Calmodulin Sequences Research Articles

Molecular identification of potentialy mycotoxigenic black Aspergili contaminating pistachio nuts in Iran

Ochratoxin A is a mycotoxin produced by Aspergillus and Penicillium species. This mycotoxin is a common contaminant of various foods including cereal products, spices, dried fruits, coffee, beer and wine. Besides species assigned to Aspergillus section Circumdati, black Aspergilli including A. niger, A. carbonarius and A. sclerotioniger are also able to produce this mycotoxin. Black Aspergilli have been found to be the predominant fungi contaminating pistachio nuts worldwide. We examined the species distribution of black Aspergilli on Iranian pistachio nuts. Sequence-based identifications have been carried out using partial calmodulin sequence data. Our data indicate that instead of the potential ochratoxin and fumonisin producing A. niger species, A. tubingensis dominates on Iranian pistachio nuts. This species is unable to produce either of these mycotoxins, consequently do not contribute to mycotoxin contamination of pistachio nuts in Iran. Further studies are in progress to clarify the role of other Aspergilli in ochratoxin contamination of pistachio in Iran.

A Region Involved in Isoform-Specific Regulation of Skeletal Muscle Ryanodine Receptor by Calmodulin

Calmodulin (CaM) regulates both skeletal (RyR1) and cardiac (RyR2) muscle ryanodine receptor calcium channels by binding to a single highly conserved CaM binding domain (RyR1 amino acid (aa) 3614-3643; RyR2 aa 3581-3610). CaM inhibits both isoforms at an elevated Ca2+ concentration (micromollar), whereas at cellular resting Ca2+ concentration CaM has opposite effects on two isoforms (activation of RyR1 and inhibition of RyR2). This implies another region of RyR is involved in isoform-specific CaM regulation at submicromollar Ca2+ concentration. To identify the regions we constructed and analyzed a series of RyR1/RyR2 chimera. RyR1/RyR2 chimera carrying RyR1 aa 1-3725 is inhibited by CaM at 0.4 µM Ca2+ (RyR2-type), whereas chimera carrying RyR1 aa 1-4301 is activated (RyR1-type). The results suggest that RyR1 aa 3726-4301 contains a region that is responsible for CaM activation. The region overlaps with a domain resembling the sequence of CaM (CaM-like domain). Replacement of RyR1 3726-4301 with the corresponding RyR2 sequence confers CaM inhibition at [Ca2+]<1 µM (RyR2 type). Furthermore, substitution of 5 non-conserved amino acids in RyR1 CaM-like domain with those of RyR2 (M4122T, I4123L, N4124D, F4125Y, N4130K) is sufficient for RyR2-type CaM inhibition. However, the reverse chimera and mutant RyR2 were not activated but inhibited by CaM. Taken together, the results suggest that 5 non-conserved amino acids of RyR1 are crucial for RyR1-specific CaM activation at submicromollar Ca2+ concentration. On the other hand, CaM inhibition of RyR2 is likely controlled by a different region. Supported by NIH (AR018687 and HL073051), NSF (EPS-0903795) and AHA (10SDG3500001).

Taxonomic studies of the Penicillium glabrum complex and the description of a new species P. subericola

A mycological survey of fungi, present in several stages of the manufacturing of cork discs for champagne stoppers in Portugal, was made. Sixty-nine strains belonging to the Glabra series of the genus Penicillium were isolated and subsequently grouped according to their partial β-tubulin gene sequences. Six groups with different partial β-tubulin gene sequences were observed, and a selection of isolates of each group was made. These selected isolates and various related ex-type strains were subjected to a taxonomical study using a polyphasic approach. This approach included analysis of macro- and microscopic features, the comparison of extrolite profiles and sequencing a part of the β-tubulin and calmodulin gene. The six β-tubulin types were reduced to three different species. One group of isolates was centred on the ex-type strain of P. glabrum, a second group accommodated the type strain of P. spinulosum and a third group contained isolates which were unique in their β-tubulin and calmodulin sequences, extrolite profiles and growth characteristics. This group of isolates is described as the new species Penicillium subericola. The type strain of P. subericola CBS 125096T was isolated from Portuguese raw cork, but additional isolates were found from soil, air and lumen.

Emerging pathogenAspergillus calidoustuscolonizes water distribution systems

Recent studies have changed the taxonomy of Aspergillus section Usti, and a novel species, Aspergillus calidoustus, has been erected. It was also demonstrated that clinical isolates previously identified as A. ustus actually belong to the emerging pathogen A. calidoustus. Aspergillus ustus were frequently isolated from Norwegian water systems, and due to the taxonomical progress, these waterborne strains could be identified more precisely. A MLST study including ITS, calmodulin, β-tubulin and actin sequences was conducted on 32 strains previously identified as A. ustus. All strains were identified as A. calidoustus, which was verified by physiological, biochemical and phylogenetic analyses. This is the first report of that A. calidoustus is able to colonize water distribution systems. In respect to the potential role of water systems as a source of nosocomial infections in patients with immunodeficiency, attention should be given to water systems in hospitals and other healthcare units, especially the heated-water installations.

First Report of Fusarium Wilt on Eremophila spp. Caused by Fusarium oxysporum in Italy.

Eremophila spp. (Myoporaceae family), endemic to Australia, are evergreen shrubs or small trees occurring in arid, semi-arid, tropical, or temperate regions. In Europe, Eremophila spp. are grown for their horticultural appeal. During 2009 and 2010, extensive wilting was observed on 2-month to 1-year-old potted plants of Eremophila laanii F. Muell., E. glabra subsp. carnosa Chinnock, and E. maculata (Ker Gawl.) F. Muell. grown in a commercial nursery near Catania (southern Italy). Internally, symptomatic plants had conspicuous vascular discoloration from the crown to the canopy. Diseased crown and stem tissues were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissues. Colonies with purple mycelia and violet reverse colors developed after 9 days. On carnation leaf agar, single-spore isolates produced microconidia on short monophialides, macroconidia that were three to five septate with a pedicellate base, and solitary and double-celled or aggregated chlamydospores. A PCR assay was conducted on two representative isolates (ITEM 12591 and ITEM 12592) by analyzing sequences of the partial CaM gene (coding calmodulin protein) and benA (coding beta-tubulin protein) using the primers as reported by O'Donnell et al. (1). Calmodulin sequences of ITEM 12951 and ITEM 12952 isolates (GenBank Nos. FR671157 and FR671158) exhibited 99.8 and 99.5% identity with Fusarium oxysporum strain ITEM 2367 (GenBank No. AJ560774), respectively, and had 99.5% homology between them. BenA gene sequences of ITEM 12951 (GenBank No. FR671426) exhibited an identity of 100% to F. oxysporum f. sp. vasinfectum strain CC-612-3 (GenBank No. AY714092.1), and benA gene sequences of ITEM 12952 (GenBank No. FR671427) exhibited an identity of 100% to F. oxysporum f. sp. vasinfectum strain LA 140 (GenBank No. FJ466740.1), whereas the homology between the two strains is 99.5%. Morphological characteristics, as well as CaM and benA sequences, identified the isolates as F. oxysporum Schlechtend:Fr. Pathogenicity tests were performed by placing 1-cm2 plugs of PDA from 9-day-old mycelial cultures near the crown on potted, healthy, 3-month-old cuttings of E. laanii, E. glabra subsp. carnosa, and E. maculata. Twenty plants for each species were inoculated with each isolate. The same number of plants served as noninoculated controls. All plants were enclosed for 4 days in plastic bags and placed in a growth chamber at 24 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 23 to 27°C. Symptoms identical to those observed in the nursery developed 20 days after inoculation with both strains. Crown and stem discoloration was detected in all inoculated plants after 45 days. Wilting was detected on 15% of plants. Control plants remained symptomless. F. oxysporum was consistently reisolated from symptomatic tissues and identified as previously above. To our knowledge, this is the first report of F. oxysporum causing disease of Eremophila spp. worldwide. Reference: (1) K. O'Donnell et al. Mycoscience 41:61, 2000.

Polyphasic taxonomy of Aspergillus section Sparsi

Aspergillus section Sparsi includes species which have large globose conidial heads with colours ranging from light grey to olive-buff. In this study, we examined isolates of species tentatively assigned to section Sparsi using a polyphasic approach. The characters examined include sequence analysis of partial β-tubulin, calmodulin and ITS sequences of the isolates, morphological and physiological tests, and examination of the extrolite profiles. Our data indicate that the revised section Sparsi includes 10 species: A. anthodesmis, A. biplanus, A. conjunctus, A. diversus, A. funiculosus, A. implicatus, A. panamensis, A. quitensis, A. sparsus, and the new taxon A. haitiensis. The recently described A. quitensis and A. ecuadorensis are synonyms of A. amazonicus based on both molecular and physiological data. The white-spored species A. implicatus has also been found to belong to this section. Aspergillus haitiensis sp. nov. is characterised by whitish colonies becoming reddish brown due to the production of conidial heads, and dark coloured smooth stipes. The taxon produces gregatins, siderin and several unknown but characteristic metabolites.

Aspergillus sect. Aeni sect, nov., a new section of the genus for A. karnatakaensis sp. nov. and some allied fungi

The new species Aspergilluskarnatakaensis sp. nov. is described and illustrated. All three isolates of this species were isolated from Indian soil; two from soil under a coconut palm in a coffee plantation in Karnataka, and one from soil in the Machrar river bed in Bansa district. This species is closely related to, but clearly distinct, from A. aeneus based on β-tubulin or calmodulin sequence data. Sequences of the ITS region of these two species are identical. Aspergillus karnatakaensis produced terrein, gregatins, asteltoxin, karnatakafurans A and B and the unknown metabolite, provisionally named NIDU. Aspergillus karnatakaensis belongs to a well-defined clade within Aspergillus subgenus Nidulantes together with eight other species including A. aeneus, A. crustosus, A. eburneocremeus, A. heyangensis, and the teleomorph producing-species Emericella bicolor, E. discophora, E. spectabilis, and E. foeniculicola. This clade is placed in a new section, Aspergillus sect. Aenei sect. nov. All teleomorph species assigned to this section are able to produce sterigmatocystin.

Taxonomy of Penicillium citrinum and related species

Penicillium citrinum and related species have been examined using a combination of partial β-tubulin, calmodulin and ITS sequence data, extrolite patterns and phenotypic characters. It is concluded that seven species belong to the series Citrina. Penicillium sizovae and Penicillium steckii are related to P. citrinum, P. gorlenkoanum is revived, Penicillium hetheringtoniisp. nov. and Penicillium tropicoidessp. nov. are described here as new species, and the combination Penicillium tropicum is proposed. Penicillium hetheringtonii is closely related to P. citrinum and differs in having slightly broader stipes, metulae in verticils of four or more and the production of an uncharacterized metabolite, tentatively named PR1-x. Penicillium tropicoides resembles P. tropicum, but differs in the slow maturation of the cleistothecia, slower growth at 30°C and the production of isochromantoxins. The type strain of P. hetheringtonii is CBS 122392T (=IBT 29057T) and the type strain of P. tropicoides is CBS 122410T (=IBT 29043T).

First Report of Fusarium Wilt of Paper Flower (Bougainvillea glabra) Caused by Fusarium oxysporum in Italy.

Paper flower (Bougainvillea glabra Choisy), native to Brazil, is the most widely and intensively cultivated species of bougainvillea as a potted plant in Sicily (Italy). During 2008 and 2009, a wilting of vegetatively produced B. glabra cv. Sanderiana was observed in several nurseries in eastern Sicily (Catania and Messina provinces). Disease incidence was higher (~10 to 30%) in the tree-shaped potted plants (standards). Occasionally, wilting was detected on plants that were not tree shaped. Internally, symptomatic plants showed conspicuous vascular orange discoloration from the crown to the canopy. Diseased crown and stem tissues were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissue. Colonies with light purple or purple mycelia and violet reverse colony colors developed after 10 days. On carnation leaf agar, single-spore isolates produced microconidia in false heads on short monophialides, macroconidia that were 3-septate with a pedicellate base, and solitary and double-celled or aggregate chlamydospores. A PCR assay was conducted on two representative strains (DISTEF-BGS1 and DISTEF-BGS2) by analyzing sequences of the parzial translation elongation factor alpha gene (TEF-1α) and CaM gene (coding calmodulin protein). The primers used are previously used by O'Donnell et al. (1,2). Calmodulin sequences of BGS1 and BGS2 strains (GenBank Nos. FN645740 and FN645741, respectively) exhibited 99% homology with Fusarium oxysporum strain ITEM 2367 (GenBank No. AJ560774), and have homology of 99.6% between them. TEF-1 gene sequences of BGS1 (GenBank No. FN645739) exhibited an identity of 100% to F. oxysporum f. sp. lycopersici MUCL 22544 GenBank No. EF056785.1) and TEF-1α gene sequences of BGS2 (GenBank No. FN655742) exhibited an identity of 100% to F. oxysporum strain NRRL 45954 (GenBank No. FJ985431.1), whereas the homology between the two strains is 98.5%. Both PCR approaches established the identity of the isolates to the F. oxysporum Schlechtend:Fr (1,2). Pathogenicity tests were performed by placing 1-cm2 plugs of PDA from 10-day-old mycelial cultures near the crown on 40 potted, healthy, 6-month-old cuttings of paper flower. Twenty plants for each isolate were used. The same number of plants served as noninoculated controls. All plants were enclosed for 5 days in plastic bags and placed in a growth chamber at 24 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 24 to 26°C. Symptoms identical to those observed in nurseries developed 1 month after inoculation with both strains. Crown and stem orange discoloration was detected in all inoculated plants after 2 months. Control plants remained symptomless. F. oxysporum was consistently reisolated from symptomatic tissues and identified as previously described. To our knowledge, F. oxysporum was previously reported on paper flower in Ghana (3). However, this is the first demonstration of the pathogenicity of F. oxysporum on paper flower and it is the first report in Europe of the disease. The presence of Fusarium wilt in Sicily is a potential threat to paper flower production in nurseries.

DNA sequence analysis of conserved genes reveals hybridization events that increase genetic diversity in Verticillium dahliae

The hybrid origin of a Verticillium dahliae isolate belonging to the vegetative compatibility group (VCG) 3 is reported in this work. Moreover, new data supporting the hybrid origin of two V. dahliae var. longisporum (VDLSP) isolates are provided as well as information about putative parentals. Thus, isolates of VDLSP and V. dahliae VCG3 were found harboring multiple sequences of actin ( Act), β-tubulin ( β-tub), calmodulin ( Cal) and histone 3 ( H3) genes. Phylogenetic analysis of these sequences, the internal transcribed sequences (ITS-1 and ITS-2) of the rRNA genes and of a V. dahliae-specific sequence provided molecular evidences for the interspecific hybrid origin of those isolates. Sequence analysis suggests that some of VDLSP isolates may have resulted from hybridization events between a V. dahliae isolate of VCG1 and/or VCG4A and, probably, a closely related taxon to Verticillium albo-atrum but not this one. Similarly, phylogenetic analysis and PCR markers indicated that a V. dahliae VCG3 isolate might have arisen from a hybridization event between a V. dahliae VCG1B isolate and as yet unidentified parent. This second parental probably does not belong to the Verticillium genus according to the gene sequences dissimilarities found between the VCG3 isolate and Verticillium spp. These results suggest an important role of parasexuality in diversity and evolution in the genus Verticillium and show that interspecific hybrids within this genus may not be rare in nature.

Tradeoff Between Stability and Multispecificity in the Design of Promiscuous Proteins

Natural proteins often partake in several highly specific protein-protein interactions. They are thus subject to multiple opposing forces during evolutionary selection. To be functional, such multispecific proteins need to be stable in complex with each interaction partner, and, at the same time, to maintain affinity toward all partners. How is this multispecificity acquired through natural evolution? To answer this compelling question, we study a prototypical multispecific protein, calmodulin (CaM), which has evolved to interact with hundreds of target proteins. Starting from high-resolution structures of sixteen CaM-target complexes, we employ state-of-the-art computational methods to predict a hundred CaM sequences best suited for interaction with each individual CaM target. Then, we design CaM sequences most compatible with each possible combination of two, three, and all sixteen targets simultaneously, producing almost 70,000 low energy CaM sequences. By comparing these sequences and their energies, we gain insight into how nature has managed to find the compromise between the need for favorable interaction energies and the need for multispecificity. We observe that designing for more partners simultaneously yields CaM sequences that better match natural sequence profiles, thus emphasizing the importance of such strategies in nature. Furthermore, we show that the CaM binding interface can be nicely partitioned into positions that are critical for the affinity of all CaM-target complexes and those that are molded to provide interaction specificity. We reveal several basic categories of sequence-level tradeoffs that enable the compromise necessary for the promiscuity of this protein. We also thoroughly quantify the tradeoff between interaction energetics and multispecificity and find that facilitating seemingly competing interactions requires only a small deviation from optimal energies. We conclude that multispecific proteins have been subjected to a rigorous optimization process that has fine-tuned their sequences for interactions with a precise set of targets, thus conferring their multiple cellular functions.

The ITS region as a taxonomic discriminator between Fusarium verticillioides and Fusarium proliferatum

The maize pathogens Fusarium verticillioides ( Fv) and Fusarium proliferatum ( Fp) are morphologically very similar to one another, so Fp isolates have been often mistaken as Fusarium moniliforme (the former name of Fv). The only presently accepted morphological discriminator between these species is the presence/absence of polyphialides. Here, a collection of 100 Fusarium strains, isolated from infected maize kernels on plants grown in north-western Italy, were assigned as Fv or Fp on the basis of the presence/absence of polyphialides. This classification was tested on a subset of isolates by sexual crosses, ITS and calmodulin sequencing and AFLP profiling. An ITS-RFLP assay was extended to the full collection and to a number of Fv and Fp isolates of different geographical origin and hosts. The ITS region is proposed as taxonomically informative for distinguishing between Fp and Fv.

B. subtilis Elongation Factor Tu binds Calcium ions

ABSTRACTAn essential step toward the understanding of the role of calcium in prokaryotes is the identification of intracellular calcium‐binding proteins. In this report we present evidence that Bacillus subtilis elongation factor EF‐Tu is a calcium‐binding protein.A genomic analysis of EF‐hand related sequences in the genus Arabidopsis showed that elongation factors have EF‐hand domains. Considering the high degree of conservation across species and based on previous findings in our laboratory suggesting EF‐Tu may be a CaBP (unpublished results) we investigated further the B. subtilis EF‐Tu. We performed multiple sequence alignment and motif discovery of bacterial EF‐Tu and D. discoideum calmodulin sequences and the results suggested the presence of a possible calcium binding site in B. subtilis EF‐Tu. Secondary structure prediction indicated that the conserved acidic amino acids were found within a loop flanked by two helices. A recombinant B. subtilis EF‐Tu was partially purified. The protein was identified by Western blot assay using anti‐EF‐Tu antibodies and two‐dimensional electrophoresis followed by liquid chromatography and mass spectrometry. Calcium binding was examined, in vivo and in vitro, by 45Ca‐autoradiography and immunodetection using monoclonal anti‐calmodulin and polyclonal anti‐EF‐hand antibodies the combined results indicated the B. subtilis EF‐Tu bindis calcium ions in vivo and in vitro. Addition of calcium chelators EGTA/BAPTA or CaCl2 to cell cultures affected expression of B. subtilis EF‐Tu.

Actin Pedestal Formation by Enteropathogenic Escherichia coli Is Regulated by IQGAP1, Calcium, and Calmodulin

During infection, enteropathogenic Escherichia coli (EPEC) injects effector proteins into the host cell to manipulate the actin cytoskeleton and promote formation of actin pedestals. IQGAP1 is a multidomain protein that participates in numerous cellular functions, including Rac1/Cdc42 and Ca(2+)/calmodulin signaling and actin polymerization. Here we report that IQGAP1, Ca(2+), and calmodulin modulate actin pedestal formation by EPEC. Infection with EPEC promotes both the interaction of IQGAP1 with calmodulin and the localization of IQGAP1 and calmodulin to actin pedestals while reducing the interaction of IQGAP1 with Rac1 and Cdc42. IQGAP1-null fibroblasts display a reduced polymerization of actin in response to EPEC. In addition, antagonism of calmodulin or chelation of intracellular Ca(2+) reduces EPEC-dependent actin polymerization. Furthermore, IQGAP1 specifically interacts with Tir in vitro and in cells. Together these data identify IQGAP1, Ca(2+), and calmodulin as a novel signaling complex regulating actin pedestal formation by EPEC.

Sequence Differences in the IQ Motifs of CaV1.1 and CaV1.2 Strongly Impact Calmodulin Binding and Calcium-dependent Inactivation

The proximal C terminus of the cardiac L-type calcium channel (Ca(V)1.2) contains structural elements important for the binding of calmodulin (CaM) and calcium-dependent inactivation, and exhibits extensive sequence conservation with the corresponding region of the skeletal L-type channel (Ca(V)1.1). However, there are several Ca(V)1.1 residues that are both identical in six species and are non-conservatively changed from the corresponding Ca(V)1.2 residues, including three of the "IQ motif." To investigate the functional significance of these residue differences, we used native gel electrophoresis and expression in intact myotubes to compare the binding of CaM to extended regions (up to 300 residues) of the C termini of Ca(V)1.1 and Ca(V)1.2. We found that in the presence of Ca(2+) (either millimolar or that in resting myotubes), CaM bound strongly to C termini of Ca(V)1.2 but not of Ca(V)1.1. Furthermore, replacement of two residues (Tyr(1657) and Lys(1662)) within the IQ motif of a C-terminal Ca(V)1.2 construct with the divergent residues of Ca(V)1.1 (His(1532) and Met(1537)) led to a weakening of CaM binding (native gels), whereas the reciprocal substitution in Ca(V)1.1 caused a gain of CaM binding. In full-length Ca(V)1.2, substitution of these same two divergent residues with those of Ca(V)1.1 (Y1657H, K1662M) eliminated calcium-dependent inactivation of the heterologously expressed channel. Thus, our results reveal that a conserved difference between the IQ motifs of Ca(V)1.2 and Ca(V)1.1 has a profound effect on both CaM binding and calcium-dependent inactivation.

Computational Design of Calmodulin Mutants with up to 900-Fold Increase in Binding Specificity

Calmodulin (CaM) is a ubiquitous second messenger protein that regulates a variety of structurally and functionally diverse targets in response to changes in Ca2+ concentration. CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are the prominent CaM targets that play an opposing role in many cellular functions including synaptic regulation. Since CaMKII and CaN compete for the available Ca2+/CaM, the differential affinity of these enzymes for CaM is crucial for achieving a balance in Ca2+ signaling. We used the computational protein design approach to modify CaM binding specificity for these two targets. Starting from the X-ray structure of CaM in complex with the CaM-binding domain of CaMKII, we optimized CaM interactions with CaMKII by introducing mutations into the CaM sequence. CaM optimization was performed with a protein design program, ORBIT, using a modified energy function that emphasized intermolecular interactions in the sequence selection procedure. Several CaM variants were experimentally constructed and tested for binding to the CaMKII and CaN peptides using the surface plasmon resonance technique. Most of our CaM mutants demonstrated small increase in affinity for the CaMKII peptide and substantial decrease in affinity for the CaN peptide compared to that of wild-type CaM. Our best CaM design exhibited an about 900-fold increase in binding specificity towards the CaMKII peptide, becoming the highest specificity switch achieved in any protein–protein interface through the computational protein design approach. Our results show that computational redesign of protein–protein interfaces becomes a reliable method for altering protein binding affinity and specificity.

Human Myosin Vc Is a Low Duty Ratio, Nonprocessive Molecular Motor

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s(-1)), rather than the ADP release step (~12.0-16.0 s(-1)). Nevertheless, the ADP affinity of actomyosin Vc (K(d) = 0.25 +/- 0.02 microm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was approximately 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of approximately 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.

Novel eye‐specific calmodulin methylation characterized by protein mapping in Drosophila melanogaster

Post-translational methylation of the epsilon-amino group of lysine residues regulates a number of protein functions. Calmodulin, a key modulator of intracellular calcium signaling, is methylated on lysine 115 in many species. Although the amino acid sequence of calmodulin is highly conserved in eukaryotes, it has been shown that lysine 115 is not methylated in Drosophila calmodulin and no other methylation site has been reported. In this study, we characterized in vivo modification states of Drosophila calmodulin using proteomic methodology involving the protein mapping of microdissected Drosophila tissues on 2-D gels. We found that Drosophila calmodulin was highly expressed in methylated forms in the compound eye, whereas its methylation was hardly detected in other tissues. We identified that lysine 94 located in an EF-hand III is the methylation site in Drosophila calmodulin. The predominance of methylated calmodulin in the compound eye may imply the involvement of calmodulin in photoreceptor-specific functions through methylation.

Adenoviral Expression of Calmodulin Antisense Reduces Hypertrophy in Cultured Cardiomyocytes

Sustained myocardial hypertrophy is associated with an increased risk of sudden death and progression to heart failure. Multiple signal pathways are involved in cardiac hypertrophy and understanding their interaction may point to new therapeutic targets. In this work, we tested the hypothesis that adenovirus-mediated calmodulin (CaM) antisense expression will reduce the intracellular availability of CaM and inhibit the hypertrophic response. Three recombinant adenoviruses were constructed: AdASCaM, containing the AntiSense sequence of CaM and the enhanced green fluorescent protein (GFP) coding sequence; AdCaM, containing the coding sequence of CaM and the GFP sequence; and the AdGFP, containing the GFP coding sequence. Neonatal rat ventricular cardiomyocytes were infected with AdASCaM, AdCaM, or AdGFP and stimulated with phenylephrine (PE, 50 microM) or angiotensin II (AngII, 10 microM) for 48 h and cell surface area measured with planimetry. After PE treatment, the surface areas of cardiomyocytes infected with AdASCaM or AdGFP were 411 +/- 174.3 micro(2) and 832.6 +/- 372.3 micro(2), respectively (P < 0.01). After AngII treatment, the surface areas of cardiomyocytes infected with AdASCaM or AdGFP were 441.5 +/- 149.2 micro(2) and 726 +/- 328.3 micro(2), respectively (P < 0.01). Adenoviral expression of the CaM antisense (AdASCaM) significantly inhibited PE or AngII-induced cardiomyocyte hypertrophy. Cardiomyocytes infected with the AdCaM showed increased area when compared with those infected with the AdGFP. These results suggest that adenovirus-mediated changes in CaM expression may alter hypertrophy in cardiac myocytes.

Cloning and characterization of a novel calmodulin‐like gene (CaM) from crayfish Procambarus clarkii

Calmodulin (CaM), the Ca2+ sensor in living cells, is essential for biological functions mediated by Ca2+dependent mechanisms. This study describes the cloning and characterization of a novel CaM from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcCaM). We also examined tissue specific distribution and expression of pcCaM mRNA as a function of molting by real-time PCR. The complete sequence of pcCaM consists of 739 bp and codes for 149 amino acid residues (molecular mass of 16.8 kDa). The deduced pcCaM protein sequence matched most closely with published CaM sequences from human CaM (96.5%) to pearl-oyster (97.9%) and killfish (98.5%). The real-time PCR analysis confirmed that pcCaM mRNA was ubiquitously expressed in all tissues tested (gill, hepatopancreas, antennal gland, axial abdominal and cardiac muscle); however it was most abundant in muscles. The real-time PCR analysis also revealed that pcCaM mRNA expression was upregulated in pre/postmolt stages as compared with intermolt expression in both epithelial and in non-epithelial tissues. The expression of pcCaM paralleled our previous findings in the trends for plasma membrane calcium ATPase (PMCA) during molting of crayfish, suggesting that their cellular function in regulating intracellular Ca2+ is linked. PMCA is thought to be regulated by calmodulin. Colocalization study of PMCA and CaM protein in tail muscle with confocal microscopy techniques confirmed the correlation of these two proteins in cellular calcium metabolism in crayfish. (Supported by Grant IBN 0445202 to MGW, YG and CMG).