METALLOTHIONEIN 2A: A POSSIBLE CANDIDATE ABLE TO INTERACT WITH LDL-R CYTOPLASMIC TAIL

caracterization of a 4.6 kb upstream region necessary for the specific alfa2 neural tubulin gene expression in p. lividus embryos

Combined effect of cadmium and sulfonamides on sea urchin development

Echinoderms play a key role in the maintenance of the integrity of the ecosystem where they live. They are constantly exposed to pollutants, particularly in their early planktonic life stages. Sulfamethoxazole with trimethoprim (TMP/SMX) is a fixed antibiotic combination whose concentration is significantly increasing in the coastal waters due to human medicine and also intensive husbandry and aquaculture activities. Previously, we studied the defense strategies activated by P. lividus embryos in response to sublethal doses of CdCl2. Although toxic effects of cadmium on embryo development are not morphologically detectable before 24 hours of exposure, we found upregulation in mRNAs related …

Effects of cadmium exposition on sea urchin development by SSH and RT-qPCR techniques

Cadmium (Cd) is a toxic heavy metal contaminating coastal environments. It has been suggested that the mechanisms of acute Cd toxicity involve the depletion of glutathione and protein-bound sulfhydryl groups, resulting in enhanced production of ROS. Cd-increased ROS in turn produces lipid peroxidation, and results in DNA damage. However, little is known about direct evidence and mechanism for Cd-generated radicals until recently. In order to study the early defense strategies activated by P. lividus 30 hours embryos, in response to exposition to sub-lethal doses of Cd (100μM), we analyzed the induced transcriptome comparing it to that of control embryos by Suppression Subtractive Hybridizat…

HPV in semen: evaluation of the link among infection, sperm parameters and viral genome integration

Human papillomaviruses (HPVs) are agents of the most common sexually transmitted diseases. Both in men and in women, HPVs cause a variety of clinical symptoms ranging from warts to cancer. It has been shown that infection with oncogenic HPV is the major cause of development of cervical cancer in women and less frequently of penile cancers in men. HPV infects epithelial cells and can bind other cell types. Its presence in semen is widely known, but the effects on fertility and reproductive function are still controversial. Partners of women with high-grade squamous intraepithelial lesions were enrolled. From each subject seminal parameters were evaluated. HPV was detected and genotyped both …

RT-PCR and in situ hybridization analysis of apolipoprotein H expression in rat normal tissues

In this study, by using different techniques (i.e. Northern blot hybridization, RT-PCR and Southern blot hybridization) on various normal rat tissues, we were able to identify liver, kidney, heart, small intestine, brain, spleen, stomach and prostate as tissues in which the ApoH gene is transcribed. Moreover, for some of these tissues, by in situ hybridization, we found a specific localization of apoH transcripts. For instance epithelial cells of the bile ducts in liver and of the proximal tubules in kidney are the major sites of apoH synthesis. Our data suggest that some of the different physiological roles proposed for apoH could correlate with its direct expression, while others could co…

Geni Neurali di tubulina, PlTalfa2 e PlTbeta3: studio dei promotori

P38 MAPK expression during development and in stress response in sea urchin embryo

Coexposure to sulfamethoxazole and cadmium impairs development and attenuates transcriptional response in sea urchin embryo

Abstract Among sulfonamides, sulfamethoxazole represents one of the most widely employed. A considerable amount of sulfamethoxazole is introduced into the marine environment after utilization in aquaculture. The cytotoxicity of sulfamethoxazole relies mainly on arylhydroxylamine metabolites and it is associated with the production of reactive oxygen species. Cadmium represents a metal largely employed in several anthropic activities and it is toxic for all living organisms even at low concentrations. Since it is not degraded, cadmium irreversibly accumulates into cells. In order to understand the mechanisms of response to changes in the chemical environment, we investigated by light microsc…

A negative cis-regulatory module restrict Talfa2 tubulin gene expression in the neural territory of the sea urchin embryo.

A metallothionein family member interacts with the intracellular domain of the low density lipoprotein (LDL) receptor

P38 MAPK plays a key role in skeletogenesis of Paracentrotus lividus embryo

Characterization of Translationally Controlled Tumour Protein from the Sea Anemone Anemonia viridis and Transcriptome Wide Identification of Cnidarian Homologues

Gene family encoding translationally controlled tumour protein (TCTP) is defined as highly conserved among organisms; however, there is limited knowledge of non-bilateria. In this study, the first TCTP homologue from anthozoan was characterised in the Mediterranean Sea anemone, Anemonia viridis. The release of the genome sequence of Acropora digitifera, Exaiptasia pallida, Nematostella vectensis and Hydra vulgaris enabled a comprehensive study of the molecular evolution of TCTP family among cnidarians. A comparison among TCTP members from Cnidaria and Bilateria showed conserved intron exon organization, evolutionary conserved TCTP signatures and 3D protein structure. The pattern of mRNA exp…

Metallothionein genes in the sea urchin Paracentrotus lividus

Metallothioneins (MTs) constitute a heterogeneous superfamily of cysteine rich proteins, which coordinate divalent (Zn2+, Cd2+) or monovalent (Cu+) metal ions. Several functions have been proposed for these peptides, ranging from toxic metal protection to physiological metal homeostasis, free radical scavenging, oxidative stress protection, antiapoptotic defense, control of the redox status of the cell and also a role during development. Regarding the MT system in vertebrates’ nearest kin, little information is available at present. Recently MTs were also characterized in cephalochordates. Hence in order to shed some light on MT origin and functional differentiation through evolution, we st…

Cis regulation analysis of the sea urchin neural tubulin gene PlT¬alpha2

INTERACTION OF THE INTRACELLULAR DOMAIN OF THE LOW DENSITY LIPOPROTEIN (LDL) RECPTOR WITH METALLOTHIONEIN2 (MT2).

A Negative Cis-Regulatory Module Induces the Paracentrotus Lividus T?2 Gene Expression in Neurogenic Territory of the Embryo

Sea urchin neural genes PlTalfa2 and PlTbeta3: gene transfer analysis.

identificazione del modulo di regolazione spaziale del gene talfa2 in paracentrotus lividus

Molecular approaches to elucidate the early response of P. lividus embryos to sublethal Cadmium exposition.

Identificazione del modulo di regolazione spaziale del gene Tα2 in Paracentrotus lividus

Il progetto Mentori per la Didattica dell’Università degli Studi di Palermo: l’estensione del numero dei partecipanti, la figura del Mentore Senior e le esperienze di innovazione didattica

L’esperienza del Progetto Mentore (PM) nasce nel 2013 dalla percezione, da parte di diversi docenti dell’Ateneo di Palermo, che l’Accademia italiana stesse abbandonando a sé stessa la funzione didattica, confidando di fatto nelle capacità di ognuno di organizzarsi, aggiornarsi e valutarsi (Felisatti, Scialdone, Cannarozzo, & Pennisi, 2019; Caradonna, Morale, Pace, Scargiali, Scialdone, & Auteri, 2020). Tale impressione era tra le altre cose basata sul fatto che nessuno dei sistemi di valutazione dei docenti universitari (il processo di abilitazione scientifica nazionale, ma anche la stessa valutazione della qualità dei corsi di studio) mettesse enfasi sulla necessità da parte del do…

Implication of p38 MAPK in skeletogenesis of Paracentrotus lividus embryo

The Paracentrotus lividus metallothionein gene family: structure and expression.

Metallothioneins are metal binding proteins that play a pivotal role in metal homeostasis and detoxification. Since their initial discovery, they have been extensively studied in a variety of organisms ranging from microbes to plants and animals. Organisms often possess multiple genes encoding metallothionein homologs with distinct properties, such as varying affinities for different metals, and in many cases different functions. Despite the plethora of available studies, very little information is known about sea urchin P. lividus MT (1, 2). We previously identified five Pl-MT embryonic cDNAs and we studied their induction after cadmium treatment (3). Now we studied their expression during…

Identification of new members of metallothionein gene family in sea urchin

Heavy metals are common marine pollutants that emanate from such sources as industrial and sewage treatment discharges and anti-fouling paints. Cadmium (Cd2+) serves no essential function in biological organisms and it is a highly toxic and carcinogenic metal. In Paracentrotus lividus sea urchin embryos cadmium causes development arrest or severe malformations. At lower doses, cadmium activates different responses (HSPs synthesis, autophagic or apoptotic processes) that can allow embryo survival. This study was conducted to elucidate the gene transcription activation/upregulation or repression/downregulation inducted in sea urchin embryos grown under cadmium stress. Comparison between trans…

Aberrant gene expression profiles in Mediterranean sea urchin reproductive tissues after metal exposures

Abstract Marine organisms are simultaneously exposed to numerous pollutants, among which metals probably represent the most abundant in marine environments. In order to evaluate the effects of metal exposure at molecular level in reproductive tissues, we profiled the sea urchin transcriptional response after non-lethal exposures using pathway-focused mRNA expression analyses. Herein, we show that exposures to relatively high concentrations of both essential and toxic metals hugely affected the gonadic expression of several genes involved in stress-response, detoxification, transcriptional and post-transcriptional regulation, without significant changes in gonadosomatic indices. Even though …

The early response to sublethal Cadmium exposition of P. lividus embryos: a molecular approach

Modulating allergic response by engineering the major Parietaria allergens.

Effects of cadmium exposure on sea urchin development assessed by SSH and RT-qPCR: metallothionein genes and their differential induction

In order to study the defense strategies activated by Paracentrotus lividus embryos in response to sub-lethal doses of CdCl2, we compared the induced transcripts to that of control embryos by suppression subtractive hybridization technique. We isolated five metallothionein (MT) cDNAs and other genes related to detoxification, to signaling pathway components, to oxidative, reductive and conjugative biotransformation, to RNA maturation and protein synthesis. RT-qPCR analysis revealed that two of the five P. lividus MT (PlMT7 and PlMT8) genes appeared to be constitutively expressed and upregulated following cadmium treatment, whereas the other three genes (PlMT4, PlMT5, PlMT6) are specifically…

Isolation and characterization of a Paracentrotus lividus cDNA encoding a stress-inducible chaperonin

Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate–dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution.

Different Kind of stress activate differnt isoforms of p38MAPK in sea urchin embryos

A FOX Binding Site is Necessary for the Activation of Pl-Tuba1a Gene in the neurogenic domains

All echinoderm larvae possess a nervous system consisting of a ciliary band and associated sensory ganglia (apical, oral and lateral ganglia) that controls swimming and feeding. Neurons of the larval nervous system first appear as neuroblasts in the thickened ectoderm of the animal plate (anterior neuroectoderm, ANE) at the late blastula – early gastrula stage and then also in the ciliary band. The neural differentiation process of sea urchin embryos has been analysed and the Gene Regulatory Network involved in the differentiation processes is extensively studied. We have previously isolated an alpha tubulin family member of the sea urchin Paracentrotus lividus (Pl-Tuba1a, formerly known as…

Sea Urchin neural gene PlTalfa2 and PlTbeta 3: gene transfer analysis

An Intronic cis-Regulatory Element Is Crucial for the Alpha Tubulin Pl-Tuba1a Gene Activation in the Ciliary Band and Animal Pole Neurogenic Domains during Sea Urchin Development

In sea urchin development, structures derived from neurogenic territory control the swimming and feeding responses of the pluteus as well as the process of metamorphosis. We have previously isolated an alpha tubulin family member of Paracentrotus lividus (Pl-Tuba1a, formerly known as Pl-Talpha2) that is specifically expressed in the ciliary band and animal pole neurogenic domains of the sea urchin embryo. In order to identify cis-regulatory elements controlling its spatio-temporal expression, we conducted gene transfer experiments, transgene deletions and site specific mutagenesis. Thus, a genomic region of about 2.6 Kb of Pl-Tuba1a, containing four Interspecifically Conserved Regions (ICRs…

A NEW INTERACTOR PARTNER FOR LDL RECEPTOR

Beta-2-glycoprotein I is growth regulated and plays a role as survival factor for hepatocytes

Beta-2-glycoprotein I (beta(2)GPI) is mainly produced by the liver and is found in plasma partially associated to lipoproteins. Although various properties have been attributed to this protein, its physiological role remains still unclear. We investigated its expression in cultured liver cells and in regenerating liver. Expression studies in HepG2 cells demonstrate that beta(2)GPI mRNA is regulated in a cell cycle-dependent manner, with very low expression in low cycling conditions and increasing levels in proliferating cells. p21 WAF-dependent growth arrest, induced by butyrate treatment, down-regulate beta(2)GPI mRNA levels. Immunolocalization in normal rat liver shows a non-homogeneous p…

Geni neurali di Tubulina, PlTalfa2 a PlTbeta 3: studio dei promotori.

Evolutionary conserved mechanisms pervade structure and transcriptional modulation of allograft inflammatory factor-1 from sea anemone Anemonia viridis.

Gene family encoding allograft inflammatory factor-1 (AIF-1) is well conserved among organisms; however, there is limited knowledge in lower organisms. In this study, the first AIF-1 homologue from cnidarians was identified and characterised in the sea anemone Anemonia viridis. The full-length cDNA of AvAIF-1 was of 913 bp with a 5' -untranslated region (UTR) of 148 bp, a 3'-UTR of 315 and an open reading frame (ORF) of 450 bp encoding a polypeptide with149 amino acid residues and predicted molecular weight of about 17 kDa. The predicted protein possesses evolutionary conserved EF hand Ca2+ binding motifs, post-transcriptional modification sites and a 3D structure which can be superimposed …

Gene expression changes after parental exposure to metals in the sea urchin affect timing of genetic programme of embryo development

Simple Summary Intergenerational and transgenerational effects, in which exposure to stressors in a parental generation affects the phenotype of the offspring have been connected to anthropic impacts on biological systems. Therefore, environmental stress experienced inside a generation, particularly during gametogenesis, may lead to erroneous patterns in their offspring just emerging at early developmental stages. In this scenario, the sea urchin embryo represents a suitable model for integrating analyses of gene expression through embryogenesis with developmental alteration induced by environmental stressors. Herein we provide pieces of evidence for the alteration of the gene regulatory ne…

A metallothionein family member interacts with the intracellular domain of the low density lipoprotein (ldl) receptor.

Effetto combinato di cadmio e sulfonammidici sullo sviluppo degli embrioni di riccio di mare: un'indagine molecolare

Chromatin dynamics of the developmentally regulated P. lividus neural alpha tubulin gene

Over 40 years ago, Allfrey and colleagues (1964) suggested that two histone modifications, namely acetylation and methylation, might regulate RNA synthesis. Nowadays it is universally accepted that activation of gene expression strictly depends on enzymatic mechanisms able to dynamically modify chromatin structure. Here, using techniques including DNaseI hypersensitive site analysis, chomatin immunoprecipitation and quantitative PCR analysis, we have analyzed the dynamics of histone post-translation modifications involved in developmentally/spatially controlled activation of the sea urchin PlTalpha2 tubulin gene. We have demonstrated that only when the PlTalpha2 core promoter chromatin is a…

STUDIO DELLA LOCALIZZAZIONE DEL MESSAGGERO DI ApoH

Metallothionein Gene Family in the Sea Urchin Paracentrotus lividus: Gene Structure, Differential Expression and Phylogenetic Analysis

Metallothioneins (MT) are small and cysteine-rich proteins that bind metal ions such as zinc, copper, cadmium, and nickel. In order to shed some light on MT gene structure and evolution, we cloned seven Paracentrotus lividus MT genes, comparing them to Echinodermata and Chordata genes. Moreover, we performed a phylogenetic analysis of 32 MTs from different classes of echinoderms and 13 MTs from the most ancient chordates, highlighting the relationships between them. Since MTs have multiple roles in the cells, we performed RT-qPCR and in situ hybridization experiments to understand better MT functions in sea urchin embryos. Results showed that the expression of MTs is regulated throughout de…

A survey on tubulin and arginine methyltransferase families sheds light on p. lividus embryo as model system for antiproliferative drug development

Tubulins and microtubules (MTs) represent targets for taxane-based chemotherapy. To date, several lines of evidence suggest that effectiveness of compounds binding tubulin often relies on different post-translational modifications on tubulins. Among them, methylation was recently associated to drug resistance mechanisms impairing taxanes binding. The sea urchin is recognized as a research model in several fields including fertilization, embryo development and toxicology. To date, some &alpha

Caracterization of a 4.6 Kb upstream region necessary for the specific a2 neural tubulin gene expression in P. lividus embryos.

A NEGATIVE CIS-REGULATORY MODULE INDUCES THE PARACENTROTUS LIVIDUS Talfa2 GENE EXPRESSION IN NEUROGENIC TERRITORY OF THE EMBRYO

Neural gene transcriptional regulation in sea urchin

Fine characterization of immunological mechanisms mediated by the major allergens of Parietaria judaica and by a hypoallergenic hybrid, rPjEDcys.

Allergy is a hypersensitivity disease IgE-mediated, affecting more than 25% of the population. Actually the only curative treatment of allergies is Allergen-Specific Immunotherapy (SIT). Recombinant hypoallergenic allergen derivatives with reduced allergenic activity have been engineered to reduce side effects during SIT. Parietaria judaica (Pj) pollen contains two major allergens, Par j 1 and expressing disulphide bond variants of Par j 1 and Par j 2, was generated. The aim of this research project is to compare the immunological mechanisms activated by the major allergens of Pj and by rPjEDcys. In vitro analysis suggested that rPjEDcys has a reduced allergenity and maintains T cells react…

I geni neurali di tubulina nello sviluppo di P. lividus

HPV infection in semen: results from a new molecular approach

AbstractHuman papillomavirus (HPV) is the agent of the most common sexually transmitted diseases causing a variety of clinical manifestations ranging from warts to cancer. Oncogenic HPV infection is the major cause of cervical cancer and less frequently of penile cancers. Its presence in semen is widely known, but the effects on fertility are still controversial. We developed a new approach to evaluate virus localisation in the different semen components. We analysed also the specific genotype localisation and viral DNA quantity by qPCR. Results show that HPV DNA can be identified in every fraction of semen: spermatozoa, somatic cells and seminal plasma. Different samples can contain the HP…

Analisi funzionale del promotore del gene Talfa2 in Paracentrotus lividus.

In silico characterization of the neural alpha tubulin gene promoter of the sea urchin embryo Paracentrotus lividus by phylogenetic footprinting

During Paracentrotus lividus sea urchin embryo development one alpha and one beta tubulin genes are expressed specifically in the neural cells and they are early end output of the gene regulatory network that specifies the neural commitment. In this paper we have used a comparative genomics approach to identify con- served regulatory elements in the P. lividus neural alpha tubulin gene. To this purpose, we have first isolated a genomic clone containing the entire gene plus 4.5 Kb of 5 0 upstream sequences. Then, we have shown by gene transfer experiments that its non-coding region drives the spatio- temporal gene expression corresponding substantially to that of the endogenous gene. In addi…

Different kind of stress activate different isoforms of p38 MAPK in sea urchin embryos

Transcriptional regulation of Tubulin α2 gene in Paracentrotus lividus.

The PlTα2 is a sea urchin Paracentrotus lividus gene coding for a neural isotype of alfa tubulin. Expression of Tα2 gene is temporally activated from the blastula stage and spatially confined to the neural territory of the embryo. In order to investigate cis-regulatory elements that control this spatio-temporal pattern we performed functional experiment microinjecting several constructs containing different portion of Tα2 promoter. Moreover quantitative chromatin immune-precipitation experiments were performed to evaluate a possible involvement of chromatin modifications in regulation of Tα2 gene expression.

Sea urchin neural alpha 2 tubulin gene: isolation and promoter analysis

Abstract Expression of Tα2 gene, during sea urchin Paracentrotus lividus development, is spatially and temporally regulated. In order to characterize this gene, we isolated the relevant genomic sequences and scanned the isolated 5 ′ -flanking region in searching for cis -regulatory elements required for proper expression. Gel mobility shift and footprinting assays, as well as reporter gene (CAT and β-gal) expression assays, were used to address cis -regulatory elements involved in regulation. Here we report that an upstream 5 ′ -flanking fragment of PlTα2 gene drives temporal expression of reporter genes congruent with that of endogenous Tα2 gene. The fragment contains cis -elements able to…

Analysis of the effects of co-exposure to antibiotics and cadmium on sea urchin embryos

In order to understand the mechanisms of responses to changes in the physical and chemical environment, as well as the mechanisms of developmental pathways, we investigated by RT-qPCR assays and light microscopy observations the impact of antibiotics and cadmium in P. lividus sea urchin embryos. In particular we inspected development and biomarkers for free radical damage and apoptosis. During development embryos were exposed to an antibiotic mix (Ab mix, usually added to sea urchin cultures) or to sulfamethoxazole/trimethoprim mix (TMP/SMX, usually added to aquacultures) and/or levels of 10-5, 10-4, 10-3 M CdCl2. Even though treatment with TMP/SMX apparently did not affect development, it …

Chromatin dynamics during sea urchin embryogenesis: effects on the neural alpha tubulin PlTa2 gene expression

Expression of PlTa2 gene during sea urchin P. lividus development, is spatially confined to the neural territory and temporally activated from the blastula stage. To evaluate a possible involvement of chromatin modifications in regulation of PlTa2 gene expression we first searched for DNaseI hypersentive sites. We found four sites localized in the introns of the gene, when we used chromatin extracted from embryos at gastrula stage but not from morula stage. This result suggests a possible functional role of the introns in the activation of the expression of PlTa2 gene. Moreover, we used specific antibodies for RNA polymerase II and for different modified form of lysine 9, lysine 27 and lysi…

Expression analysis and chromatin dynamics of tubulin PlT-alpha2 gene in Paracentrotus lividus.

The Repertoire of Tissue Inhibitors of Metalloproteases: Evolution, Regulation of Extracellular Matrix Proteolysis, Engineering and Therapeutic Challenges

Tissue inhibitors of metalloproteases (TIMPs) belong to a fascinating protein family expressed in all Metazoa. They act as regulators of the turnover of the extracellular matrix, and they are consistently involved in essential processes. Herein, we recapitulate the main activities of mammalian TIMPs (TIMP1–4) in the control of extracellular-matrix degradation and pathologies associated with aberrant proteostasis. We delineate the activity of TIMPs in the control of extracellular matrix (ECM) homeostasis and discuss the diversity of TIMPs across metazoans taking into account the emergence of the components of the ECM during evolution. Thus, the TIMP repertoire herein analysed includes the ho…

The nucleic acid-binding protein PcCNBP is transcriptionally regulated during the immune response in red swamp crayfish Procambarus clarkii

Gene family encoding cellular nucleic acid binding proteins (CNBP) is well conserved among vertebrates; however, there is limited knowledge in lower organisms. In this study, a CNBP homolog from the red swamp crayfish Procambarus clarkii was characterised. The full-length cDNA of PcCNBP was of 1257 bp with a 5′-untranslated region (UTR) of 63 bp and a 3′-UTR of 331 bp with a poly (A) tail, and an open-reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids with the predicted molecular weight of about 33 kDa. The predicted protein possesses 7 tandem repeats of 14 amino acids containing the CCHC zinc finger consensus sequence, two RGG-rich single-stranded RNA-binding domain an…

Hsp40 Is Involved in Cilia Regeneration in Sea Urchin Embryos

In a previous paper we demonstrated that, in Paracentrotus lividus embryos, deciliation represents a specific kind of stress that induces an increase in the levels of an acidic protein of about 40 kD (p40). Here we report that deciliation also induces an increase in Hsp40 chaperone levels and enhancement of its ectodermal localization. We suggest that Hsp40 might play a chaperoning role in cilia regeneration.