII with subsequent stimulation of CaMKII-dependent pathways (54) and even apoptosis (55).

The first reports regarding human heart failure a few years ago described that CaMKII activity was increased almost 2-3-fold (2). It was suggested that this increase was compensatory because it correlated positively with the cardiac index and ejection fraction of patients with greatly impaired left ventricular function. In a different study, a ~2-fold increase in CaMKIId was reported in failing human myocardium (2). Phosphatase activity is also enhanced in human heart failure (2), such that the net effect is unclear with respect to the phosphorylation state of individual target proteins. For example, while many PKA targets are relatively dephosphorylated in heart failure, the RyR may be hyperphosphorylated, a fact initially thought to be solely due to reduction of local phosphatase bound directly to the RyR (56).

Animal models of cardiac hypertrophy induced by transverse aortic constriction also showed an increase in CaMKII expression (28,47) whereas CaMKI and IV expression levels were not changed (47). Ramirez et al. (57) reported that the specific nuclear isoform of CaMKII ( dB) caused transcriptional activation of atrial natriuretic factor gene expression (a hypertrophic signaling marker) in neonatal rat ventricular myocytes. Similarly, transgenic overexpression of CaMKII dB in mice induces cardiac hypertrophy and modest ventricular dilation (58). A similar phenotype is induced by transgenic expression of CaMKIV (49), which also enters the nucleus. However, CaMKIV is expressed at very low levels in the heart (2,4).

To investigate the effects of CaMKII d on cellular Ca regulation we have overexpressed the cytoplasmic dC isoform in mouse heart and reported in two accompanying papers the phenotype and cellular basis for hypertrophy and heart failure found in this model (28,29). The transgenic line studied exhibited a 3-fold increase in CaMKIId activity, profound dilated hypertrophy and severe ventricular dysfunction (Figure 1). Since cytoplasmic CaMKII targets numerous Ca-handling proteins, we compared Ca regulation in cardiomyocytes isolated from 3-month-old transgenic CaMKII dC mice vs wild-type littermates. We found major alterations in intracellular Ca handling with marked reductions in twitch D[Ca]i and SR Ca content (Figure 2). This was associated with decreased SERCA2 and PLB expression, but enhanced NCX function and expression, all typical features which are widely accepted for heart failure (37).

Interestingly, possible acute effects of CaMKII on RyR phosphorylation and function were also detected which do not fit the usual findings in heart failure. Thus, the frequency of Ca sparks (indicative of diastolic spontaneous SR Ca release events or opening of RyR clusters), width, and duration were greatly enhanced, demonstrating increased diastolic SR Ca leak (Figure 3). Therefore, in addition to disturbed SR Ca uptake, increased Ca loss from the SR by means of a leaky RyR might be important in CaMKII-induced heart failure. Also, the increased Ca spark frequency was interesting since this is usually decreased when SR Ca load is reduced. We found that this most likely is due to increased RyR phosphorylation directly by CaMKII, resulting in increased and prolonged openings of the RyR. Most importantly, the increased Ca spark frequency could be reduced back to normal levels by blocking CaMKII with KN-93, and backphosphorylation indeed showed increased RyR phosphorylation in transgenic compared to wild-type mice. These results showing a novel mechanism of CaMKII-dependent SR Ca leak through hyperphosphorylated RyR in heart failure were recently confirmed by two independent groups. They showed that the peptide inhibitor AIP depresses Ca spark frequency in rabbit hearts due to decreased CaMKII binding to RyR (30) and that CaMKII-dependent RyR phosphorylation increases RyR open probability using single channel measurements in lipid bilayers (31). Reduced contractile function in this model of heart failure could be explained by a combination of higher SR Ca leak, weaker SR Ca pumping, and greater NCX function due to RyR hyperphosphorylation.


Figure 1. Transgenic Ca/calmodulin-dependent protein kinase IId (TG CaMKII dC) overexpression leads to cardiac hypertrophy and heart failure. The Southern blots show three different transgenic lines, with CaMKII activity being investigated in the low (TGL) and medium (TGM) overexpressing lines (the founder mice of the high (TGH) overexpressing line died a few weeks after birth). Cytosolic overexpression was measured by immunofluorescence. Macroscopic pictures show the extent of cardiac dilation and the echocardiograms depict decreased contractile function (adapted from Refs. 28 and 29). WT = wild type. *P<0.05 compared to WT.

[View larger version of this image (127 K JPG file)]


Figure 2. CaMKII dC overexpression and excitation-contraction coupling. CaMKII phosphorylates several Ca-handling proteins including phospholamban (PLB), SR Ca release channels (RyR), and L-type Ca channels responsible for Ca influx (ICa). Action potential (AP) duration is prolonged, and Ca influx is increased. However, SR Ca content and Ca transients are greatly decreased leading to contractile dysfunction (adapted from Refs. 28 and 29). TG and WT = transgenic and wild-type mice, respectively; NCX = Na/Ca-exchanger; SR = sarcoplasmic reticulum; SERCA = SR Ca-ATPase.

[View larger version of this image (125 K JPG file)]


Figure 3. Elementary SR Ca release events (Ca sparks). Original confocal images showing significantly increased Ca spark frequency in CaMKII dC transgenic (TG) mouse myocytes leading to a CaMKII-dependent diastolic SR Ca leak. This leak is significantly (~4 times) higher in TG than in wild type (WT). In contrast, CaMKII inhibition using KN-93 decreases Ca spark frequency back to control levels. An explanation for this increased Ca spark frequency most likely is the CaMKII-dependent hyperphosphorylation of SR Ca release channels (RyR; adapted from Refs. 28 and 29). CaMKII = Ca/calmodulin-dependent protein kinase II; au = arbitrary units. *P<0.05 compared to WT.

[View larger version of this image (215 K JPG file)]


Final considerations

CaMKII has regained attention during the last few years. Its involvement in cardiac hypertrophy and heart failure is novel and interesting. New mechanisms were proposed and confirmed by independent groups. Exact hypertrophic signaling pathways still need to be investigated, but a couple of internationally well-recognized groups are on their way. Also, the new findings of CaMKII in apoptosis and arrhythmias open a new field of research. We are just starting to understand the subcellular mechanisms responsible for it. The next few years will bring more insight into these mechanisms and possibly therapeutic approaches on experimental levels might be initiated thereafter.


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Acknowledgments

I would cordially like to thank FESBE for the invitation to this meeting and Stefan Wagner, Michael Kohlhaas, and Can-Martin Sag, University of Göttingen, Göttingen, Germany, for comments on the manuscript.


Correspondence and Footnotes

Address for correspondence:L.S. Maier, Abteilung Kardiologie and Pneumologie, Herzzentrum, Georg-August-Universität Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. Fax: +49-551-39-8941. E-mail: lmaier@med.uni-goettingen.de

Internet: www.lmaier.de Part of a talk presented at the XIX Annual Meeting of the Federação de Sociedades de Biologia Experimental, Águas de Lindóia, SP, Brazil, August 25-29, 2004. L.S. Maier is the recipient of a grant from the Deutsche Forschungsgemeinschaft (DFG, Emmy Noether-program, MA 1982/2-1 and MA 1982/4-1), a Young Investigator Award from the GlaxoSmithKline Research Foundation, and a grant from the University of Göttingen Anschubfinanzierung). Received January 31, 2005. Accepted May 5, 2005.

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