HCV viraemia is more important than genotype as a predictor of response to interferon in sicily (Southern Italy)
Abstract Background/Aims: To investigate host- and virus-related factors predictive of early and sustained alanine aminotransferase normalization after interferon therapy for HCV-related chronic liver disease, in an area where genotype 1 is highly prevalent. Methods: We studied 100 patients with HCV-RNA positive chronic liver disease (73 chronic hepatitis and 27 cirrhosis) undergoing alpha-interferon treatment. Thirty-four patients had an early response but relapsed, 15 patients remained into sustained response for at least 12 months after therapy, and 51 patients did not respond. Serum HCV-RNA levels were assessed by bDNA (Chiron), and genotype by LiPA (Innogenetics) and by sequencing of t…
A hybrid expressing genetically engineered major allergens of the Parietaria pollen as a tool for Specific Allergy Vaccination
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 …
Plp38MAPK activity in embryogenesis, morphogenesis and stress response: an indicator of perfect health?
P38 conveys a variety of signals, including conventional growth, migratory and death signals, as well as responding to environmental and mechanical stimuli. These signals induce phosphorylation of p38, which triggers both its translocation to the nucleus and the activation of its catalytic function. In Paracentrotus lividus we have shown that for a correct cilia re-generation the p38 activation is essential; we also showed, by time course Western blotting, two activation peaks during development (morula and early gastrula stages) with a precise localization of activated p38 in micromere and micromere derived cells (Nichel treatment higlighted this feature), and in the oral apical region. Th…
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…
Implication of specific isoforms of p38 in termothollerance and in the response to different stresses in sea urchin embryo
ENGINEERING OF p65(-1)-/- KNOCK-IN MOUSE, A NEW ALTERNATIVE SPLICED ISOFORM OF p65, A PROTEIN OF NF-KB COMPLEX.
Morphological response of the larvae of Arbacia lixula to near-future ocean warming and acidification
The distribution of the sea urchin Arbacia lixula, a warm affinity species, has been expanding in the Mediterranean Sea. To address questions on potential for future success of this species in the region, the thermotolerance of larval development was investigated in context of regional warming. The larvae were reared in present day spawning period (20 °C) and warming conditions (+4 = 24 and +6 = 26 °C). As the calcifying larvae of sea urchins are vulnerable to stunted growth caused by ocean acidification, the impact of lower pH (−0.3 pH units) on larval development was also investigated in combination with warming. Morphological traits of the larvae, post-oral length arms, overall length of…
Fattore anti-lps da Parietaria Judaica
L’endotossina è una tossina microbica, parte integrante della membrana esterna della parete di batteri Gram-negativi che viene rilasciata completamente con la lisi del batterio. È costituita da sub-unita’ molecolari che hanno dimensioni comprese tra 10.000 e 20.000 Daltons, mentre le aggregazioni hanno dimensioni di circa 100.000 Daltons. Un esempio tipico di endotossina è rappresentato dal lipopolisaccaride (LPS) che è presente nella membrana esterna dei batteri Gram-negativi. Le endotossine sono le principali responsabili delle conseguenze cliniche delle infezioni da batteri gram-negativi. Infatti, l’endotossina è ritenuta responsabile della patogenesi della sepsi, dello shock settico e d…
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 new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19
AbstractInflammation is a physiological process whose deregulation causes some diseases including cancer. Nuclear Factor kB (NF-kB) is a family of ubiquitous and inducible transcription factors, in which the p65/p50 heterodimer is the most abundant complex, that play critical roles mainly in inflammation. Glucocorticoid Receptor (GR) is a ligand-activated transcription factor and acts as an anti-inflammatory agent and immunosuppressant. Thus, NF-kB and GR are physiological antagonists in the inflammation process. Here we show that in mice and humans there is a spliced variant of p65, named p65 iso5, which binds the corticosteroid hormone dexamethasone amplifying the effect of the glucocorti…
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
L'allergene maggiore di Parietaria judaica Parj 1.0101 contiene una regione capace di legare LPS e con attività immunomodulatoria
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…
The major allergen of the Parietaria pollen contains an LPS-binding region with immuno-modulatory activity
Background The major allergens in Parietaria pollen, Par j 1 and Par j 2, have been identified as lipid transfer proteins. The family of the Par j 1 allergens is composed of two isoforms, which differ by the presence of a 37 amino acid peptide (Par37) exclusive to the Par j 1.0101 isoform. The goal of this study was to elucidate the biological properties of the Par37 peptide. Methods In silico analysis, spectrofluorimetric experiments and in vitro cell culture assays were used to identify the biological properties of Par37. In addition, a mouse model of sensitization was used to study the influence of Par37 in the murine immune response. Results In silico analysis predicted that Par37 displ…
The operational sex ratio of the sea urchin Paracentrotus lividus populations: the case of the Mediterranean marine protected area of Ustica Island (Tyrrhenian Sea, Italy)
We investigated, with a series of field and laboratory observations, the possible effect of the starfish Marthasteria glacialis predation on the operational sex ratio (OSR), i.e. the number of sexually mature males divided by the total number of sexually mature adults of both sexes at any one time, of the edible sea urchin Paracentrotus lividus. The OSR was estimated three times during the sea urchin summer spawning period (July 2004, June 2005 and July 2006) on barren substrates of Ustica Island Marine Protected Area (Southern Tyrrhenian Sea, Italy). Four sites were selected: two characterized by high M. glacialis density (take zone C) and two controls with low starfish density (no-take zo…
The rise of thermophilic sea urchins and the expansion of barren grounds in the Mediterranean Sea
Recent ecological studies have shown a strong relation between temperature, echinoids and their grazing effects on macro-algal communities. In this study, we speculate that climate warming may result in an increasingly favourable environment for the reproduction and development of the sea urchin Arbacia lixula. The relationship between increased A. lixula density and the extent of barren grounds in the Mediterranean Sea is also discussed.
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).
From allergen back to antigen: a rational approach to new forms of immunitherapy
FROM ALLERGEN BACK TO ANTIGEN: A RATIONAL APPROACH TO NEW FORMS OF IMMUNOTHERAPY
Sea urchin neural genes PlTalfa2 and PlTbeta3: gene transfer analysis.
identificazione del modulo di regolazione spaziale del gene talfa2 in paracentrotus lividus
DNA sequence and pattern of expression of the sea urchin (Paracentrotus lividus) alpha-tubulin genes.
To study the molecular aspects of the regulation of transcription of a multigene family, we have isolated and sequenced cDNA and genomic clones coding for the alpha-tubulin of the sea urchin Paracentrotus lividus. Two cDNA clones, P alpha 10 and P alpha 4, contain respectively the coding information for 391 C-terminal and for 338 N-terminal amino acids of the 452 residues that constitute the complete protein. They show silent nucleotide substitutions only, suggesting that P alpha 10 and P alpha 4 represent the cloned copies of two allelic gene transcripts, which encode for two alpha-tubulin isoforms with identical amino acid sequence in the region of the overlap. The comparison of the predi…
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
Implication of p38 MAPK in skeletogenesis of Paracentrotus lividus embryo
Deciliation: A stressful event for Paracentrotus lividus embryos.
In this report, by using mono- and two-dimensional electrophoretic analysis, we demonstrate that deciliation on sea urchin embryos induces a stress response. Deciliation indeed causes not only the activation of ciliary subroutine, but also a transient decrease of bulk protein synthesis. This decrease is in agreement with our previous results on heat shock response in sea urchin, although deciliation does not induce the expression of the same main hsp set. We were able to characterize one main deciliation-stress protein of 40 kDa whose expression is transiently induced by deciliation and whose localisation is likely to be nuclear.
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…
The early response to sublethal Cadmium exposition of P. lividus embryos: a molecular approach
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…
Effects of temperature rising and ocean acidification on reproductive success in the sea urchin Arbacia lixula (L.)
Sea urchin neural genes PlTalpha2 and PlT beta 3: gene tranfer analysis
Hsp56 expression in Paracentrotus lividus embryos
Chromatographic Characterization of Soluble Collagen in Sea Urchin Embryos (Paracentrotus Lividus)
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.
Cross-reactive allergens of the Parietaria judaica pollen
Different Kind of stress activate differnt isoforms of p38MAPK in sea urchin embryos
Sex ratio operativa della popolazione di Paracentrotus lividus (Lam.) nell’AMP Isola di Ustica
Nell’AMP ”Isola di Ustica” l'instaurazione del regime di protezione, e quindi del divieto di prelievo, ha provocato un aumento della densità del riccio edule Paracentrotus lividus (Lam.) con conseguenze negative sulle comunità algali. L’intenso pascolo di questi echinidi regolari ha infatti trasformato la fascia superficiale sommersa in un barren ground, area a bassa complessità e diversità. Una buona stima della sex ratio operativa (OSR) di P. lividus può chiarire aspetti importanti della dinamica di popolazione di questa specie ed essere di notevole aiuto in una logica di intervento gestionale. Scopo del lavoro è stato quello di valutare la OSR della popolazione di P. lividus presente nel…
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…
p38 MAPK activity during sea urchin embryo development (Paracentrotus lividus)
Characterization of small HSPs from Anemonia viridis reveals insights into molecular evolution of alpha crystallin genes among cnidarians.
Gene family encoding small Heat-Shock Proteins (sHSPs containing α-crystallin domain) are found both in prokaryotic and eukaryotic organisms; however, there is limited knowledge of their evolution. In this study, two small HSP genes termed AvHSP28.6 and AvHSP27, both organized in one intron and two exons, were characterised in the Mediterranean snakelocks anemone Anemonia viridis. The release of the genome sequence of Hydra magnipapillata and Nematostella vectensis enabled a comprehensive study of the molecular evolution of α-crystallin gene family among cnidarians. Most of the H. magnipapillata sHSP genes share the same gene organization described for AvHSP28.6 and AvHSP27, differing from …
Localization of mitochondrial Hsp56 chaperonin during sea urchin development.
We have previously demonstrated that Paracentrotus lividus nuclear genome encodes for the heat shock inducible chaperonin homolog Hsp 56 (1) and that the mature protein is localized in the mitochondrial matrix (2). In this paper we report that constitutive Hsp56 is maternally inherited, in fact it is present in the in unfertilized eggs, and that it has a perinuclear specific localization during cleavage. In the later stages both the constitutive and the heat shock inducible chaperonin has a specific territorial distribution. Moreover following heat shock, the Hsp56 appears in the cytoplasm and in the postmitochondrial supernatant beside the mitochondrial fraction.
Attività immunomodulatoria del peptide PAR37 derivato dall'allergene maggiore di Parietaria judaica ParJ1.0101
A NEW INTERACTOR PARTNER FOR LDL RECEPTOR
Complete decontamination and regeneration of DNA purification silica colum
Silica columns are among the most used DNA purification systems, allowing a good yield of high-quality nucleic acids without organic extractions. Silica column regeneration protocols reported up to now to remove DNA traces are time-consuming, and their effectiveness on genomic DNA has not been demonstrated. Here we report a very rapid regeneration procedure that ensures no DNA carryover, independent of its size, without impairing column efficiency. The method takes advantage of the improved DNA removal by low concentrations of Triton X-100.
Serum hepatitis C virus (HCV)-RNA and response to alpha-interferon in anti-HCV positive chronic hepatitis
Hepatitis C virus (HCV) replication was assessed before and during alpha-interferon (IFN) treatment in 22 anti-HCV positive patients with posttransfusion or sporadic chronic hepatitis (CH). Eleven patients were “responders” and 11 patients “non-responders” to IFN. Thirteen anti-HCV negative healthy subjects and five anti-HCV negative patients with autoimmune CH served as controls. Serum HCV-RNA was detected by the polymerase chain reaction (PCR) in all untreated anti-HCV positive patients but in none of the anti-HCV negative subjects. PCR primers from the 5′-non-coding (NC) region were more sensitive than primers from a non-structural (NS5) region in detecting HCV-RNA (21/22, 95% vs. 7/22, …
Identificazione e caratterizzazione funzionale di isoforme di p65 del complesso NF-kB
Sea urchin deciliation induces thermoresistance and activates the p38 mitogen-activated protein kinase pathway.
In this study, we demonstrate by a variety of approaches (ie, morphological analysis, Western blots, immunolocalization, and the use of specific antibodies) that hyperosmotic deciliation stress of sea urchin embryos induces a thermotolerant response. Deciliation is also able to activate a phosphorylation signaling cascade the effector of which might be the p38 stress-activated protein kinase because we found that the administration of the p38 inhibitor SB203580 to sea urchin deciliated gastrula embryos makes the hyperosmotic deciliation stress lethal.
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 …
Successo di fecondazione di Arbacia lixula: limitazione da sperma e longevità dei gameti
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…
Six novel mutations of the LDL receptor gene in FH kindred of Sicilian and Paraguayan descent
Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in the gene coding for the low density lipoprotein receptor (LDL-R). It is characterized by a high concentration of low density lipoprotein (LDL), which frequently gives rise to premature coronary artery disease. We studied the probands of five FH Sicilian families with 'definite' FH and one proband of Paraguayan descent with homozygous FH who has been treated with an effective living-donor liver transplantation. In order to seek the molecular defect in these six families, we used direct sequencing to define the molecular defects of the LDL-R gene responsible for the disease. We described three…
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…
Arbacia lixula pluteus size is shaped by temperature.
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
HCV NS5A mutations in Europeans infected by genotype 1b.
A hybrid expressing engineered major allergens of the Parietaria pollen as a tool for Specific Allergy Vaccination
Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years
Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria…
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
p38 MAPK activation is required for Paracentrotus lividus skeletogenesis
We investigated the p38 MAPK role during sea urchin, Paracentrotus lividus, development. We found that at the morula stage, before the onset of skeletogenesis, p38 MAPK shows a peak of activity, and we tested whether p38 MAPK activity has any effect on skeletogenesis. By immunohistochemistry on whole-mount embryos we show the preferential localization of the active p38 form both in the presumptive PMCs and bilateral spiculo- genesis centers in control embryos, and in the radialized supernumerary spiculogenesis centers induced by NiCl2 treatment. By using SB203580, a p38 MAPK specific inhibitor, we show that p38 activity is required both for the initial triradiate spicule rudiments formation…
I geni neurali di tubulina nello sviluppo di P. lividus
Analisi funzionale del promotore del gene Talfa2 in Paracentrotus lividus.
Temperature modulates the response of the thermophilous sea urchin Arbacia lixula early life stages to CO2-driven acidification
The increasing abundances of the thermophilous black sea urchin Arbacia lixula in the Mediterranean Sea are attributed to the Western Mediterranean warming. However, few data are available on the potential impact of this warming on A. lixula in combination with other global stressors such as ocean acidification. The aim of this study is to investigate the interactive effects of increased temperature and of decreased pH on fertilization and early development of A. lixula. This was tested using a fully crossed design with four temperatures (20, 24, 26 and 27 °C) and two pH levels (pHNBS 8.2 and 7.9). Temperature and pH had no significant effect on fertilization and larval survival (2d) for te…
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
Different micrococcal nuclease cleavage patterns characterize transcriptionally active and inactive sea-urchin histone genes.
Micrococcal nuclease (MNase) and DNaseI have made a great contribution to our present understanding of the structural organization of the eucaryotic genome [l - 31. The enhanced sensitivity of active portions of the genome to DNaseI gave, in fact, the first indication of differences in the nucleoproteic arrangement of the transcribed as compared to the silent DNA regions [4]. The use of MNase, as a probe of the chromatin organization of specific genes in the active and inactive state, provided additional evidence for this and further showed that the packing of several coding segments of DNA in a regular array of nucleosomal particles is severely but reversibly affected by the transcriptiona…
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 …
Isoforme specifiche di p38 implicate nella termotolleranza e nella risposta a differenti stress nell'embrione di riccio di mare
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 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…
Larval Development of Arbacia lixula (Linneo, 1758) in two Marine Protected Area: preliminary data.
The sea urchin Arbacia lixula is a crucial species in driving the algal assemblages dynamic of the infralittoral zone. A. lixula is considered a thermophilic species and its increase in abundance in western Mediterranean waters, probably is due to an increase in seawater temperature. The aim of this study was to examine the reproductive success of A. lixula by larval development. The study was conducted within two Sicilian MPA (Capo Gallo-Isola delle Femmine and Ustica), characterized by photophilic algae, the same exposure and seawater temperature. In each area, twenty random specimens was sampled from May to September 2010, and mature gametes were collected from six males and six females …
Transcriptional Activity Analysis of p65(-1), a new p65 Isoform of Nf-kb Complex
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.