Never conclude with a negative result, explore all possibilities before changing your hypothesis. An interview with Dr. Catherine Figarella, former Director Groupe de Recherche sur les Glandes Exocrines, Faculté de Médecine, Marseille, France; Active Member of the Board of the French Cystic Fibrosis Association: 'Vaincre la Mucoviscidose'. Interview by Martín E. Fernández-Zapico.

Dr. Catherine Figarella is a world expert in the isolation and characterization of human exocrine pancreatic proteins (enzymatic and non-enzymatic ones). She was a pioneer in the identification and characterization of the numerous zymogens present in pancreatic juice. In particular, her discovery of a peculiar behavior of one of the main proteolytic zymogens: human trypsinogen 1, which was more readily activated into active trypsin than human trypsinogen 2 and trypsinogens of other species led her to propose that a premature intracellular activation of this zymogen may play a role in the pathogenesis of chronic pancreatitis. She demonstrated that a similar phenomenon may occur in cystic fibrosis (CF) and has applied this knowledge of pancreatic zymogens to follow the evolution of the pancreatic disease in CF. With this brief but keen biographical article Dr. Figarella shares her life experience as an innovative medical and biochemical investigator of human exocrine pancreatic function.

Mechanism of restriction of normal and cystic fibrosis transmembrane conductance regulator-deficient human tracheal gland cells to adenovirus infection and ad-mediated gene transfer.

CF-KM4 (cystic fibrosis transmebrane conductance regulator-deficient) and MM-39 (healthy) cells, two serous cell lines from submucosal tracheal glands, were found to be poorly susceptible to adenovirus (Ad)5 infection and Ad5-mediated gene transduction. The major limiting steps apparently resided in the primary events of Ad5 interaction, i.e., cell attachment and entry. Both CF-KM4 and MM-39 cells failed to express the Coxsackie-Ad receptor (CAR), and experimental data suggested that alpha[2-->6]-linked sialic acid residues of sialoglycoproteins (SAGP) in CF-KM4 cells, and heparan sulfate glycosaminoglycans (HS-GAG) in MM-39, were used as receptors by Ad5 virions. Ad5 attached to SAGP and HS-GAG receptors via its fiber knob domain, but entered the cells via a penton base- and Arg-Gly-Asp (RGD)-integrin-independent pathway. The block to Ad5-mediated gene transfer in MM-39 and KM4 cells could be overcome by conferring to the vector a novel cell-binding specificity. Thus, Ad5 vectors carrying a stretch of 7-lysine residues genetically inserted at the C-terminus of the fiber knob were found to transduce MM-39 cells with a 10- to 20-fold higher efficiency than the original vectors, but the transduction of CF-KM4 was not significantly improved. Retargeting Ad5 to integrin receptors via RGD peptide ligands, inserted at the extremity of the fiber shaft, resulted in a transducing efficiency of 20- and 50-fold higher in MM-39 and KM4 cells, respectively, compared with Ad5 vectors carrying fibers terminated by their natural knob domain.

Uptake of plasmid/glycosylated polymer complexes and gene transfer efficiency in differentiated airway epithelial cells.

BACKGROUND: We have studied gene transfer efficiency of glycosylated polylysines and glycosylated polyethylenimines as vectors in immortalized differentiated airway gland serous cells and primary cultures of human airway surface epithelial cells. METHODS AND RESULTS: In both cell types, lactosylated PEI was more efficient for gene transfer than unsubstituted PEI and lactosylated polylysine which requires the presence of endosomolytic agents. However, for all the vectors tested, gene transfer efficiency was lower in differentiated cells as compared with poorly differentiated cells. The presence of membrane lectins, i.e. cell surface sugar-specific receptors, was evaluated using fluorescein-conjugated neoglycoproteins and microscopy or flow cytometry. In differentiated airway surface epithelial cells, membrane lectins were not expressed and plasmid DNA/fluorescein-conjugated glycosylated polymer complexes were not incorporated. This accounted in part for the lack of gene transfer efficiency in these cells. In contrast, in differentiated airway gland serous cells, expression of lectins and their endocytotic properties appeared to be similar to that observed in undifferentiated cells, and plasmid DNA/fluorescein-conjugated glycosylated polymer complexes were incorporated in similar amounts by cells in both differentiated states CONCLUSIONS: Glycosylated PEI appears to be a promising gene delivery system since it is more efficient than the sugar-free polymer and does not require endosomolytic agents. However, in differentiated airway gland serous cells, a low gene transfer efficiency was observed that could not be attributed to low expression of membrane lectins or low uptake of glycosylated complexes. An impaired intracellular trafficking of glycosylated complexes in differentiated airway gland serous cells is suggested.

Implication of Reg I in human pancreatic duct-like cells in vivo in the pathological pancreas and in vitro during exocrine dedifferentiation.

A feature associated frequently with the pathologic pancreas is the presence of tubular complexes produced by a phenotypic modulation of acinar cells that take on the characteristics of ductular cells. Since the type I Reg gene, an acinar cell product, is increased in the pancreas following an acinar injury, we aimed to evaluate whether the Reg I protein might be involved in this dedifferentiation process in the human pancreas. We studied duct-like structures in fixed human pathologic pancreatic tissues and human cells with a ductal phenotype obtained by culturing human exocrine preparations. Immunocytochemistry, Western blotting, and RT-PCR were applied for detection of type I Reg. Reg I was observed not only in acinar cells but also in the duct-like cells and dilated duct cells, both positive for cytokeratin 19. However, none of the other acinar markers was observed in these cells. In vitro, human acinar cells dedifferentiated, losing their acinar phenotype, but expression of Reg I remained constant throughout the culture duration. Furthermore, Reg I was not associated with proliferation. We demonstrated that Reg I expression was linked to acinar cell dedifferentiation. We postulate that Reg I might be used as a marker to understand the events leading to phenotypic changes of acinar cells to address the physiological role of Reg I in the pancreas.

Clonamiento y expresión heterologa en saccharomyces cerevisiae de la acuaporina 2 de trypoanosoma cruzy (TcAQP2) / Cloning and heterologous expression in saccharomyces cerevisiae of the aquaporin 2 of trypanosoma cruzi (TcAQ2)

Trypanosoma cruzi es el agente causal de la Enfermedad de Chagas, una entidad endémica no controlada y en crecimiento en Latinoamérica. El parásito, durante su ciclo de vida, se ve sometido a cambios bruscos en la osmolaridad. Tales situaciones de estrés osmótico ameritan sistemas fisiológicos que le permitan a estas células adaptarse y sovrevivir a la nueva situación. En distintos organismos, no de los elementos primordiales que intervienen en osmorregulación son las acuaporinas, proteínas de membrana pertenecientes a la familia MIP ("major intrinsec protein"), que permiten el paso selectivo de agua (acuaparinas clásicas) y otras moléculas no cargadas (acuagliceroporinas) a través de membranas biológicas, en función de gradientes osmóticos. En el genoma de T. cruzi existen cinco genes que presentan similitud con proteínas de la familia MIP. Una de estás ya ha sido caracterizada, TcAQP1 (Trypanosoma cruzi, acuaporina 1), un canal permeable solo al agua. En este artículo de investigación se inició el estudio de un segundo gen con similitud a acuaporina, al cual hemos denominado TcAQP2. El gen TcAQP2 codifica para una proteína de 276 aminoácios. Por medio de herramientas de bioinformática se realizó la predicción de las estructura de dicha proteína; posee características que permiten ubicarla dentro de la familia MIP. Se realizó el clonamiento y secuenciación de la TcAQP2, detectándose diez mutaciones silentes. Este hecho sugiere que se trata de mutaciones propias de la cepa de T. cruzi CL Brener utilizada en el desarrollo experimental de este trabajo. Posteriormente se realizó el subclonamiento de TcAQP2 en el plásmido de expresión en el modelo de levadura pYES.2 y se transformó en las células de saccharomyces cerevisiae. La expresión funcional de la TcAQP2 en dichas células, muestra evidencias de que está proteína interviene en osmorregulación cuando las células son expuestas a choques hiper-osmóticos e hipo-osmóticos...

Trypanosoma cruzi, the etiological agent of changes disease, es prevalent throughout Latin America. During its life cycle, the parasite undergoes extreme fluctuations in osmolarity, consequentrly physiological systems are required to assure its survival. In many organisms one of the more important systems involved in osmoregulation are aquaporins. These proteins are members of the major intrinsic protein family (MIP). Aquaporins can be divided in two groups according to its permeablity: the first one is only permeable to water (orthodox aquiaporins and the second one is permeable to water, glycerol, and other small, uncharged molecules (aquaglyceroporins). In the T cruzi genome there are 5 genes that encode proteins similar to aquaporins. One of these genes (Trypanosoma cruzi aquaporin 1, TcAQp1) has been already characerized as a channel that is only permeable to water. It was also demonstrated that TcAQP1 is involved in parasite osmoregulation. In this work, we started to investigate other gene with aquaporin similarity, which we named Trypanosoma cruzi aquaporin 2 (TcAQP2). The gene TcAQP2 encodes a protein of 276 aminoacids. The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA), which is the classical conserve motif in the MIP family proteins. Cloning and sequencing of the TcAQP2 gene allowed the detection of ten silent mutations in all clones analyzed, as compare to the sequence reported in data bank of the parasite, suggesting that they belong to the CL Brener strain used in this study. In order to analyze the TcAQP2 function, subcloning in the plasmid for heterologous expression in yeast (pYES.2) of this gene was performed. After transformation and functional expression alf TcAQP2 in Saccharomyces cerevisiae, cells were exposed to osmotic stress. The results indicate that this protein is involved in osmoregulation, and suggest that TcAQP2 participate as a channer for water and/or glycerol in...