Soluble calcium-binding proteins (SCBPs) of the earthworm Lumbricus terrestris: possible role as relaxation factors in muscle.

The soluble Ca2+-binding protein (SCBP) from the earthworm Lumbricus terrestris was analyzed with regard to its role as a soluble muscle relaxation factor. The actomyosin ATPase activity was inhibited by the addition of decalcified SCBP as it binds Ca2+ stronger than the regulatory proteins associated with the actomyosin. Competitive 45Ca2+-binding assays with decalcified actomyosin and SCBP showed that 45Ca2+ is first bound to actomyosin and is subsequently taken over by SCBP with increasing incubation time. Ca2+ competition experiments carried out with 45Ca2+ loaded SCBP and fragmented sarcoplasmic reticulum vesicles revealed that 45Ca2+ bound to SCBP can be deprived by the ATP-dependent Ca2+ uptake of the sarcoplasmic reticulum. Furthermore, experiments in a diffusion chamber showed that the addition of SCBP significantly enhances the 45Ca2+ flux in a concentration dependent manner. The amount of the Ca2+ flux increase tends to reach a maximum value of about 70%. With all protein components isolated from the obliquely striated muscle, our in vitro experiments consistently show that SCBP may accelerate muscle relaxation similar as assumed for vertebrate parvalbumin.

Four paralog gelsolin genes are differentially expressed in the earthworm Lumbricus terrestris.

We have identified and characterized four distinct variants of the gelsolin-related protein (EWAM P1-P4) in the earthworm L. terrestris. All of these proteins biochemically qualify as gelsolins since they sever actin filaments in a calcium dependent manner. P1, P2 and P3 are present in the Lumbricus body wall muscle whereas in the gizzard muscle P3 and P4 were found. P1-P4 are encoded by four paralog genes and are differentially expressed in various muscle cell tissues. While the genes for P1 and P2 contain one intron, there was no intron in both P3 and P4 genes. The coding sequences consist of 1104bp (368 amino acids) for P1/P4 and 1101bp (367 amino acids) for P2/P3. Corresponding genes were confirmed by northern blot analysis which revealed three (calculated lengths: 3100, 2300 and 2100 nucleotides) and two (calculated lengths: 2300 and 1700 nucleotides) mRNA transcripts in the body wall and the gizzard, respectively. EWAM mRNA was localized by fluorescence in situ hybridization in the body wall and the gizzard muscle. P1 mRNA was detected in the inner proximal layers of both the circular and longitudinal muscle of the body wall whereas in the gizzard no significant staining was observed for P1. P2-P4 mRNAs were abundant in the outer distal layers of both the circular and the longitudinal muscles of both body wall and gizzard. The differential expression of four paralog gelsolin genes suggests a functional adaptation of different muscle cells with respect to actin filament turnover and modulation of its polymer state.

Isolation and characterization of human CapG expressed and post-translationally modified in Pichia pastoris.

CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification. Ca2+-dependent exposure of hydrophobic sites allowed single step and selective elution from a Phenyl Sepharose™ matrix. 3.5 mg CapG/10 g wet biomass were isolated and showed a Ca2+-sensitive and dose-dependent capping activity of actin in a fluorometric assay. In P. pastoris, CapG is located at actin patches, actin cables and arranges along the budding neck. The proliferation rate and morphology of the yeast cells are not influenced by the interaction of CapG with actin. The modification pattern of human CapG from P. pastoris and human carcinoma cells is highly similar. We validated most of the known post-translational modifications and found three new phosphorylation and nine new acetylation sites by mass spectrometry. The N-terminus is acetylated or truncated. Truncated CapG is not phosphorylated at the residues S10, T212 and S337. First mutagenesis experiments indicate an N-terminal acetylation dependent C-terminal phosphorylation.

Gelsolin in Onychophora and Tardigrada with notes on its variability in the Ecdysozoa.

Rearrangements of the filamentous actin network involve a broad range of actin binding proteins. Among these, the gelsolin proteins sever actin filaments, cap their fast growing end and nucleate actin assembly in a calcium-dependent manner. Here, we focus on the gelsolin of the onychophoran Peripatoides novaezealandiae and the eutardigrade Hypsibius dujardini. From the cDNA of P. novaezealandiae we obtained the complete coding sequence with an open reading frame of 2178bp. It encodes a protein of 726 amino acids with a calculated molecular mass of 82,610.9Da and a pI of 5.57. This sequence is comprised of six segments (S1-S6). However, analysis of data from TardiBase reveals that the gelsolin of the eutardigrade Hypsibius dujardini has only three segments (S1-S3). The coding sequence consist of 1119bp for 373 amino acids with a calculated molecular mass of 42,440.95Da and a pI of 6.17. The Peripatoides and Hypsibius gelsolin revealed both conserved binding motifs for G-actin, F-actin and phosphatidylinositol 4,5-bisphosphate (PIP2), along with a full set of type-1 and type-2 Ca2+-binding sites which could result in the binding of eight and four calcium ions, respectively. Both gelsolin proteins lack a C-terminal latch-helix indicating a more rapid activation in the submicromolar Ca2+ range. We suggest that a gelsolin with three segments was present in the last common ancestor of the ecdysozoan clade Panarthropoda (Onychophora, Tardigrada, Arthropoda), primarily because the gelsolin of all non-Ecdysozoa studied so far (except Chordata) reveals this number of segments. Mapping of our molecular data onto a well-established phylogeny revealed that the number of gelsolin segments does not correlate with the phylogenetic lineage but rather with particular functional demands to alter the kinetics of actin polymerization.

Soluble calcium-binding proteins (SCBPs) of the earthworm Lumbricus terrestris: molecular characterization and localization by FISH in muscle and neuronal tissue.

Soluble calcium-binding proteins (SCBPs) of invertebrates probably serve like their vertebrate counterpart-the parvalbumins-as soluble relaxing factors in muscles. Three SCBP isoforms (SCBP1-3) have been isolated and biochemically characterized in the earthworm Lumbricus terrestris (Huch et al. in J Comp Physiol B 158:325-334, 1988). For SCBP2, we found two isoforms named SCBP2a/2b. Both of them together with SCBP3 are present in the body wall muscle. In the gizzard solely, SCBP2b and no SCBP2a or SCBP3 could be detected. The coding sequences of all three isoforms consist of 534 bp for 178 amino acids and contain four EF-hand motifs, of which the second EF-hands are truncated. Recombinant proteins show heat stability and a Ca2+-dependent mobility shift similar to the native proteins, indicating comparable calcium-binding properties. All three isoforms are encoded by three distinct and differentially expressed genes. The genes for SCBP2a, SCBP2b, and SCBP3 are interrupted by only one intron, inserting at nearly the same positions. Northern blot analysis revealed two mRNA transcripts for SCBP2 of approximately 1250 and 1500 kb and one transcript for SCBP3 of approximately 1250 kb. SCBP mRNA was localized by fluorescent in situ hybridization in the body wall and the gizzard. The distribution of the staining intensities resembles that for the myosin ATPase activity and indicates a correlation between the amount of SCBP and speed of muscle contraction. In addition, SCBP mRNA was localized within the nervous tissue, the cerebral and subesophageal ganglia and the ventral nerve cord.

EF-hand proteins and the regulation of actin-myosin interaction in the eutardigrade Hypsibius klebelsbergi (tardigrada).

Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships.

Involvement of a gelsolin-related protein in spermatogenesis of the earthworm Lumbricus terrestris.

A gelsolin-related protein was isolated from seminal vesicles of the annelid Lumbricus terrestris. Compared with the isoforms of the gelsolin-related protein previously found in the muscle of the annelid body wall, the isolated protein was assigned to the first isoform (EWAM-P1) because of its electrophoretic mobility, chromatographic elution behaviour, immunological cross-reactivity and identical nucleotide sequence of segments obtained by reverse transcription/polymerase chain reaction. Immunofluorescence studies with smear preparations of developing male germ cells revealed characteristic changes of the local distribution of actin and EWAM-P1 during spermatogenesis. These changes were correlated with the developmental transport processes and structural alterations. F-actin, as revealed by rhodamine-phalloidin staining, formed a toroid-shaped structure in cytoplasmic bridges connecting the germ cells to a central cytophore during the developmental stages. An actin antibody reacting with both G- and F-actin demonstrated that actin was concentrated at the proximal and distal parts of the spermatocytes. EWAM-P1 was also localized in these regions, with intense staining in the distal part of spermatocytes and young spermatids in which the Golgi complex and proacrosome resided. The anti-actin antibody further stained the periphery of the nucleus. This staining gradually reduced during sperm maturation and covered about half of the length of the nucleus in elongated spermatids. Co-localization of EWAM with actin implied a functional significance of this gelsolin-related protein for the rearrangement of the actin cytoskeleton during earthworm spermiogenesis.

Effects of Bayofly on specimens of Culicoides species when incubated in hair taken from the feet of previously treated cattle and sheep.

Sheep and cattle were treated with Bayofly pour-on containing 1 g cyfluthrin per 100 ml ready-to-use solution. Seven, 14, 21 and 28 days after treatment, hair was clipped off from the back and feet and mixed for 10-15 s, 30 s, 1 or 2 min with freshly caught Culicoides midges. It was found that the insecticide on hair from the legs--the predominant biting site of midges--had a significant killing effect on Culicoides for 3-4 weeks, even after a short-term contact.

Butox 7.5 pour on: a deltamethrin treatment of sheep and cattle: pilot study of killing effects on Culicoides species (Ceratopogonidae).

Topical treatment (at the neck and along the vertebral column) with deltamethrin (Butox 7.5 pour on) of cattle (30 ml/400-kg body weight) and sheep (10 ml/60-kg body weight) was done to find out, whether the insecticide may reach in a sufficient dosage the legs, which are known to be the main biting site of Culicoides specimens that are the vectors of the recently introduced Bluetongue virus in central Europe. At days 7, 14, 21, 28, and 35 after treatment, some hair was cut off from the legs--close to the claws. Freshly (the night before) caught Culicoides obsoletus specimens were then exposed for 15, 30, 60, or 120 s to such hair and afterwards transferred to a filter paper within plastic Petri dishes to observe their fate. It turned out that even a short contact of 15 s of the Culicoides specimens with deltamethrin-treated hair of cattle or sheep was sufficient to paralyze and kill Culicoides specimens within a reasonable short time even when the hair were cut off at day 28 after treatment. While the results obtained in cattle and sheep were rather similar for days 7 and 14 after treatment, the speed of the killing effect of treated hair of cattle on Culicoides considerably slowed down beginning from day 21 after treatment. However, all the experiments clearly showed that the insecticide deltamethrin may reach the feet of cattle and may kill Culicoides specimens when the product is poured along the vertebral column. Such a treatment may considerably reduce the risk of transmission of the agents of disease. However, in the case of the thick fleece of sheep, the insecticide must be poured directly into the skin to reach full activity.

Repetitive sequences in the ITS1 region of the ribosomal DNA of Tunga penetrans and other flea species (Insecta, Siphonaptera).

Different Tunga penetrans isolates from various hosts obtained from South America (Fortaleza. Brazil) have been studied by nucleotide sequence comparison of the first and the second internal transcribed spacer (ITS1, ITS2) of the ribosomal deoxyribonucleic acid (rDNA) and part of the mitochondrial 16S rDNA. Results show no significant host-dependent sequence differences. No indication for intraindividual and intraspecific polymorphisms could be detected. Comparing the ITS1 spacer region of T. penetrans from South America with that from Africa (Togo, Cameroon), distinct length variations have been observed caused by a repetitive sequence motif of 99 bp. The ITS1 from the South American T. penetrans contain two tandemly repeated copies, whereas four of these units are present in the spacer of the African T. penetrans. The absence of homogenization of these units indicates a recent separation of both populations. However, the different number of repetitions together with two base substitutions put the evolutionary distance of only 135 years as postulated for the transfer of T. penetrans from South America to Africa into question. Repetitive sequences could also be detected within the ITS1 rDNA region of other flea species Ctenocephalides felis, Echidnophaga gallinacea, Pulex irritans, Spilopsyllus cuniculi, and Xenopsylla cheopis. The repeat units with lengths from 10 to 99 bp are arranged in pure tandem or interspersed. The repetitive elements observed in the ITS1 of various flea species may serve as a valuable tool for phylogeographic studies.