Response of the thyroid gland of the lizard Podarcis sicula to endothelin-1

Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET) induced hormonal functions in various tissues. We have studied the interaction of ET-1 with thyroid gland of the lizard Podarcis sicula. The effects of ET-1 administration on the plasma levels of the thyroid hormones (T3 and T4) and TSH were stimulatory. Morphological changes in the thyroid after treatment with ET-1 were also detected: the height of the epithelial cells slightly increased and the apical surface acquired microvilli protruding into the follicular lumen. The colloid filled up the lumen and showed a rich peripheral vacuolation. In conclusion, a modulatory role in the control of the reptilian thyroid gland is suggested for ET-1. This is the first report on the interaction of ET-1 with the thyroid gland of reptilian.     

Association between GM3 and CD4-Ick complex in human peripheral blood lymphocytes

The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56^lck in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56^lck complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-immunoprecipitation experiments revealed that CD4 and p56^lck were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56^lck complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56^lck, we analyzed this association in U937, a CD4+and p56^lck negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58[emsp4 ]kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56^lck. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.     

New approaches to the study of sphingolipid enriched membrane domains: The use of microscopic autoradiography to reveal metabolically tritium labeled sphingolipids in cell cultures

This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture.To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3×10^–7 M [1-³H]sphingosine. [1-³H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-³H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy.Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis.     

The effects of sialoadenectomy and exogenous EGF on taste bud morphology and maintenance

Taste buds on the dorsal tongue surface are continually bathedin saliva rich in epidermal growth factor (EGF). In the followingexperiment, taste bud number and morphology were monitored followingsubmandibular and sublingual salivary gland removal (sialoadenectomy),to determine if EGF plays a role in the maintenance and formationof taste buds. Adult male rats were divided into four groups:sialoadenectomized (SX, n = 4); sialoadenectomized with EGFreplacement (SX + EGF, n = 5); sham-operated (SH, n = 4); andsham-operated with exogenous EGF (SH + EGF, n = 5). After a3 week recovery, SX + EGF and SH + EGF animals were given 50µg/day EGF in their drinking water for 14 days. At day14, saliva was collected, the animals were killed and the presenceof EGF determined by radioligand-binding assay. Tongues wereremoved and histologically examined for the presence and morphologyof taste buds on fungiform and circumvallate papillae, or immunostainedfor the presence of EGF, TGF     

Cyclization of the Urokinase Receptor-Derived Ser-Arg-Ser-Arg-Tyr Peptide Generates a Potent Inhibitor of Trans-Endothelial Migration of Monocytes

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR_88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR_88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC₅₀ value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.     

A GFP-Tagged Gross Deletion on Chromosome 1 Causes Malignant Peripheral Nerve Sheath Tumors and Carcinomas in Zebrafish

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft-tissue sarcomas, characterized by complex karyotypes. The molecular bases of such malignancy are poorly understood and efficient targeted molecular therapies are currently lacking. Here we describe a novel zebrafish model of MPNSTs, represented by the transgenic mutant line Tg(-8.5nkx2.2a:GFP) ^ia2. ia2 homozygous animals displayed embryonic lethality by 72 hpf, while the heterozygotes develop visible tumor masses with high frequency in adulthood. Histological and immunohistochemical examination revealed aggressive tumors with either mesenchymal or epithelial features. The former (54% of the cases) arose either in the abdominal cavity, or as intrathecal/intraspinal lesions and is composed of cytokeratin-negative spindle cells with fascicular/storiform growth pattern consistent with zebrafish MPNSTs. The second histotype was composed by polygonal or elongated cells, immunohistochemically positive for the pan-cytokeratin AE1/AE3. The overall histologic and immunohistochemical features were consistent with a malignant epithelial neoplasm of possible gastrointestinal/pancreatic origin. With an integrated approach, based on microsatellite (VNTR) and STS markers, we showed that ia2 insertion, in Tg(-8.5nkx2.2a:GFP) ^ia2 embryos, is associated with a deletion of 15.2 Mb in the telomeric portion of chromosome 1. Interestingly, among ia2 deleted genes we identified the presence of the 40S ribosomal protein S6 gene that may be one of the possible drivers for the MPNSTs in ia2 mutants. Thanks to the peculiar features of zebrafish as animal model of human cancer (cellular and genomic similarity, transparency and prolificacy) and the GFP tag, the Tg(-8.5nkx2.2a:GFP) ^ia2 line provides a manageable tool to study in vivo with high frequency MPNST biology and genetics, and to identify, in concert with the existing zebrafish MPNST models, conserved relevant mechanisms in zebrafish and human cancer development.     

Structural characterization of heterodimeric laccases from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

The subfamily of POXA3 laccase isoenzymes produced by the fungus Pleurotus ostreatus has been characterized as an example of the complexity and heterogeneity of fungal isoenzyme patterns. Two isoenzymes, POXA3a and POXA3b, were previously purified, exhibiting an unusual heterodimeric structure composed of a large (67 kDa) and a small (18 or 16 kDa) subunit. A unique gene encodes the large subunit of both POXA3a and POXA3b, but alternative splicing produces two variants—differing for an insertion of four amino acids—for each isoenzyme. Two genes encoding POXA3a and POXA3b small subunits have been identified, and the corresponding amino acid sequences show only two amino acid substitutions. The 18- and 16-kDa subunits of both POXA3a and POXA3b differ for N-glycosylation at Asn150 of the 16-kDa subunit. The POXA3 large subunit 3D model allows us to highlight peculiarities of this molecule with respect to the laccases whose 3D structures are known.