Non-viral Gene Therapy for Melanoma Using Lysenin from Eisenia Foetida.

Earthworms, long utilized in traditional medicine, serve as a source of inspiration for modern therapeutics. Lysenin, a defensive factor in the coelom fluid of the earthworm Eisenia fetida, has multiple bioactivities. However, the inherent toxicity of Lysenin as a pore-forming protein (PFP) restricts its application in therapy. Here, a gene therapy strategy based on Lysenin for cancer treatment is presented. The formulation consists of polymeric nanoparticles complexed with the plasmid encoding Lysenin. After transfection in vitro, melanoma cells can express Lysenin, resulting in necrosis, autophagy, and immunogenic cell death. The secretory signal peptide alters the intracellular distribution of the expressed product of Lysenin, thereby potentiating its anticancer efficacy. The intratumor injection of Lysenin gene formulation can efficiently kill the transfected melanoma cells and activate the antitumor immune response. Notably, no obvious systemic toxicity is observed during the treatment. Non-viral gene therapy based on Lysenin derived from Eisenia foetida exhibits potential in cancer therapy, which can inspire future cancer therapeutics.

The Protective Effect of Lithium Against Rotenone may be Evolutionarily Conserved: Evidence from Eisenia fetida, a Primitive Animal with a Ganglionic Brain.

Chronic exposure to stress is a non-adaptive situation that is associated with mitochondrial dysfunction and the accumulation of reactive oxygen species (ROS), especially superoxide anion (SA). This accumulation of ROS produces damage-associated molecular patterns (DAMPs), which activate chronic inflammatory states and behavioral changes found in several mood disorders. In a previous study, we observed that an imbalance of SA triggered by rotenone (Ro) exposure caused evolutionarily conserved oxi-inflammatory disturbances and behavioral changes in Eisenia fetida earthworms. These results supported our hypothesis that SA imbalance triggered by Ro exposure could be attenuated by lithium carbonate (LC), which has anti-inflammatory properties. The initial protocol exposed earthworms to Ro (30 nM) and four different LC concentrations. LC at a concentration of 12.85 mg/L decreased SA and nitric oxide (NO) levels and was chosen to perform complementary assays: (1) neuromuscular damage evaluated by optical and scanning electron microscopy (SEM), (2) innate immune inefficiency by analysis of Eisenia spp. extracellular neutrophil traps (eNETs), and (3) behavioral changes. Gene expression was also evaluated involving mitochondrial (COII, ND1), inflammatory (EaTLR, AMP), and neuronal transmission (nAchR α5). LC attenuated the high melanized deposits in the circular musculature, fiber disarrangement, destruction of secretory glands, immune inefficiency, and impulsive behavior pattern triggered by Ro exposure. However, the effects of LC and Ro on gene expression were more heterogeneous. In summary, SA imbalance, potentially associated with mitochondrial dysfunction, appears to be an evolutionary component triggering oxidative, inflammatory, and behavioral changes observed in psychiatric disorders that are inhibited by LC exposure.

A new species of Enchytraeus (Enchytraeidae, Clitellata) from sewage sludge of a plum processing plant in Japan.

Enchytraeus ohtakai sp. nov. (Enchytraeidae, Clitellata, Oligochaeta) was discovered in the organic matter of a wastewater treatment facility of a plums processing plant in Honshu, Japan. The wastewater is characterized by high organic matter content and low salt concentration. Morphological analysis and DNA-sequencing of a fragment of the COI barcoding gene show that the new species belongs to the E. albidus species group. Within this group it differs in: vasa deferentia restricted to XII, preclitellar bundles with mostly three chaetae, postclitellar bundles with two or three, dorsal blood vessel from XII or XIII, spermathecal ectal duct completely glandular. spermatheca with a large diverticulum, accessory sexual glands present in XII, clitellum ventrally almost absent. The individual gene trees of COI analysis recovered this new species as a monophyletic group within the genus Enchytraeus, closely related to E. albidus species group.

A rapid and simple signature peptides-based method for species authentication of three main commercial Pheretima.

Pheretima with various activities is a commonly used animal-derived traditional medicine in Asia countries. However, almost half of them are non-pharmacopoeia species in the market due to the similar morphological characteristics between medicinal and non-medicinal species. This study aims to establish an effective method based on signature peptides for species authentication of three main commercial Pheretima, including two major Pheretima species (Amynthas aspergillum, Metaphire vulgaris) and one main adulteration (Metaphire magna). Firstly, the species of 52 batches of commercial Pheretima were authenticated based on DNA barcodes. Secondly, proteomic analysis was performed for protein characterization of three main commercial Pheretima. Furthermore, their signature peptides were screened and validated using ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, a simplified sample processing method was developed. Finally, large quantities of commercial Pheretima samples were analyzed for further verifying the feasibility of the signature peptides-based method. The result showed that the established method had a great application potential for authenticity identification of commercial Pheretima. SIGNIFICANCE: The authenticity assessment of medicinal materials is a main issue in the quality control process as deceptive practices could imply severe health risks. In this study, a rapid and simple method based on signature peptides was established for species authentication of three main commercial Pheretima, which can be an effective alternative to complex DNA barcoding and difficult morphological identification, and provided a reference for improvement of Pheretima quality standards.

Marker peptide screening and species-specific authentication of Pheretima using proteomics.

Pheretima is a common and valuable animal-derived medication used in traditional Chinese medicine. There are four species of Pheretima specified in the Chinese Pharmacopoeia (2015 edition), i.e. Pheretima aspergillum, P. vulgaris, P. guillelmi, and P. pectinifera. A recent report revealed ~ 55% of Pheretima in the commercial marketplace may be adulterated by other species, contrary to the Pharmacopoeia standard. The safety, efficacy, and authenticity of Pheretima is an important issue. Currently, the availability of specific quality-markers for the various species and effective identification methods are still limited. In this study, label-free quantification proteomics of species from Pheretima and Amynthas was carried out using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS), and marker peptides were identified based on their ion intensities using multivariate data analysis (principal component analysis and supervised partial least-squares discriminant analysis). A total of 48,476 peptides with high confidence corresponding to 13,397 proteins were identified from all samples. The marker peptides were validated by comparison with synthetic peptide reference standards using LC-MS/MS operating in a multiple-reaction monitoring mode. A multiple-peptide identification strategy was proposed for the authentication of Pheretima and subsequently applied to samples obtained from retail outlets in various regions of China. The results showed that eight out of the 15 samples tested were deemed authentic Pheretima.

Combination of c oxidase subunit I based deoxyribonucleic acid barcoding and HPLC techniques for the identification and quality evaluation of Pheretima aspergillum.

This study aimed to identify Pheretima aspergillum (Guang-Pheretima) and its adulterants using the cytochrome c oxidase subunit I based deoxyribonucleic acid barcoding technology, and further to evaluate their quality using an optimized high-performance liquid chromatography method. For deoxyribonucleic acid barcoding identification, the Kimura-2-Parameter model was used to analyze genetic distance, and phylogenetic neighbor-joining tree was constructed for species identification of 20 labeled Guang-Pheretima samples. A high-performance liquid chromatography method was developed for the simultaneous determination of seven nucleoside components for quality evaluation. Compared with the GenBank database, 10 samples were identified as real Guang-Pheretima (P. aspergillum), and the others as the adulterants-Metaphire magna. The maximum intraspecific genetic distances of c oxidase subunit I sequence for P. aspergillum were smaller than the minimum interspecific genetic distances between P. aspergillum and M. magna. Ten P. aspergillum and 10 M. magna samples were clearly clustered in the neighbor-joining tree. The contents of seven nucleosides components in P. aspergillum were significantly higher than that in its adulterant-M. magna. The incidence of adulterants for Guang-Pheretima was high (up to 50%) with an alarming quality. This study provided a powerful idea for the quality evaluation of other highly valuable plant- or animal-derived products for safety concerns to avoid misidentification.

Ecotoxicological effects of petroleum-contaminated soil on the earthworm Eisenia fetida.

Petroleum is an important industrial raw material that enters the soil during production and use and is harmful to soil organisms. To evaluate the toxicity of petroleum-contaminated soil, earthworms (Eisenia fetida) were used as model organisms for soil ecotoxicity studies. We found that earthworm weight and cocoon production decreased significantly after exposure to petroleum-contaminated soil. In addition, soil contaminated with high concentrations of petroleum can cause damage to the DNA within earthworm seminal vesicles. Superoxide dismutase (SOD), catalase, and peroxidase activities were significantly inhibited when earthworms were exposed to petroleum-contaminated soil, indicating that oxidative stress was induced by petroleum pollutants. The mRNA levels of annetocin precursor, a reproduction-related gene, was significantly inhibited after petroleum exposure. The mRNA levels of translationally controlled tumour protein (TCTP) and SOD exhibited a concentration-dependent relationship, and their relative expression increased with petroleum concentration.

Transcriptome analysis of genes expressed in the earthworm Eisenia fetida in response to cadmium exposure.

Eisenia fetida earthworm is an ecotoxicologically important test species to monitor various pollutants. However, there is a little knowledge about the effects of cadmium (Cd) on earthworms at the transcriptional level. Firstly, we exposed E. fetida to soils supplemented with different concentrations (10, 30, 60 mg/kg soil) of Cd. Moreover, we depicted the characterization of gene expressions with E. fetida using high-throughput profiling of gene expression. In addition, a comparison of the gene expression profiles between each Cd treatment group and the control group suggested that differential expressional genes (DEGs) mainly enriched in enzyme activity, metabolism, oxidative stress, regeneration and apoptosis pathways. 8 DEGs from these pathways had been selected randomly to confirm the data of RNA-seq. Among these DEGs, six genes (metallothionein-2, phytochelatin synthase 1a, CuZn superoxide dismutase, sex determining region Y-box 2, sex determining region Y-box 4b, TP53-regulated inhibitor of apoptosis 1-like) up-regulated and 2 genes (beta-1,4-endoglucanase, apoptosis-stimulating of p53 protein 2-like) down-regulated in response to Cd exposure. The alteration of them indicated that earthworms could reduce the toxicity and bioavailability of Cd in polluted soil ecosystems through different pathways. This work lays an important foundation for linking earthworm transcriptional level with the ecological risk of Cd in soil ecosystem.

Transcriptomic-proteomics-anticoagulant bioactivity integrated study of Pheretima guillemi.

ETHNOPHARMACOLOGICAL RELEVANCE: Earthworms, a type of animal drugs from traditional Chinese medicine, have been used to treat coagulation for many years with less adverse effects and similar anticoagulant effects compared to the commonly used anticoagulants. There are four species of earthworms recorded in Chinese Pharmacopoeia, while few of them were studied and deficient information were involved in the NCBI and UniProt earthworm protein database. We have adopted a transcriptomic-proteomics-anticoagulant bioactivity integrated approach to investigate a seldom-studied Chinese Pharmacopoeia recorded species, Pheretima guillelmi. AIM OF THE STUDY: In the present study, we aimed to reveal the anticoagulant bioactivity of Pheretima guillelmi, and identify its functional proteins via LC-MS/MS-transcriptome cross identification. METHODS AND RESULTS: With the aid of fibrinogen-thrombin time assay, Pheretima guillelmi was found to possess strong anticoagulant activity, and the bioactivity was quite stable under 30-50 °C and near-neutral conditions. A comprehensive non-reference transcriptome assembly of P. guillelmi was first established to supplement the currently inadequate earthworm protein database and to illustrate the active proteins. Illumina RNA sequencing generated 25,931,175 of clean reads with over 97% high-quality clean reads (Q20) and assembled an average of 133,228 of transcript and 106,717 of unigenes. A total of 11,259 coding sequences were predicted via ESTScan (3.0.3). The P. guillelmi unigenes were searched and annotated against public database. The bioactive proteins in P. guillelmi were with broad distribution of molecular weight. With bottom-up proteomics analysis, ten proteins were identified against UniProt and NCBI earthworm database; and 31 proteins with high-confidence were matched against transcriptomic established P. guillelmi database. CONCLUSION: This study illuminated the therapeutic potency of P. guillelmi for antithrombus and provide a new strategy to investigate animal drugs of Chinese materia medica.

Complete mitochondrial genome of an Amynthas earthworm, Amynthas aspergillus (Oligochaeta: Megascolecidae).

We have determined the mitochondrial genome of the first Amynthas earthworm, Amynthas aspergillus (Perrier, 1872), which is a natural medical resource in Chinese traditional medicine. Its mitogenome is 15,115 bp in length containing 37 genes with the same contents and order as other sequenced earthworms. All genes are encoded by the same strand, all 13 PCGs use ATG as start codon. The content of A + T is 63.04% for A. aspergillus (33.41% A, 29.63% T, 14.56% G and 22.41% C). The complete mitochondrial genomes of A. aspergillus would be useful for the reconstruction of Oligochaeta polygenetic relationships.

Toxicological effects of soil contaminated with spirotetramat to the earthworm Eisenia fetida.

The aim of this study was to evaluate the potential toxicity of spirotetramat to the earthworm Eisenia fetida in a natural soil environment. Many biochemical markers, viz., superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione S-transferase (GST), cellulase, and malondialdehyde (MDA) contents were measured after exposure to 0.25, 1.25, and 2.5mgkg(-1) for 2, 7, 14, 21, and 28days. In addition, the comet assay was performed on earthworm coelomocytes to assess the level of genetic damage. The results demonstrate that the SOD activity and MDA content were significantly stimulated by the highest dose (2.5mgkg(-1)) of spirotetramat for the entire period of exposure. The activities of CAT and POD increased significantly by 2d and 21d, respectively, but the activities of both were significantly inhibited after prolonged exposure (28d). After an initial increase on the 2nd day, the cellulase activity in the high-dose treatment group was significantly inhibited for the entire remaining exposure period. The comet assay results demonstrate that spirotetramat (⩽2.5mgkg(-1)) can induce low and intermediate degrees of DNA damage in earthworm coelomocytes. The results indicate that spirotetramat may pose potential biochemical and genetic toxicity to earthworms (E. fetida), and this information is helpful for understanding the ecological toxicity of spirotetramat on soil invertebrate organisms.

Multilevel assessment of Cry1Ab Bt-maize straw return affecting the earthworm Eisenia fetida.

Non-target effects of two varieties of Bacillus thuringiensis (Bt)-maize straw (5422Bt1 [event Bt11] and 5422CBCL [MON810]) return on the Eisenia fetida were investigated by using multilevel assessments, compared to near-isogenic non-Bt-maize (5422). 5422Bt1 straw return had no deleterious effects on adult earthworms and had significantly positive effects on juveniles over three generations. Negative, no, and positive effects on adults treated with 5422CBCL straw were observed in the 1st, 2nd and 3rd generation, respectively. Negative and positive effects were observed on juveniles produced from the 1st- and 2nd-generation adults treated with 5422CBCL straw, respectively. Glutathione peroxidase activity of earthworms from Bt-maize treatments was significantly higher than that of control on the 90th d. Translationally controlled tumour protein (TCTP) and superoxide dismutase (SOD) genes were down-regulated, while annetocin (ANN) expression was up-regulated in 5422Bt1 treatments. TCTP and SOD genes were up-regulated, while ANN and heat shock protein 70 were down-regulated in E. fetida from 5422CBCL treatments. Enzyme-linked immunosorbent assay revealed that Cry1Ab released from 5422Bt1 and 5422CBCL straw degraded rapidly on the 15th and 30th d and had a slow decline in the rest testing time. Cry1Ab concentrations in the soil, casts and guts of earthworm significantly decreased over the course of the experiment. This study was the first to evaluate generational effects of Bt-maize straw return on earthworms under laboratory conditions. The responses of enzymes activity and genes expression may contribute to better understand above different effects of Bt-maize straw return on earthworms from the 1st generation.

SSH gene expression profile of Eisenia andrei exposed in situ to a naturally contaminated soil from an abandoned uranium mine.

The effects of the exposure of earthworms (Eisenia andrei) to contaminated soil from an abandoned uranium mine, were assessed through gene expression profile evaluation by Suppression Subtractive Hybridization (SSH). Organisms were exposed in situ for 56 days, in containers placed both in a contaminated and in a non-contaminated site (reference). Organisms were sampled after 14 and 56 days of exposure. Results showed that the main physiological functions affected by the exposure to metals and radionuclides were: metabolism, oxireductase activity, redox homeostasis and response to chemical stimulus and stress. The relative expression of NADH dehydrogenase subunit 1 and elongation factor 1 alpha was also affected, since the genes encoding these enzymes were significantly up and down-regulated, after 14 and 56 days of exposure, respectively. Also, an EST with homology for SET oncogene was found to be up-regulated. To the best of our knowledge, this is the first time that this gene was identified in earthworms and thus, further studies are required, to clarify its involvement in the toxicity of metals and radionuclides. Considering the results herein presented, gene expression profiling proved to be a very useful tool to detect earthworms underlying responses to metals and radionuclides exposure, pointing out for the detection and development of potential new biomarkers.

Evaluation of the sensitivity of genotoxicity and cytotoxicity endpoints in earthworms exposed in situ to uranium mining wastes.

Earthworms were exposed for 56 days to a contaminated soil from an abandoned uranium mine and to the natural reference soil LUFA 2.2. The exposure occurred in situ: the containers with contaminated soil were placed near the mine pit; the containers with reference soil were placed in a reference site. For the assessment of metals bioaccumulation, DNA damages, cell-to-cell variation in DNA content, Median Fluorescence Intensity (MFI), coelomocytes frequency and proliferation, organisms were sampled after 0, 1, 2, 7, 14 and 56 days of exposure. For the assessment of radionuclides bioaccumulation, animals were sampled after 0, 14 and 56 days of exposure. As for growth, organisms were sampled after 0, 14, 28 and 56 days of exposure. The reproduction assay was performed according to the OECD (2004) guideline. DNA damages were assessed by comet assay and flow cytometry was used to determine cell-to-cell variation in DNA content, Median Fluorescence Intensity (MFI), coelomocytes frequency and proliferation. Results have shown a myriad of effects in the organisms exposed to the contaminated soil, namely: the inhibition of reproduction, growth reduction, DNA damages, cytotoxicity, changes in eleocytes fluorescence intensity, coelomocytes proliferation and bioaccumulation of metals and radionuclides. Our results showed that the evaluation of genotoxicity and cytotoxicity endpoints, along with other parameters at an individual level in standard reproduction assays conducted in situ, are important to improve the risk assessment process of areas contaminated with uranium and other radioactive mining wastes.

Molecular cloning, characterization of copper/zinc superoxide dismutase and expression analysis of stress-responsive genes from Eisenia fetida against dietary zinc oxide.

The full length cDNA of copper/zinc superoxide dismutase (Cu/Zn-SOD) from Eisenia fetida (E. fetida) was cloned (GenBank accession no. JN579648). Sequence characterization revealed that the cDNA contained characteristic Cu/Zn-SOD family signatures ((45)GFHVHEFGDNT(55) and (138)GNAGGRLACGVI(149)), cysteines (Cys-58 and-146) predicted to form one disulphide bond, Cu-binding (His-47, -49, -64 and -120) and Zn-binding (His-64, -72, -81 and Asp-84). They were essential for the structure and function of Cu/Zn-SOD. Differential expression of stress-responsive genes like Cu/Zn-SOD, catalase (CAT), heat shock protein 70 (Hsp70) and metallothionein (MT) was applied as potential biomarkers to assess their efficacy for the ecotoxicological effects of dietary zinc oxide (ZnO) on E. fetida. The results showed that the expression of Cu/Zn-SOD and MT increased to reach the highest levels of 6.22 and 7.68 fold in a dose-dependent manner at day 10 respectively. The highest expression of 3.03 fold of CAT was registered at day 10. The transient expression of Hsp70 without consistent time- or/and dose-dependent was observed. It implied that the transcriptional patterns of Cu/Zn-SOD, CAT and MT could serve as early warning signals in ecotoxicological assessment of dietary ZnO on earthworms while the expression of Hsp70 was not well done, which is helpful to monitoring and regulation of ZnO in veterinary application.

Molecular characterization of the iron binding protein ferritin in Eisenia andrei earthworms.

Ferritin is a storage protein that plays a key role in iron metabolism. In this study, we report on the sequence characterization of a ferritin-coding cDNA in Eisenia andrei earthworms isolated by RT-PCR using degenerated primers, and we suggest the presence of a putative IRE in the 5'-UTR of ferritin mRNA. The obtained ferritin sequence was compared with those of other animals showing sequence and structure homology in consensus sites, including the iron-responsive element (IRE) and ferroxidase centers. Despite the sequence homology in the E. andrei mRNA of ferritin with the sequences of other animals in consensus IRE sites, the presented cytosine in the IRE of E. andrei ferritin in the expected position does not form a conventional bulge. The presence of ferritin in the coelomic fluid of E. andrei was proven by iron staining assay. Moreover, aconitase activity in the coelomic fluid was assessed by aconitase assay, suggesting the presence of an iron regulatory protein. Quantitative analysis revealed changes in the gene expression levels of ferritin in coelomocytes in response to bacterial challenge, reaching the maximum level 8h after the stimulation with both Gram-positive and Gram-negative bacteria.

Acute toxicity, biochemical and gene expression responses of the earthworm Eisenia fetida exposed to polycyclic musks.

AHTN (Tonalide) and HHCB (galaxolide) are recognized as ubiquitous contaminants in soil and have potential adverse impacts on soil organisms. The aim of this study is to investigate the effects of AHTN and HHCB on the earthworm (Eisenia fetida) as an important soil animal with attention to the acute toxicity, biochemical and transcriptional changes of representative antioxidant enzymatic (SOD, CAT) and stress-response gene (Hsp70). The 48 h-LC(50) value was 20.76 µg cm(-2) for AHTN and 11.87 µg cm(-2) for HHCB respectively in the acute lethal studies. The time-dependent elevation in the level of malondialdehyde (MDA) suggests that reactive oxygen species (ROS)-induced cellular oxidative injury of E. fetida might be one of the main toxic effects of AHTN and HHCB. SOD and CAT were both up-regulated at low exposure dose (0.6 µg cm(-2) AHTN and 0.3 µg cm(-2) HHCB) during 48 h testing period, which protected earthworms from oxidative stresses. However, the down-regulation of SOD and CAT after 48 h exposure to high dose contaminants might be caused by the extreme oxidative stress levels (maximum up-regulation 1.70-fold and 1.40-fold for MDA levels at 6.0 µg cm(-2) AHTN and 3.0 µg cm(-2) HHCB compared to the controls, respectively). The Hsp70 gene expression did not show variation during 48 h, except that it had a significant down-regulation (P<0.05) after 48 h of exposure to high doses of contaminants. These results showed that the dermal contact of AHTN and HHCB could result in pronounced biochemical and physiological responses to earthworms, and the transcriptional level changes in antioxidant genes could be potential molecular biomarkers for the stress of the pollutants.

Characterization of genes expressed in response to cadmium exposure in the earthworm Eisenia fetida using DDRT-PCR.

The transition metal cadmium is a pervasive and persistent environmental contaminant that is both a human toxicant and a carcinogen. To inhibit cadmium-induced damage, cells increase the expression of genes encoding stress-response proteins. The transcription of many stress-responsive genes, including those that encode metallothioneins, glutathione-S-transferases (GSTs) and heat shock proteins have been reported. The aim of this work was to investigate in Eisenia fetida the genes whose expressions are regulated following exposure to cadmium. mRNA differential display reverse transcription-polymerase chain reaction was used to analyze gene expression in E. fetida exposed to 50mg/l cadmium solution. Among the derived cDNA clones sequenced, we found 15 genes up-regulated and 12 down-regulated in response to cadmium exposure. The translated amino acid sequences of eight clones were similar to the Lumbricus terrestris hemoglobin dodecamer, Tribolium castaneum membrane protein, Escherichia coli UMN026 DNA-binding transcriptional activator, Brugia malayi immunoglobulin, Homo sapiens cell growth-inhibiting protein, Apis mellifera poly U binding factor, Escherichia fergusonii copper transporter, and the mRNA that encodes E. coli K-12 cytoplasmic insertase into membrane protein. Five cDNA fragments presented no homology with known gene sequences, suggesting that these sequences may either encode proteins not yet identified or correspond to untranslated regions of mRNA molecules. In-depth functional analyses of these genes are needed to reveal their exact roles.

Cloning, expression, and characterization of cadmium-induced metallothionein-2 from the earthworms Metaphire posthuma and Polypheretima elongata.

In this study we report the sequences of MT-2 cDNA from two species of Megascoleidae earthworms, Metaphire posthuma and Polypheretima elongata, by mRNA differential display after exposure of the organisms to cadmium. Complementary (c)DNA was verified as the MT-2 gene by the characteristics of its predicted translation product, namely a high cysteine content, conserved CXC motifs, and a molecular weight of around 8 kDa. Amino acid sequence alignment revealed a conserved TKCCG in the cloned MT-2 of both megascolecid earthworms instead of the corresponding conserved TQCCG found in lumbricid earthworms. The cDNAs corresponding to the two megascolecid MT-2 genes were expressed, and the MT-2 proteins were purified for biochemical characterization. The binding of Cu2+ exhibited monophasic kinetics and those of Zn2+ and Cd2+ biphasic kinetics. The proteins bound more tightly to Cd2+ than to Zn2+ and more tightly still to Cu2+. Zn-MT and apo-MT were the most effective at scavenging free radicals, followed by Cd-MT. In conclusion, MT-2s from M. posthuma and P. elongata showed unique sequence features compared to those of lumbricid earthworms. These earthworms could be used to evaluate heavy-metal pollution in soil due to the inducible MT-2 by cadmium exposure.

Characterization, molecular cloning and localization of calreticulin in Eisenia fetida earthworms.

Calreticulin is a highly conserved calcium-binding protein affecting many cellular processes inside and outside of the endoplasmic reticulum (ER). It participates in the regulation of Ca(2+) homeostasis, acts as a chaperone and modulates gene transcription, integrin-mediated cell signalling as well as cell adhesion. Here we report on the sequence characterization of a calreticulin-coding cDNA of Eisenia fetida earthworms. The neighbor-joining phylogeny tree constructed based on the deduced amino acid sequence indicates a common origin of the E. fetida calreticulin molecule and that of mollusks. A polyclonal anti-calreticulin antibody used for immunocytochemistry and immunohistochemistry localized the protein in the mesenchymal lining of the coelomic cavity and in coelomocytes of E. fetida. In situ hybridization revealed high expression of E. fetida calreticulin in various cells and tissues, namely epidermis, neurons of the ventral nerve cord, intestine, sperms, body wall muscles and some coelomocytes. Real-time PCR confirmed the strong expression of calreticulin in the nervous system, particularly in cerebral ganglia, in body wall muscles and in seminal vesicles. Moreover, a high calreticulin expression was measured in the muscular pharynx.