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1.
Sci Rep ; 11(1): 22913, 2021 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-34824310

RESUMO

Inflammation 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 glucocorticoid receptor and is expressed in the liver of patients with hepatic cirrhosis and hepatocellular carcinoma (HCC). Furthermore, we have quantified the gene expression level of p65 and p65 iso5 in the PBMC of patients affected by SARS-CoV-2 disease. The results showed that in these patients the p65 and p65 iso5 mRNA levels are higher than in healthy subjects. The ability of p65 iso5 to bind dexamethasone and the regulation of the glucocorticoid (GC) response in the opposite way of the wild type improves our knowledge and understanding of the anti-inflammatory response and identifies it as a new therapeutic target to control inflammation and related diseases.


Assuntos
Inflamação/imunologia , Receptores de Glucocorticoides/metabolismo , Fator de Transcrição RelA/metabolismo , Corticosteroides/metabolismo , Adulto , Processamento Alternativo , Animais , COVID-19/imunologia , Carcinoma Hepatocelular/metabolismo , Dexametasona/metabolismo , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Glucocorticoides/metabolismo , Hepatite/metabolismo , Humanos , Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Fígado/metabolismo , Hepatopatias/imunologia , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Isoformas de Proteínas , Receptores de Glucocorticoides/imunologia , SARS-CoV-2/patogenicidade , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/fisiologia
2.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052191

RESUMO

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 α- and ß-tubulin genes have been identified in P. lividus, while no data are available in echinoderms for arginine methyl transferases (PRMT). To evaluate the exploiting of the sea urchin embryo in the field of antiproliferative drug development, we carried out a survey of the expressed α- and ß-tubulin gene sets, together with a comprehensive analysis of the PRMT gene family and of the methylable arginine residues in P. lividus tubulins. Because of their specificities, the sea urchin embryo may represent an interesting tool for dissecting mechanisms of tubulin targeting drug action. Therefore, results herein reported provide evidences supporting the P. lividus embryo as animal system for testing antiproliferative drugs.


Assuntos
Citostáticos/toxicidade , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Ouriços-do-Mar/efeitos dos fármacos , Testes de Toxicidade/métodos , Moduladores de Tubulina/toxicidade , Tubulina (Proteína)/metabolismo , Animais , Embrião não Mamífero/efeitos dos fármacos , Metilação , Processamento de Proteína Pós-Traducional , Ouriços-do-Mar/embriologia
3.
Fish Shellfish Immunol ; 67: 86-94, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28579525

RESUMO

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 with human members of AIF-1 family. The AvAIF-1 transcript was constitutively expressed in all tested tissues of unchallenged sea anemone, suggesting that AvAIF-1 could serve as a general protective factor under normal physiological conditions. Moreover, we profiled the transcriptional activation of AvAIF-1 after challenges with different abiotic/biotic stresses showing induction by warming conditions, heavy metals exposure and immune stimulation. Thus, mechanisms associated to inflammation and immune challenges up-regulated AvAIF-1 mRNA levels. Our results suggest its involvement in the inflammatory processes and immune response of A. viridis.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Anêmonas-do-Mar/genética , Anêmonas-do-Mar/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/química , Evolução Molecular , Filogenia , Anêmonas-do-Mar/classificação , Alinhamento de Sequência
4.
Chemosphere ; 180: 275-284, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28411544

RESUMO

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 microscopy observations and RT-qPCR assays the impact of sulfamethoxazole and cadmium in P. lividus sea urchin embryos. During development, embryos were exposed to sulfamethoxazole amount comparable to that usually used in aquaculture procedures and/or sublethal levels of cadmium chloride. Impairment of development and biomarkers for inflammation, detoxification, metal scavenging and cell death were inspected. Even though treatment with sulfamethoxazole apparently did not affect development, it stimulated a remarkable molecular response to oxidative stress. Moreover, combined exposure seriously compromised development and the defense mechanisms to cadmium were blocked. This study leads to the conclusion that coexposure to sulfamethoxazole and cadmium induces neutralizing effects on sea urchin embryos. Thus, in marine areas nearby aquaculture farms, where sulfamethoxazole discharge represents an important environmental contaminant, cadmium occurrence may alter population dynamics of P. lividus.


Assuntos
Cádmio/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Sulfametoxazol/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Aquicultura , Cloreto de Cádmio/farmacologia , Ouriços-do-Mar/embriologia
5.
Int J Mol Sci ; 18(4)2017 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-28417916

RESUMO

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 development in a cell type-specific manner and in response to various metals. The MT7 transcript is expressed in all tissues, especially in the stomach and in the intestine of the larva, but it is less metal-responsive. In contrast, MT8 is ectodermic and rises only at relatively high metal doses. MT5 and MT6 expression is highly stimulated by metals in the mesenchyme cells. Our results suggest that the P. lividus MT family originated after the speciation events by gene duplications, evolving developmental and environmental sub-functionalization.


Assuntos
Metalotioneína/genética , Família Multigênica , Paracentrotus/classificação , Paracentrotus/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Desenvolvimento Embrionário/genética , Éxons , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ordem dos Genes , Metalotioneína/química , Metais/farmacologia , Modelos Moleculares , Filogenia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
6.
PLoS One ; 12(1): e0170969, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28141828

RESUMO

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), was identified as responsible for proper gene expression. An enhancer role was ascribed to ICR1 and ICR2, while ICR3 exerted a pivotal role in basal expression, restricting Tuba1a expression to the proper territories of the embryo. Additionally, the mutation of the forkhead box consensus sequence binding site in ICR3 prevented Pl-Tuba1a expression.


Assuntos
Cílios/metabolismo , Desenvolvimento Embrionário/genética , Íntrons/genética , Neurogênese/genética , Paracentrotus/genética , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética , Tubulina (Proteína)/genética , Animais , Sítios de Ligação/genética , Sequência Conservada/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Modelos Genéticos , Mutagênese Sítio-Dirigida , Paracentrotus/embriologia , Deleção de Sequência/genética , Especificidade da Espécie , TATA Box/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transgenes , Tubulina (Proteína)/metabolismo
7.
Genome Biol Evol ; 8(4): 1056-71, 2016 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-26957029

RESUMO

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 phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations.


Assuntos
Cnidários/química , Cnidários/genética , Evolução Molecular , Inibidores Teciduais de Metaloproteinases/química , Inibidores Teciduais de Metaloproteinases/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Modelos Moleculares , Filogenia , Conformação Proteica , Alinhamento de Sequência
8.
Cell Stress Chaperones ; 21(3): 535-46, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26939892

RESUMO

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 and a nuclear localization signal, strongly suggesting that PcCNBP was a homolog of vertebrate CNBP. The PcCNBP transcript was constitutively expressed in all tested tissues of unchallenged crayfish, including hepatopancreas, gill, eyestalk, haemocytes, intestine, stomach and cuticle with highest expression in haemocytes, intestine, gills and hepatopancreas. The mRNA expression of PcCNBP in haemocytes was modulated at transcriptional level by different immune challenges, suggesting its involvement in the immune response of P. clarkii during both bacteria and viruses infection.


Assuntos
Sequência de Aminoácidos/genética , Astacoidea/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Animais , DNA Complementar/genética , Expressão Gênica , Hemócitos/metabolismo , Hepatopâncreas/metabolismo , Imunidade Inata/genética , Anotação de Sequência Molecular , Proteínas de Ligação a RNA/metabolismo , Distribuição Tecidual/genética
9.
PLoS One ; 9(9): e105908, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25251681

RESUMO

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 the sHSP genes of N. vectensis which mainly show an intronless architecture. The different genomic organization of sHSPs, the phylogenetic analyses based on protein sequences, and the relationships among Cnidarians, suggest that the A.viridis sHSPs represent the common ancestor from which H. magnipapillata genes directly evolved through segmental genome duplication. Additionally retroposition events may be considered responsible for the divergence of sHSP genes of N. vectensis from A. viridis. Analyses of transcriptional expression profile showed that AvHSP28.6 was constitutively expressed among different tissues from both ectodermal and endodermal layers of the adult sea anemones, under normal physiological conditions and also under different stress condition. Specifically, we profiled the transcriptional activation of AvHSP28.6 after challenges with different abiotic/biotic stresses showing induction by extreme temperatures, heavy metals exposure and immune stimulation. Conversely, no AvHSP27 transcript was detected in such dissected tissues, in adult whole body cDNA library or under stress conditions. Hence, the involvement of AvHSP28.6 gene in the sea anemone defensome is strongly suggested.


Assuntos
Cnidários/genética , Evolução Molecular , Proteínas de Choque Térmico Pequenas/genética , Anêmonas-do-Mar/genética , alfa-Cristalinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cnidários/classificação , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Metais Pesados/farmacologia , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Temperatura
10.
Mar Environ Res ; 93: 70-7, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23962538

RESUMO

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 temperature <27 °C. At 27 °C, the fertilization success was very low (<1%) and all larvae died within 2d. Both temperature and pH had effects on the developmental dynamics. Temperature appeared to modulate the impact of decreasing pH on the % of larvae reaching the pluteus stage leading to a positive effect (faster growth compared to pH 8.2) of low pH at 20 °C, a neutral effect at 24 °C and a negative effect (slower growth) at 26 °C. These results highlight the importance of considering a range of temperatures covering today and the future environmental variability in any experiment aiming at studying the impact of ocean acidification.


Assuntos
Arbacia/crescimento & desenvolvimento , Água do Mar/química , Animais , Arbacia/embriologia , Arbacia/fisiologia , Dióxido de Carbono/química , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização , Concentração de Íons de Hidrogênio , Masculino , Temperatura
11.
Mol Biol Rep ; 40(3): 2157-67, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23212613

RESUMO

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 switched-on in response to cadmium treatment. Moreover, we found that this transcriptional induction is concentration dependent and that the cadmium concentration threshold for the gene activation is distinct for every gene. RT-qPCR experiments showed in fact that, among induced genes, PlMT5 gene is activated at a very low cadmium concentration (0.1 µM) whereas PlMT4 and PlMT6 are activated at intermediate doses (1-10 µM). Differently, PlMT7 and PlMT8 genes increase significantly their expression only in embryos treated with the highest dose (100 µM CdCl2). We found also that, in response to a lethal dose of cadmium (1 µM), only PlMT5 and PlMT6 mRNA levels increased further. These data suggest a hierarchical and orchestrated response of the P. lividus embryo to overcome differential environmental stressors that could interfere with a normal development.


Assuntos
Cádmio/toxicidade , Metalotioneína/genética , Ouriços-do-Mar/efeitos dos fármacos , Ouriços-do-Mar/genética , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Metalotioneína/química , Dados de Sequência Molecular , Hibridização de Ácido Nucleico/métodos , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Ouriços-do-Mar/embriologia , Alinhamento de Sequência , Análise de Sequência de DNA
12.
Mol Biol Rep ; 39(3): 2633-44, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21678058

RESUMO

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 conserved 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' 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 addition, we have identified by genome and EST sequence analysis the S. purpuratus alpha tubulin orthologous gene and we propose a revised annotation of some tubulin family members. Moreover, by computational techniques we delineate at least three putative regulatory regions located both in the upstream region and in the first intron containing putative binding sites for Forkhead and Nkx transcription factor families.


Assuntos
Embrião não Mamífero , Regulação da Expressão Gênica/genética , Proteínas do Tecido Nervoso/genética , Paracentrotus/genética , Regiões Promotoras Genéticas/genética , Tubulina (Proteína)/genética , Animais , Sítios de Ligação/genética , Biologia Computacional , Pegada de DNA , Primers do DNA/genética , Etiquetas de Sequências Expressas , Técnicas de Transferência de Genes , Genes Reporter/genética , Genômica , Proteínas de Fluorescência Verde/metabolismo , Microinjeções , Anotação de Sequência Molecular
13.
Int J Dev Biol ; 55(6): 591-6, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21948706

RESUMO

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 acetylated on H3K9, tri-methylated on H3K4 and not di-methylated on H3K27, RNA pol II can be enrolled. In contrast, we have shown that when chromatin is methylated both on H3K9 (me2/3) and H3K27 (me2) and mono methylated on H3K4 the promoter is not accessible to RNA pol II. Our results suggest that, during P. lividus embryogenesis, both HAT/HDAC and HMT/HDM activities, which are able to regulate accessibility of the PlTalpha2 basal promoter to RNA polymerase II, are coordinately switched-on.


Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Paracentrotus/embriologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Histona Desmetilases/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Histonas/genética , Metilação , Sistema Nervoso/embriologia , Paracentrotus/genética , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo
14.
Mol Med Rep ; 3(2): 341-5, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21472245

RESUMO

Familial hypercholesterolemia (FH) is a genetic disease mainly caused by mutations in the low density lipoprotein receptor (LDL-R) gene. However, FH-like phenotypes may also arise from mutations occurring in other genes, the products of which normally interact with the LDL receptor. Although several FH-associated proteins have been discovered, many FH-like phenotypes cannot be linked to mutations in already characterized genes, suggesting the existence of other genes still to be identified, the mutations of which may be directly linked to the FH disorder. In order to identify new putative LDLr interactors possibly involved in its internalization and/or sorting, the cytoplasmic tail of the receptor was used as 'bait' in a two-hybrid assay. We identified an 85-amino acid protein able to bind the LDLr intracellular domain through the last 14 C-terminal amino acids. The novel protein is probably derived from the translation of an alternative open reading frame of the human MT2A gene.

15.
Anal Biochem ; 385(1): 182-3, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19000895

RESUMO

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.


Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , DNA Fúngico/isolamento & purificação , Dióxido de Silício/química , Genoma Fúngico/genética , Octoxinol/química , Tamanho da Partícula , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética
16.
Int J Mol Med ; 18(3): 449-55, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16865229

RESUMO

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 correlate with its absorption from bloodstream or adjacent cells.


Assuntos
Perfilação da Expressão Gênica/métodos , Glicoproteínas/metabolismo , Hibridização In Situ/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Células Cultivadas , Humanos , Jejuno/metabolismo , Rim/metabolismo , Fígado/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Distribuição Tecidual , beta 2-Glicoproteína I
17.
Int J Mol Med ; 17(3): 539-46, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16465405

RESUMO

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 novel missense mutations (C100Y, C183Y and G440C), two frameshift mutations (g.1162delC in exon 8 and g.2051delC in exon 14) and one mutation (g.2390-1Gright curved arrow A) at splicing acceptor consensus sequences located in intron 16 of the LDL-R gene; the analysis of cDNA of this splicing mutation showed the activation of a cryptic splice site in intron 16 and the binding studies showed a reduction in internalisation of LDL-DIL in the proband's cultured fibroblasts. Moreover, a g.2051delC in exon 14 was identified in the proband of Paraguayan ancestry with clinical features of homozygous FH. The mutation identified in the South American patient represents the first description of a variant in South American patients other than Brazilian FH patients. The 5 mutations identified in the Sicilian patients confirm the heterogeneity of LDL-R gene mutations in Sicily.


Assuntos
Hiperlipoproteinemia Tipo II/etnologia , Hiperlipoproteinemia Tipo II/genética , Mutação/genética , Receptores de LDL/genética , Adulto , Bioensaio , Células Cultivadas , Criança , Pré-Escolar , Análise Mutacional de DNA , Éxons/genética , Humanos , Lipídeos/sangue , Pessoa de Meia-Idade , Paraguai/etnologia , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sicília/etnologia
18.
J Histochem Cytochem ; 51(12): 1581-7, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14623926

RESUMO

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.


Assuntos
Cílios/metabolismo , Proteínas de Choque Térmico/biossíntese , Chaperonas Moleculares/biossíntese , Regeneração , Estresse Fisiológico/metabolismo , Animais , Centrossomo/metabolismo , Cílios/fisiologia , Cílios/ultraestrutura , Técnicas de Cultura , Ectoderma/metabolismo , Eletroforese em Gel Bidimensional , Embrião não Mamífero/metabolismo , Proteínas de Choque Térmico HSP40 , Ouriços-do-Mar
19.
Cell Stress Chaperones ; 8(1): 70-5, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12820656

RESUMO

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.


Assuntos
Cílios/fisiologia , Gástrula/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ouriços-do-Mar/embriologia , Animais , Anticorpos Monoclonais , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Gástrula/enzimologia , Temperatura Alta , Imidazóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação , Piridinas/farmacologia , Transdução de Sinais/fisiologia , Estresse Fisiológico , Proteínas Quinases p38 Ativadas por Mitógeno
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