215 research outputs found
Rapsodia Io. Petri Cermenati de recta regnorum ac rerumpublicarum administratione ...
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Nfix Induces a Switch in Sox6 Transcriptional Activity to Regulate MyHC-I Expression in Fetal Muscle
No full textSox6 belongs to the Sox gene family and plays a pivotal role in fiber type differentiation, suppressing transcription of slow-fiber-specific genes during fetal development. Here, we show that Sox6 plays opposite roles in MyHC-I regulation, acting as a positive and negative regulator of MyHC-I expression during embryonic and fetal myogenesis, respectively. During embryonic myogenesis, Sox6 positively regulates MyHC-I via transcriptional activation of Mef2C, whereas during fetal myogenesis, Sox6 requires and cooperates with the transcription factor Nfix in repressing MyHC-I expression. Mechanistically, Nfix is necessary for Sox6 binding to the MyHC-I promoter and thus for Sox6 repressive function, revealing a key role for Nfix in driving Sox6 activity. This feature is evolutionarily conserved, since the orthologs Nfixa and Sox6 contribute to repression of the slow-twitch phenotype in zebrafish embryos. These data demonstrate functional cooperation between Sox6 and Nfix in regulating MyHC-I expression during prenatal muscle development.</p
A megalin-like receptor is involved in protein endocytosis by Bombyx mori midgut cells in culture
No full textProtein absorption by the insect midgut currently attracts increasing research efforts, in part fostered by the need to develop efficient strategies for oral delivery of bioinsecticides targeting haemocoelic receptors. Gut absorption of undegraded proteins has been unequivocally demonstrated in a number of insect species in vivo but only recently the mechanism involved has been clarified: we have proved that the isolated midgut of Bombyx mori larvae performs the transepithelial translocation of the model protein albumin by transcytosis.
Single columnar cells in culture, isolated from the larval midgut, represent the best tool to identify the mechanism involved in protein endocytosis, the fist step of the transcytotic process. Mature columnar cells were obtained from the proliferation and differentiation of stem cells detached from B. mori midgut epithelium and maintained in culture. We investigated FITC-albumin internalisation by confocal laser scanning microscopy. The protein uptake was time-dependent and strongly reduced by low temperatures and by metabolic inhibitors. Labelled albumin uptake as a function of increasing protein concentration showed a saturation kinetics and was inhibited by native albumin in a concentration-dependent manner. These data prove that albumin uptake is an active process and indicate that a receptor mediates the internalisation of the protein. The internalisation takes place by clathrin-mediated endocytosis, since two specific inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalised in the intermicrovillar areas of the apical plasma membrane. The integrity of the cytoskeletal organisation is essential for the correct functioning of the endocytic machinery because preincubations with nocodazole or cytochalasin D induced a significant reduction of FITC-albumin uptake. RT-PCR analysis and colocalisation experiments indicated that the receptor involved is a putative homologue of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein (LDL)-receptor family, responsible for the uptake of a variety of molecules, albumin included, in many mammalian epithelial cells. Although the specific role of the putative megalin homologue in protein absorption by the midgut of lepidopteran larvae needs to be further clarified, our study indicates that this receptor is highly conserved during evolution
A megalin-like receptor is involved in protein endocytosis in the midgut of an insect (Bombyx mori, Lepidoptera).
No full textA megalin-like receptor is involved in protein endocytosis in the midgut of an insect (Bombyx mori, Lepidoptera)
No full textThe mechanism responsible for fluorescein isothiocyanate (FITC)-albumin internalization
by columnar cells in culture obtained from the midgut of Bombyx mori larvae was examined by confocal laser scanning microscopy. Protein uptake changed over time, and it appeared to be energy dependent,
since it was strongly reduced by both low temperatures and metabolic inhibitors. Labeled albumin uptake as a function of increasing protein concentration showed a saturation kinetics with a Michaelis constant
value of 2.0 +/- 0.6 microM. These data are compatible with the occurrence of receptor-mediated endocytosis. RT-PCR analysis and colocalization experiments with an anti-megalin primary antibody indicated that
the receptor involved was a putative homolog of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein receptor family, responsible for the uptake of various molecules,albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required Ca2+ for albumin internalization and was inhibited by gentamicin. FITC-albumin internalization
was clathrin mediated, since two inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalized in the intermicrovillar areas of the apical plasma membrane.
The integrity of actin and microtubule organization was essential for the correct functioning of the endocytic machinery
Primary cultures of insect midgut cells : a system to study membrane permeability to proteins and specific tools to enhance permeation
No full textRecent studies have shown that Bombyx mori larval midgut can transport proteins unaltered following the transcellular pathway by transcytosis. In insects, the steps involved in this complex process are still unknown. Recently, this topic attracts increasing research efforts because of the demand of new and efficient strategies for the oral delivery of bioinsecticides targeting haemocoelic receptors. A promising tool to investigate these aspects is represented by midgut cells in culture. We developed a culture of B. mori midgut cells following the procedure of Sadrud-Din et al. (1994; 1996). We analysed the characteristics of the culture, showing that up to 60% of the stem cells isolated from the midgut, differentiated after three weeks in culture into columnar and goblet cells, the two predominant cell types in the epithelium. These cells presented in vitro the same shape, morphology and polarity recorded in vivo, even if their dimensions were slightly reduced. Moreover, cultured cell homogenates displayed aminopeptidase N and alkaline phosphatase activity, proving that these two enzymes, involved in vivo in the intermediate and final digestion of nutrients, are expressed also in vitro. We used this cell culture to assess the specific mechanism involved in the endocytosis and the sequence of intracellular events implicated in the movement of endocytic vesicles, of fluorescein isothiocyanate (FITC)-albumin, a protein absorbed by transcytosis in the midgut of B. mori larvae. We demonstrated that FITC-albumin uptake increased over time and was energy dependent, since it was strongly reduced by both low temperature and metabolic inhibitors. Labelled albumin uptake as a function of increasing protein concentration showed a saturation kinetics and was inhibited by extracellular unlabelled albumin in a concentration dependent manner. These data are compatible with the occurrence of a receptor-mediated endocytosis. FITC-albumin internalization was clathrin mediated, since two inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalised in the intermicrovillar areas of the apical plasma membrane. RT-PCR analysis and colocalisation experiments with an anti-megalin primary antibody indicated that the receptor involved was a putative homologue of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein (LDL)-receptor family, responsible for the uptake of various molecules, albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required Ca2+ for albumin internalisation. Albumin uptake was also inhibited by gentamycin, insulin and transferrin, proteins known to be megalin ligands. We demonstrated also that the integrity of actin and microtubule organisation was essential for the correct functioning of the endocytic machinery. Once internalised, albumin colocalized with early endosomes and lysosomes, suggesting that only a part of the protein is transcytosed, since a conspicuous amount is directed to the degradative pathway.
Searching for enhancers of protein transport, we evaluated the ability of Cell-Penetrating Peptides (CPPs) to cross the intestinal cell membrane delivering a fused model protein. One of the best known and more often used CPPs is Tat, a peptide formed by 11 amino acid residues, that derives from the human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) peptide, Tat-(47-57). Tat-EGFP (Enhanced Green Fluorescent Protein) fusion protein, produced in the laboratory of Prof. Rosa Rao (University of Naples Federico II), was used in the experiments. Tat-EGFP was internalised by columnar cells in culture more efficiently than EGFP alone and its uptake increased over time. The internalisation process appeared to be energy-independent, since it was not reduced by either low temperature or metabolic inhibitors. The ability of Tat to translocate EGFP entering the cell across the brush border membrane was observed from experiments performed in the midgut isolated in a perfusion apparatus and incubated with the fusion protein added to the mucosal side of the epithelium.
Densoviruses (DNVs) are insect parvoviruses which are lethal for several insects at larval stages, including agronomical pests and insects vector-borne diseases. During my permanence at the “Laboratoire de Biologie Intégrative et Virologie” in Montpellier (France), under the supervision of Dr. Mylene Ogliastro, we studied the interaction between Junonia coenia Densovirus (JcDNV) and the permissive host Spodoptera frugiperda. Cultures of S. frugiperda midgut cells were prepared to detect the sites of penetration of JcDNV and to analyse the internalisation mechanism. The virus was unable to infect stem cells and differentiated goblet cells. In columnar cells with well developed microvilli, whether at an early phase of differentiation or fully differentiated, the virus was visible after 10 minutes on the cell surface at both the apical and the basolateral side, and after 30 minutes within the cytoplasm, but never in the nucleus. Virus particles were apparent as spots, suggesting a distribution in intracellular compartments. Virus internalisation appeared to be energy dependent, since it was strongly reduced by low temperature
A morphological and functional characterization of Bombyx mori larval midgut cells in culture
No full textRecent studies have shown that Bombyx mori larval midgut can transport proteins unaltered following the transcellular pathway by transcytosis. The numerous steps involved in this complex process are still unknown in the insect midgut, and a promising tool to elucidate this aspect is the availability of single midgut cells in culture suitable for transport experiments. Mature midgut cells in culture were obtained from stem cells isolated from B. mori larvae cultured in Grace’s medium supplemented with 20-hydroxyecdysone (20-HE) and α-arylphorin. After three weeks, up to 60 % of the cultured cells were differentiated into columnar and goblet cells, the two predominant cell types in the midgut epithelium. These cells presented in vitro the same shape, morphology and polarity recorded in vivo, even if their dimensions were slightly reduced. Columnar cells displayed a well developed cytoskeletal arrangement, with actin filaments highly organized within the thick brush border and distributed in faint filaments in the cell cytoplasm. Microtubules formed a substantial net just beneath the brush border and ran longitudinally from the apical to the basal pole of the cell. Cultured cells homogenates displayed aminopeptidase N and alkaline phosphatase activity, proving that these two enzymes, involved in vivo in the intermediate and final digestion, are expressed also in vitro. The columnar cells differentiated in culture were able to internalize two model proteins with quite different transport rates
Cell penetration and electrophysiological effects of an insect parvovirus in the midgut of Spodoptera frugiperda
No full textDensoviruses (DNV) are insect parvoviruses sharing with that group a non enveloped 20-25 nm icosaedric capsid and a single stranded DNA genome. Since they are lethal for several insects at larval stages, including agronomical pests and insects vector-borne diseases, the question of their use as biopesticides is revisited. As a model, we studied the interaction of Junonia coenia Densovirus (JcDNV) and one permissive host, Spodoptera frugiperda (Lepidoptera, Noctuidae). The larvae get infected by the oral route, ingesting viral particles contaminating food. The success of the infection first depends on the penetration of the virus through the midgut epithelium. In order to study this early step, we performed a cellular and physiological analysis of the entry of a Cy3-labelled JcDNV through S. frugiperda midgut in vitro.
Primary cultures of midgut cells were developed to detect the sites of penetration and the intracellular pathway followed by Cy3-JcDNV. The virus was unable to infect stem cells and differentiated goblet cells, while in incubated columnar cells, whether at an early phase of differentiation or fully differentiated, the virus was visible after 10 minutes in the basolateral membrane and in microvilli, and after 30 minutes within the cytoplasm. Virus particles were apparent as spots, suggesting that they could be distributed in intracellular compartments.
S. frugiperda midguts were then isolated from fifth instar larvae, mounted between Ussing chambers and incubated with JcDNV added to the luminal side of the epithelium. After 10 minutes of exposition, the virus induced a significant decrease of the paracellular electrical resistance, an indication that ions moved more rapidly through the aqueous channels formed by the intercellular junctions. The confocal image of whole-mount midguts, isolated and incubated for 10 minutes with luminal Cy3-JcDNV, clearly showed the presence of the virus in the enterocytes’ cytoplasm
The HMGB protein gene family in zebrafish : evolution and embryonic expression patterns
No full textThe High-Mobility Group Box (HMGB) proteins are highly abundant proteins with both nuclear and extracellular roles in key biological processes. In mammals, three family members are present: HMGB1, HMGB2 and HMGB3. We characterized the HMGB family in zebrafish and report a detailed phylogenetic analysis of HMGB proteins. The B1, B2, and B3 subfamilies are present in cartilaginous fish, bony fish, and tetrapods, while jawless fish sequences emerge as basal to the gene family expansion. Two co-orthologs of each mammalian HMGB gene are present in zebrafish. All six zebrafish hmgb genes are maternally expressed, but huge differences in expression levels exist during embryonic development. The hmgb2a/hmgb2b genes are the most highly expressed, while hmgb3b is expressed at the lowest level. Remarkably, hmgb3 genes are not present in fugu, medaka, Tetraodon and stickleback. Our analysis highlights substantial overlaps, but also subtle differences and specificities in the expression patterns of the zebrafish hmgb genes
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