Ionic, paramagnetic and photophysical properties of a new biohybrid material incorporating copper perchlorate

The sol-gel method was employed in the synthesis of di-urethane cross-linked poly(-caprolactone) (d-PCL(530)/siloxane biohybrid ormolytes incorporating copper perchlorate, (Cu(ClO4)2). The highest ionic conductivity of the d PCL(530)/siloxanenCu(ClO4)2 system is that with n = 10 (1.4 x 10-7 and 1.4 x 10-5 S cm-1, at 25 and 100 ºC, respectively). In an attempt to understand the ionic conductivity/ionic association relationship, we decide to inspect the chemical environment experienced by the Cu2+ ions in the d-PCL(530)/siloxane medium. The observed EPR spectra are typical of isolated monomeric Cu2+ ions in axially distorted sites. The molecular orbital coefficients obtained from the EPR spin Hamiltonian parameters and the optical absorption band suggests that bonding between the Cu2+ and its ligand in the ormolytes are moderately ionic. Investigation by photoluminescence spectroscopy did not evidence or allow selective excitation of transitions corresponding to complexed Cu2+ species.

Protein-based nanodevices for therapeutic applications

Tese de Doutoramento em Biologia Molecular e Ambiental (área de especialização em Biologia Celular e Saúde).

Ultrasound enhances lipase-catalyzed synthesis of poly (ethylene glutarate)

The present work explores the best conditions for the enzymatic synthesis of poly (ethylene glutarate) for the first time. The start-up materials are the liquids; diethyl glutarate and ethylene glycol diacetate, without the need of addition of extra solvent. The reactions are catalyzed by lipase B from Candida antarctica immobilized on glycidyl methacrylate-ter-divinylbenzene-ter-ethylene glycol dimethacrylate at 40 °C during 18 h in water bath with mechanical stirring or 1 h in ultrasonic bath followed by 6 h in vacuum in both the cases for evaporation of ethyl acetate. The application of ultrasound significantly intensified the polyesterification reaction with reduction of the processing time from 24 to 7 h. The same degree of polymerization was obtained for the same enzyme loading in less time of reaction when using the ultrasound treatment. The degree of polymerization for long-term polyesterification was improved approximately 8-fold due to the presence of sonication during the reaction. The highest degree of polymerization achieved was 31, with a monomer conversion of 96.77%. The ultrasound treatment demonstrated to be an effective green approach to intensify the polyesterification reaction with enhanced initial kinetics and high degree of polymerization.

PDMS biofunctionalization study for the development of a microfluidic device: Application to salivary cortisol

The authors acknowledge to Sofia Neves from ICVS for her help in the antibodies selection.

Poly(ester-urethane) scaffolds: effect of structure on properties and osteogenic activity of stem cells

The present study aimed to investigate the effect of structure (design and porosity) on the matrix stiffness and osteogenic activity of stem cells cultured on poly(ester-urethane) (PEU) scaffolds. Different three-dimensional (3D) forms of scaffold were prepared from lysine-based PEU using traditional salt-leaching and advanced bioplotting techniques. The resulting scaffolds were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), mercury porosimetry and mechanical testing. The scaffolds had various pore sizes with different designs, and all were thermally stable up to 300â °C. In vitrotests, carried out using rat bone marrow stem cells (BMSCs) for bone tissue engineering, demonstrated better viability and higher cell proliferation on bioplotted scaffolds compared to salt-leached ones, most probably due to their larger and interconnected pores and stiffer nature, as shown by higher compressive moduli, which were measured by compression testing. Similarly, SEM, von Kossa staining and EDX analyses indicated higher amounts of calcium deposition on bioplotted scaffolds during cell culture. It was concluded that the design with larger interconnected porosity and stiffness has an effect on the osteogenic activity of the stem cells.

Prevalencia de infección por virus hepatitis C, B y VIH en pacientes alcoholistas de la ciudad de Córdoba.

La Hepatitis C y B, junto al alcoholismo, continúan siendo un verdadero problema de Salud Pública. Sin embargo, actualmente no existen datos locales que nos permitan estimar la prevalencia de infección por virus hepatotropos en pacientes alcoholistas, sus genotipos, distrubución geográfica, ni su asociación con determinado tipo de alcoholismo. Además, la co-infección del virus de la inmunodeficiencia humana (VIH) con los virus de hepatitis supone un impacto muy importante desde el punto de vista sanitario y estadístico en nuestro país, ya que alrededor del 50 por ciento de los pacientes VIH positivos presentan dicha coinfección. Es así que, por ser de interés sanitario y por compartir vías de transmisión con el virus de hepatitis B y C, nos parece adecuado estudiar también la presencia de VIH-1 en esta población. Nuestro centro de atención pública (IPAD), atiende a pacientes con trastornos por el consumo de sustancias; deshabitúa y rehabilita alcoholistas y a otros trastornos por consumo. Dichos pacientes, que viven en la ciudad capital, serán evaluados serológicamente para la detección de virus hepatotropos C-B y el VIH. Considerando que en nuestra institución se atiende a un 70 % de Alcoholistas Puros (con un promedio de 7 pacientes nuevos por día), nos resulta importante pesquisar la prevalencia de Virus C, B y VIH en nuestra población de alcoholistas. Toda esta problemática, es la propuesta de mi tesis doctoral. Hipótesis: estimamos encontrar en nuestra población de estudio cifras superiores a la prevalencia de estos virus publicada en bancos de sangre, lo cual se toma como referencia. Objetivos: -Conocer la prevalencia de infección por Virus de Hepatitis C, B y VIH-1 en pacientes alcoholistas de la ciudad de Córdoba, determinar si existe asociación de estos virus con algún tipo de alcoholismo, e identificar genotipos prevalentes y su distribución geográfica en Córdoba. Se incluirán en forma prospectiva y aleatorea, pacientes que concurren por primera vez, de ambos sexos, mayores de 21 años, alcoholistas puros (Gama-Delta-Epsilon de Jellinek). Se confeccionará una ficha, previo consentimiento informado, que permitirá categorizar al "tipo de bebedor". Se les realizará Serología para HCV, Ag HBs (en caso de reactividad se adicionará el Anti HBcore) y VIH. En caso de la positividad serológica, se procederá al frisado de los mismos, para la Genotipificación correspondiente. La recolección, captura y procesamiento de los datos se realizarán en una planilla o ficha. Luego se reubicarán en una base electrónica de datos y se harán los análisis estadísticos de los mismos. Resultados esperados: estimamos encontrar, coincidiendo con la bibliografía, un aumento en la prevalencia de estos virus. Creemos que pueden existir diferencias en los distintos tipos de alcoholismo debido a las diversas situaciones de riesgo a las que se exponen (más exposición en el Gama de Jellineck). Por esto esperamos encontrar un aumento en la prevalencia del Virus C, especialmente en el tipo consuetudinario (delta de Jellineck) por los trastornos nutritivos derivados del modo de consumo. Posiblemente esto pueda ser la llave de otros estudios que puedan esclarecer una vía de transmisión desconocida para este virus. De la misma manera, identificar los distintos genotipos existentes en nuestra ciudad y su distribución, y que como sabemos tiene implicancia en la evolución, y en los costos por el tiempo de tratamiento. Esta información será un aporte para programar medidas de vigilancia epidemiológica adecuada, elaborar estrategias preventivas además de aplicar el tratamiento correspondiente a los pacientes infectados que se detecten como tal durante el desarrollo del proyecto.

PREVALENCIA DE INFECCION POR VIRUS HEPATITIS C, B Y VIH EN PACIENTES ALCOHOLISTAS DE LA CIUDAD DE CORDOBA

La Hepatitis C y B, junto al alcoholismo, continúan siendo un verdadero problema de Salud Pública. Sin embargo, actualmente no existen datos locales que nos permitan estimar la prevalencia de infección por virus hepatotropos en pacientes alcoholistas, sus genotipos, distrubución geográfica, ni su asociación con determinado tipo de alcoholismo. Además, la co-infección del virus de la inmunodeficiencia humana (VIH) con los virus de hepatitis supone un impacto muy importante desde el punto de vista sanitario y estadístico en nuestro país, ya que alrededor del 50 por ciento de los pacientes VIH positivos presentan dicha coinfección. Es así que, por ser de interés sanitario y por compartir vías de transmisión con el virus de hepatitis B y C, nos parece adecuado estudiar también la presencia de VIH-1 en esta población. Nuestro centro de atención pública (IPAD), atiende a pacientes con trastornos por el consumo de sustancias; deshabitúa y rehabilita alcoholistas y a otros trastornos por consumo. Dichos pacientes, que viven en la ciudad capital, serán evaluados serológicamente para la detección de virus hepatotropos C-B y el VIH. Considerando que en nuestra institución se atiende a un 70 % de Alcoholistas Puros (con un promedio de 7 pacientes nuevos por día), nos resulta importante pesquisar la prevalencia de Virus C, B y VIH en nuestra población de alcoholistas. Toda esta problemática, es la propuesta de mi tesis doctoral. Hipótesis: estimamos encontrar en nuestra población de estudio cifras superiores a la prevalencia de estos virus publicada en bancos de sangre, lo cual se toma como referencia. Objetivos: -Conocer la prevalencia de infección por Virus de Hepatitis C, B y VIH-1 en pacientes alcoholistas de la ciudad de Córdoba, determinar si existe asociación de estos virus con algún tipo de alcoholismo, e identificar genotipos prevalentes y su distribución geográfica en Córdoba. Se incluirán en forma prospectiva y aleatorea, pacientes que concurren por primera vez, de ambos sexos, mayores de 21 años, alcoholistas puros (Gama-Delta-Epsilon de Jellinek). Se confeccionará una ficha, previo consentimiento informado, que permitirá categorizar al "tipo de bebedor". Se les realizará Serología para HCV, Ag HBs (en caso de reactividad se adicionará el Anti HBcore) y VIH. En caso de la positividad serológica, se procederá al frisado de los mismos, para la Genotipificación correspondiente. La recolección, captura y procesamiento de los datos se realizarán en una planilla o ficha. Luego se reubicarán en una base electrónica de datos y se harán los análisis estadísticos de los mismos. Resultados esperados: estimamos encontrar, coincidiendo con la bibliografía, un aumento en la prevalencia de estos virus. Creemos que pueden existir diferencias en los distintos tipos de alcoholismo debido a las diversas situaciones de riesgo a las que se exponen (más exposición en el Gama de Jellineck). Por esto esperamos encontrar un aumento en la prevalencia del Virus C, especialmente en el tipo consuetudinario (delta de Jellineck) por los trastornos nutritivos derivados del modo de consumo. Posiblemente esto pueda ser la llave de otros estudios que puedan esclarecer una vía de transmisión desconocida para este virus. De la misma manera, identificar los distintos genotipos existentes en nuestra ciudad y su distribución, y que como sabemos tiene implicancia en la evolución, y en los costos por el tiempo de tratamiento. Esta información será un aporte para programar medidas de vigilancia epidemiológica adecuada, elaborar estrategias preventivas además de aplicar el tratamiento correspondiente a los pacientes infectados que se detecten como tal durante el desarrollo del proyecto.

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans.

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was approximately 35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-alpha, were recovered from AS (MTL-a/MTL-alpha), while a single type of PAP1 allele (PAP1-alpha) was recovered from AR isolates (MTL-alpha/MTL-alpha). Among the heterozygous deletions of PAP1-a (Deltapap1-a/PAP1-alpha) and PAP1-alpha (PAP1-a/Deltapap1-alpha), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

A viscous bioerodible poly(ortho ester) as a new biomaterial for intraocular application.

The biocompatibility of a viscous, hydrophobic, bioerodible poly(ortho ester) (POE) intended for intraocular application was investigated. POE was evaluated as a blank carrier and as containing modulators of degradation. Each formulation was injected intracamerally and intravitreally in rabbit eyes, and clinical and histological examinations were performed postoperatively for 2 weeks. In the case of intracameral injections, polymer biocompatibility appeared to depend on the amount injected in the anterior chamber. When 50 microL was administered, the polymer degraded within 2 weeks, and clinical observations showed good biocompatibility of POE with no toxicity to the ocular tissues or increase in intraocular pressure. The injection of a larger volume, 100 microL, of POE, appeared inappropriate because of direct contact of polymeric material with the corneal endothelium, and triggered reversible edema and inflammation in the anterior chamber of the eye that regressed after a few days. After intravitreal administration, POE was well tolerated and no inflammatory reaction developed during the observation period. The polymer degraded slowly, appearing as a round whitish bubble in the vitreous cavity. The presence of modulators of degradation both improved POE biocompatibility and prolonged polymer lifetime in the eye. POE appears to be a promising biomaterial for clinical intraocular application.

Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity.

Voltage-gated Na(+) channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca(2+) permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca(2+) or Na(+) ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca(2+) permeability, suggesting that ion-toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.

The pathogenesis of diabetic complications: the role of DNA injury and poly(ADP-ribose) polymerase activation in peroxynitrite-mediated cytotoxicity

Recent work has demonstrated that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron-transport chain triggers several pathways of injury [(protein kinase C (PKC), hexosamine and polyol pathway fluxes, advanced glycation end product formation (AGE)] involved in the pathogenesis of diabetic complications by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Increased oxidative and nitrosative stress activates the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP). PARP activation, on one hand, depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport and ATP formation. On the other hand, PARP activation results in inhibition of GAPDH by poly-ADP-ribosylation. These processes result in acute endothelial dysfunction in diabetic blood vessels, which importantly contributes to the development of various diabetic complications. Accordingly, hyperglycemia-induced activation of PKC and AGE formation are prevented by inhibition of PARP activity. Furthermore, inhibition of PARP protects against diabetic cardiovascular dysfunction in rodent models of cardiomyopathy, nephropathy, neuropathy, and retinopathy. PARP activation is also present in microvasculature of human diabetic subjects. The present review focuses on the role of PARP in diabetic complications and emphasizes the therapeutic potential of PARP inhibition in the prevention or reversal of diabetic complications.

Ocular biocompatibility of a poly(ortho ester) characterized by autocatalyzed degradation.

The biocompatibility of autocatalyzed poly(ortho ester) (POE(70)LA(30)), a viscous, hydrophobic, bioerodible polymer, was investigated. POE(70)LA(30) was synthesized, sterilized by gamma irradiation, and injected in rabbit eyes at adequate volumes through subconjunctival, intracameral, intravitreal, and suprachoroidal routes. Clinical examinations were performed postoperatively at regular time points for 6 mo, and histopathologic analysis was carried out to confirm tissular biocompatibility. After subconjunctival injection, the polymer was well tolerated and persisted in the subconjunctival space for about 5 weeks. In the case of intracameral injections, polymer biocompatibility was good; the POE(70)LA(30) bubble was still present in the anterior chamber for up to 6 mo after injection. No major histopathologic anomalies were detected, with the exception of a localized Descemet membrane thickening. After intravitreal administration, POE(70)LA(30) biocompatibility was excellent, and no inflammatory reaction could be detected during the observation period. The polymer was degraded in approximately 3 mo. Suprachoroidal injections of POE(70)LA(30) were reproducible and well tolerated. POE(70)LA(30) triggered a slight elevation of the retina and choroid upon clinical observation. The polymer was detectable in the suprachoroidal space for about 6 mo. No inflammatory reaction and no major retinal anomalies could be detected by histology. In conclusion, POE(70)LA(30) appears to be a promising biomaterial for intraocular application, potentially providing sustained drug delivery over an extended period of time, with a good tolerance.

Genetic analysis of c-Myc in mouse bone marrow and liver

1. Summary The transcription factor and proto-oncogene c-myc plays an important role in integrating many mitogenic signals within the cell. The consequences are both broad and varied and include the regulation of apoptosis, cellular differentiation, cellular growth and cell cycle progression. It is found to be mis-regulated in over 70% of all cancers, however, our knowledge about c-Myc remains limited and very little is known about its physiological role in mammalian development and in adulthood. We have addressed the physiological role of c-Myc in both the bone marrow and the liver of mice by generating adult c-myc flox/flox mice that lacked c-myc in either the bone marrow or the liver after conversion of the c-myc flox alleles into null alleles by the inducible Mx¬Cre transgene with polyI-polyC. In investigating the role of c-Myc in the haematopoietic system, we concentrated on the aspects of cellular proliferation, cellular differentiation and apoptosis. Mice lacking c-Myc develop anaemia between 3-8 weeks and all more differentiated cell types are severely depleted leading to death. However in addition to its role in driving proliferation in transient amplifying cells, we unexpectedly discovered a new role for c-Myc in controlling haematopoietic stem cell (HSC) differentiation. c-Myc deficient HSCs are able to proliferate normally in vivo. In addition, their differentiation into more committed progenitors is blocked. These cells expressed increased adhesion molecules, which possibly prevent HSCs from being released from the special stem cell supporting stromal niche cells with which they closely associate. Secondly we used the liver as a model system to address the role of c-Myc in cellular growth, meaning the increase in cell size, and also cellular proliferation. Our results revealed c-Myc to play no role in metabolic cellular growth following a period of fasting. Following treatment with the xenobiotic TCPOBOP, c-Myc deficient hepatocytes increased in cell size as control hepatocytes and could surprisingly proliferate albeit at a reduced rate demonstrating a c-Myc independent proliferation pathway to exist in parenchymal cells. However, following partial hepatectomy, in which two-thirds of the liver was removed, mutant livers were severely restricted in their regeneration capacity compared to control livers demonstrating that c-Myc is essential for liver regeneration. Résumé Le facteur de transcription et proto-oncogène c-myc joue un rôle important dans l'intégration de nombreux signaux mitogéniques dans la cellule. Les conséquences de son activation sont étendues et variées et incluent la régulation de l'apoptose, de la différenciation, de la croissance et de la progression du cycle cellulaire. Même si plus de 20% des cancers montrent une dérégulation de c-myc, les connaissances sur ce facteur de transcription restent limitées et ses rôles physiologiques au cours du développement et chez l'adulte sont très peu connus. Nous avons étudié le rôle physiologique de c-Myc dans la molle osseuse et le foie murin en générant des souris adultes c-myc flox/flox. Dans ces souris, les allèles c-myc flox sont convertis en allèles nuls par le transgène Mx-Cre après induction avec du Poly-I.C. Pour notre étude du rôle de c-Myc dans le système hématopoiétique, nous nous sommes concentrés sur les aspects de la prolifération et de la différenciation cellulaire, ainsi que sur l'apoptose. Les souris déficientes pour c-Myc développent une anémie 3 à 8 semaines après la délétion du gène; tous les différents types cellulaires matures sont progressivement épuisés ce qui entraîne la mort des animaux. Néanmoins, outre sa capacité à induire la prolifération des cellules transitoires de la molle osseuse, nous avons inopinément découvert un nouveau rôle pour c-Myc dans le contrôle de la différenciation des cellules souches hématopoiétiques (HSC). Les HSC déficientes pour c-Myc prolifèrent normalement in vivo mais leur différenciation en progéniteurs plus engagés dans une voie de différenciation est bloquée. Ces cellules surexpriment certaines molécules d'adhésion ce qui empêcherait les HSC d'être relachées du stroma spécialisé, ou niche, auquel elles sont étroitement associées. D'autre part, nous avons utilisé le foie comme système modèle pour étudier le rôle de c-Myc dans la prolifération et dans la croissance cellulaire, c'est à dire l'augmentation de taille des cellules. Nos résultats ont révélé que c-Myc ne joue pas de rôle dans le métabolisme cellulaire qui suit une période de jeûne. L'augmentation de la taille cellulaire des hépatocytes déficients pour c-Myc suite au traitement avec l'agent xénobiotique TCPOBOP est identique à celle observée pour les cellules de contrôle. Le taux de prolifération des hépatocytes mutants est par contre réduit, indiquant qu'une voie de différenciation indépendante de c-Myc existe dans les cellules parenchymales. Néanmoins, après hépatectomie partielle, où deux-tiers du foie sont éliminés chirurgicalement, les foies mutants sont sévèrement limités dans leur capacité de régénération par rapport aux foies de contrôle, montrant ainsi que c-Myc est essentiel pour la régénération hépatique.

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.