Parasitisation by Bathycreadium elongatum (Digenea, Opecoelidae) in pyloric caeca of Trachyrincus scabrus (Teleostei, Macrouridae).

A novel process of transmural passive displacement of a digenean parasite was studied in the digestive tract of the roughsnout grenadier Trachyrincus scabrus, which is found in the northwestern Mediterranean Sea. This mechanism seems to facilitate the elimination of a significant portion of intestinal parasites. The digenean parasite Bathycreadium elongatum was found in the intestine, mainly within pyloric caeca, in 74.4% of T. scabrus, with a mean abundance of 44 individuals per fish. Nodule-like lesions were also found in the mesentery of pyloric caeca of infected T. scabrus. Histological sections of the nodules revealed granulomatous inflammatory responses surrounding degraded digeneans. Partial nucleotide sequences of the 28S rRNA gene obtained from intracaecal B. elongatum and from the core of the nodules of the mesentery of pyloric caeca showed 100% mutual identity with an overlap of 971 bp. The greatest abundance of both intracaecal B. elongatum and nodules occurred in spring. During summer, and especially autumn, the abundance of intracaecal B. elongatum decreased. Prevalence and abundance of nodules increased in autumn. In winter intracaecal parasite abundance and prevalence began to increase, but decreased again in nodules. During spring and summer, parasites pass into the visceral cavity, hypothetically owing to the fragility of the wall of pyloric caeca in their apical zone, and become degraded through a granulomatous inflammatory response. This process seems to have a detrimental effect on the B. elongatum cycle since some of parasites are trapped and degrade in the connective tissue in which they are unable to complete their life cycle.

WNT/beta-catenin increases the production of incretins by entero-endocrine cells.

AIMS/HYPOTHESIS: Glucose-dependent insulinotropic peptide (GIP) plays a pivotal role in the regulation of glucose homeostasis. Rates of diet-induced obesity, insulin resistance and type 2 diabetes are decreased when GIP signalling is disturbed in mice, suggesting that GIP plays a role in the onset of type 2 diabetes. WNT signalling is linked to type 2 diabetes and induces synthesis of the other incretin, glucagon-like peptide 1 (GLP-1). GLP-1 analogues improve treatment of type 2 diabetes patients in whom GLP-1 signalling is intact and have captured clinical attention. GIP levels are altered at the onset of type 2 diabetes and later on, while GIP signalling is impaired. Thus, GIP is not a candidate for treatment but might be an important target from a prevention perspective. Hypothesising that hypersecretion of GIP links altered WNT signalling to the onset of type 2 diabetes, we sought to determine whether WNT signalling induces GIP production by entero-endocrine cells. METHODS: RT-PCR and chromatin immunoprecipitation (ChIP) were used to study Gip gene induction. Gip promoter elements mediating WNT/lithium induction were identified (electrophoretic mobility shift assay, co-transfection of deletion mutants, ChIP). RESULTS: Lithium or WNT/beta-catenin signalling enhanced GIP production by entero-endocrine cells through a conserved site in the proximal Gip promoter. Lithium favours lymphoid enhancer factor-1/beta-catenin binding to Gip promoter and diminishes ChIP through T cell factor-4 and histone deacetylase 1. CONCLUSIONS/INTERPRETATION: Lithium and WNT are incretin inducers in general. This work provides a novel link between WNT signalling, obesity and diabetes.

La tiroides como modelo de mecanismos moleculares en enfermedades geneticas / The thyroid as a model for molecular mechanisms in genetic diseases

Las enfermedades tiroideas constituyen una heterogénea colección de anormalidades asociadas a mutaciones en los genes responsables en el desarrollo de la tiroides: factor de transcripción tiroideo 1 (TTF-1), factor de transcripción tiroideo 2 (TTF-2) y PAX8, o en uno de los genes que codifican para las proteínas involucradas en la biosíntesis de hormonas tiroideas como tiroglobulina (TG), tiroperoxidasa (TPO),sistema de generación de peróxido de hidrógeno (DUOX2), cotransportdor de Na/I– (NIS), pendrina (PDS), TSH y receptor de TSH. El hipotiroidismo congénito ocurre con una prevalencia de 1 en 4.000 nacidos. Los pacientes coneste síndrome pueden ser divididos en dos grupos: con hipotiroidismo congénito sin bocio (disembriogénesis) o con bocio (dishormonogénesis). El grupo de disembriogénesis, que corresponde al 85% de los casos, resulta de ectopía,agenesia o hipoplasia. En una minoría de estos pacientes, el hipotiroidismo congénito está asociado con mutaciones en los genes TTF-1, TTF-2, PAX-8, TSH o TSHr. La resencia de bocio congénito (15% de los casos) se ha asociado a mutaciones en los genes NIS, TG, TPO, DUOX2 o PDS. El hipotiroidismo congénito por dishormonogénesis es trasmitido en forma autonómica recesiva. Mutaciones somáticas en el TSHr han sido identificadas en adenomas tiroideos hiperfuncionantes. Otra enfermedad tiroidea bien establecida es la resistencia a hormonas tiroideas(RTH). Es un síndrome de reducida respuesta tisular a la acción hormonal causado por mutaciones localizadas en el gen del receptor de hormonas tiroideas (TR). Mutantes de TRinterfieren con la función del receptor normal por un mecanismo de dominancia negativa. En conclusión, la identificación de mutaciones en los genes de expresión tiroidea ha permitido un mayor entendimiento sobre la relación estructura-función de los mismos. La tiroides constituye un excelente modelo para el estudio molecular de las enfermedades genéticas.

La tiroides como modelo de mecanismos moleculares en enfermedades geneticas / The thyroid as a model for molecular mechanisms in genetic diseases

Las enfermedades tiroideas constituyen una heterogénea colección de anormalidades asociadas a mutaciones en los genes responsables en el desarrollo de la tiroides: factor de transcripción tiroideo 1 (TTF-1), factor de transcripción tiroideo 2 (TTF-2) y PAX8, o en uno de los genes que codifican para las proteínas involucradas en la biosíntesis de hormonas tiroideas como tiroglobulina (TG), tiroperoxidasa (TPO),sistema de generación de peróxido de hidrógeno (DUOX2), cotransportdor de Na/I¹ (NIS), pendrina (PDS), TSH y receptor de TSH. El hipotiroidismo congénito ocurre con una prevalencia de 1 en 4.000 nacidos. Los pacientes coneste síndrome pueden ser divididos en dos grupos: con hipotiroidismo congénito sin bocio (disembriogénesis) o con bocio (dishormonogénesis). El grupo de disembriogénesis, que corresponde al 85% de los casos, resulta de ectopía,agenesia o hipoplasia. En una minoría de estos pacientes, el hipotiroidismo congénito está asociado con mutaciones en los genes TTF-1, TTF-2, PAX-8, TSH o TSHr. La resencia de bocio congénito (15% de los casos) se ha asociado a mutaciones en los genes NIS, TG, TPO, DUOX2 o PDS. El hipotiroidismo congénito por dishormonogénesis es trasmitido en forma autonómica recesiva. Mutaciones somáticas en el TSHr han sido identificadas en adenomas tiroideos hiperfuncionantes. Otra enfermedad tiroidea bien establecida es la resistencia a hormonas tiroideas(RTH). Es un síndrome de reducida respuesta tisular a la acción hormonal causado por mutaciones localizadas en el gen del receptor de hormonas tiroideas (TR). Mutantes de TRinterfieren con la función del receptor normal por un mecanismo de dominancia negativa. En conclusión, la identificación de mutaciones en los genes de expresión tiroidea ha permitido un mayor entendimiento sobre la relación estructura-función de los mismos. La tiroides constituye un excelente modelo para el estudio molecular de las enfermedades genéticas. (AU)

Genomic organization of the human thyroglobulin gene: the complete intron-exon structure.

OBJECTIVE: In order to complete the knowledge of the genomic organization of the human thyroglobulin gene, the present work was designed to establish the intron-exon organization from exon 24 to exon 35 and to construct a more complete physical map of the gene. DESIGN: Screening of two genomic libraries, and subsequent restriction mapping, hybridization and sequencing were used to characterize the recombinant phages. METHODS: Two human genomic DNA libraries were screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. The Long PCR method was used to amplify the genomic DNA region containing exon 24. Intron-exon junction sequences were determined by using the Taq polymerase-based chain termination method. RESULTS: We isolated and characterized five lambda phage clones that include nucleotides 4933 to 6262 of the thyroglobulin mRNA, encompassing exons 25-35 of the gene. The remaining exon 24 (nucleotides 4817-4932) was sequenced from the amplified fragment. In total, 8010 intronic bases were analyzed. CONCLUSIONS: The present study shows that the five phages isolated and the amplified fragment include 59.4 kb genomic DNA, covering 1446 nucleotides of exonic sequence distributed over 12 exons, from exon 24 to exon 35. Using previous studies and our current data, 220 kb of the human thyroglobulin gene was analyzed, a physical map was constructed, and all exon-intron junctions were sequenced and correlated with the different domains of the protein. In summary, the thyroglobulin gene contains 48 exons ranging in size from 63 nucleotides to 1101 nucleotides.

Congenital goiter with hypothyroidism caused by a 5' splice site mutation in the thyroglobulin gene.

In this work we have extended our initial molecular studies of a consanguineous family with two affected goitrous siblings (H.S.N. and Ac.S.N.) with defective thyroglobulin (Tg) synthesis and secretion because of a homozygotic deletion of a fragment of 138 nucleotides (nt) in the central region of the Tg mRNA, identified previously in H.S.N. In order to identify the intron/exon boundaries and to analyze the regions responsible for pre-mRNA processing corresponding to a 138 nt deletion, we performed a screening of a human genomic library. The intron/exon junction sequences were determined from one positive clone by sequencing both strands of the DNA template. The results showed that the deletion mapped between positions 5549 and 5686 of the Tg mRNA and corresponded to exon 30. The positions of the exon limits differed by three nucleotides from the previously reported data obtained from direct sequencing of the deleted reverse transcriptase-polymerase chain reaction fragment from H.S.N. These variations are because the intron/exon junctions in this region were not available at the time when the deletion was first described. The deletion does not affect the reading frame of the resulting mRNA and is potentially fully translatable into a Tg polypeptide chain that is shortened by 46 residues. The same 138 nt deletion was observed in reverse transcriptase-polymerase chain reaction studies performed in the thyroid tissues from Ac.S.N. Genomic DNA analysis showed that a G to T transversion was observed at position +1 in the donor site of intron 30. Both affected patients (H.S.N. and Ac.S.N.) are homozygous for the mutation whereas the normal sister (At.S.N.) had a normal allele pattern. The functional consequences of the deletion are related to structural changes in the protein molecule that either could modify the normal routing of the translation product through the membrane system of the cell or could impair the coupling reaction. Probably the mutant Tg polypeptide might be functionally active in the production of thyroid hormone, because in the presence of a normal iodine ingestion (approximately 150 microg/day), Ac.S.N. was able to maintain normal serum levels of total triiodothyronine (T3) associated with relatively low serum total thyroxine (T4) with normal somatic development without signs of brain damage.

Genomic organization of the 5' region of the human thyroglobulin gene.

OBJECTIVE: The purpose of the present work is to establish the intron-exon organization from exon 12 to exon 23 of the human thyroglobulin gene and to construct a physical map of the 5' terminal half of the gene. DESIGN: Screening of a genomic library and subsequent restriction map, hybridization and sequencing methods have been employed to characterize the recombinant positive phages. METHODS: A human genomic DNA library was screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. Intron/exon junction sequences were determined by the Taq polymerase-based chain terminator method. Finally, the thyroglobulin gene was mapped using the Gene Bridge 4 radiation hybrid clone panel. RESULTS: We isolated and characterized four lambda phage clones that include nucleotides 3002 to 4816 of the thyroglobulin mRNA, encompassing exons 12 to 23 of the gene. The exon sizes range between 78 and 219 nucleotides. We found that the GT-AG splicing sequences rule was perfectly respected in all the introns. A total of 7302 intronic bases was analyzed. Hormogenic tyrosine 5 and 1291 are encoded by exons 2 and 18. Also, seven alternative spliced variants are associated with the 5' region. Thyroglobulin gene maps to 5,5 centiRays from the AFMA053XF1 marker, in chromosome 8. CONCLUSIONS: The present study shows that the first 4857 bases of thyroglobulin mRNA are divided into 23 exons and the four phages isolated include 32.6 kb genomic DNA, covering 1815 nucleotides of exonic sequence distributed in 12 exons, from exon 12 to 23.