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Living organisms have evolved the ability to perceive and respond to light of different wavelengths within the visible spectrum by the generation of photoreceptor proteins. Recent studies revealed the participation of these proteins in the virulence of plant pathogenic bacteria. Pseudomonas syringae pv. tomato DC3000 (Pto) is responsible for the bacterial speck, which affects tomato crops. Pto genome contains two genes encoding red/far-red light photoreceptors (BphP1: PSPTO_1902 and BphP2: PSPTO_2652). This work demonstrates the participation of Pto phytochromes and light in the bacterial physiology and during the interaction with tomato plants. We found that Pto phytochromes are implicated in the control of some features related with the bacteria capability to enter into the plant apoplast and cause bacterial speck disease, such as motility, biofilm formation, adhesion and emulsification capability. Red light and bacteriophytochromes are important during the early colonization stage of tomato phyllosphere, affecting Pto virulence. In addition, the development of disease symptoms in infiltrated leaflets is affected by light, which may be the consequence of type-two secretion system regulation.

Ralstonia pseudosolanacearum GMI1000 (Rpso GMI1000) is a soil-borne vascular phytopathogen that infects host plants through the root system causing wilting disease in a wide range of agro-economic interest crops, producing economical losses. Several features contribute to the full bacterial virulence. In this work we study the participation of light, an important environmental factor, in the regulation of the physiological attributes and infectivity of Rpso GMI1000. In silico analysis of the Rpso genome revealed the presence of a Rsp0254 gene, which encodes a putative blue light LOV-type photoreceptor. We constructed a mutant strain of Rpso lacking the LOV protein and found that the loss of this protein and light, influenced characteristics involved in the pathogenicity process such as motility, adhesion and the biofilms development, which allows the successful host plant colonization, rendering bacterial wilt. This protein could be involved in the adaptive responses to environmental changes. We demonstrated that light sensing and the LOV protein, would be used as a location signal in the host plant, to regulate the expression of several virulence factors, in a time and tissue dependent way. Consequently, bacteria could use an external signal and Rpsolov gene to know their location within plant tissue during the colonization process. Fil: Tano, María Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Ripa, Maria Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Tondo, Maria Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina Fil: Carrau, Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Petrocelli, Silvana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina Fil: Rodriguez, María Victoria. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina Fil: Ferreira, Virginia. Universidad de la República. Facultad de Química; Uruguay Fil: Siri, María Inés. Universidad de la República. Facultad de Química; Uruguay Fil: Piskulic, Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Light modulates almost every aspect of plant physiology, including plant-pathogen interactions. Among these, the hypersensitive response (HR) of plants to pathogens is characterized by a rapid and localized programmed cell death (PCD), which is critical to restrict the spread of pathogens from the infection site. The aim of this work was to study the role of light in the interaction between Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) and non-host tobacco plants. To this end, we examined the HR under different light treatments (white and red light) by using a range of well-established markers of PCD. The alterations found at the cellular level included: i) loss of membrane integrity and nuclei, ii) RuBisCo and DNA degradation, and iii) changes in nuclease profiles and accumulation of cysteine proteinases. Our results suggest that red light plays a role during the HR of tobacco plants to Pto DC3000 infection, delaying the PCD process.

Type IV pili (Tfp) are widely distributed adhesins of bacterial surfaces. In plant pathogenic bacteria, Tfp are involved in host colonization and pathogenesis. Xanthomonas citri subsp. citri (Xcc) is the phytopathogen responsible for citrus canker disease. In this work, three Tfp structural genes, fimA, fimA1, and pilA from Xcc were studied. A pilA mutant strain from Xcc (XccΔpilA) was constructed and differences in physiological features, such as motilities, adhesion, and biofilm formation, were observed. A structural study of the purified Tfp fractions from Xcc wild-type and Xcc∆pilA showed that pilins are glycosylated in both strains and that FimA and FimA1 are the main structural components of the pili. Furthermore, smaller lesion symptoms and reduced bacterial growth were produced by Xcc∆pilA in orange plants compared to the wild-type strain. These results indicate that the minor pilin-like gene, pilA, is involved in Tfp performance during the infection process. Fil: Petrocelli, Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Arana, Maite Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Cabrini, Marcela N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Casabuono, Adriana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Moyano, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Beltramino, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Moreira, Leandro M.. Universidade Federal de Ouro Preto; Brasil Fil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Citrus, one of the most valuable fruit crops around the world, are severely damaged by biotic stress and huge economical losses are caused by pathogen infections. Non-host response is an essential plant defense mechanism against pathogen attack, however is still not completely characterized and is poorly studied in non-model plants. In previous reports, we characterized C. sinensis non-host response to Xanthomonas campestris pv. vesicatoria (Xcv), in comparison to infection caused by Xanthomonas citri subsp. citri (Xcc). This was described as a hypersensitive response with structural and physiological modifications, and transcriptional reprogramming of pathogen related proteins and transcription factors, among others. Phytohormones serve as key regulators in plant response to stress, by means of interconnected complex pathways. Here, we study the participation of phytohormone pathways during Citrus non-host response to Xcv. Our results indicate a decrease in abscisic acid, cytokinins, gibberellins, brassinosteroids, auxins and nitric oxide, an increase in ethylene and salicylic acid, constant levels of jasmonic acid, whereas polyamine levels change in a very specific pattern. The present work provides a first broad approach to hormone participation during non-host response in a non-model plant of the Citrus genus, also representative of woody plants. Fil: Petrocelli, Silvana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Pizarro, María Dolores. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alet, Analía Inés. Universidad Nacional de Rosario; Argentina Fil: De Ollas, Carlos. Universitat Jaume I; España Fil: Talón, Manuel. Instituto Valenciano de Investigaciones Agrarias; Fil: Tadeo, Francisco R.. Instituto Valenciano de Investigaciones Agrarias; Fil: Gómez Cadenas, Aurelio. Universitat Jaume I; España Fil: Arbona, Vicent. Universitat Jaume I; España Fil: Orellano, Elena Graciela. Universidad Nacional de Rosario; Argentina. Instituto de Biología Molecular y Celular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Daurelio, Lucas Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; Argentina

Summary Plants, when exposed to certain pathogens, may display a form of genotype-independent resistance, known as non-host response. In this study, the response of Citrus sinensis (sweet orange) leaves to Xanthomonas campestris pv. vesicatoria (Xcv), a pepper and tomato pathogenic bacterium, was analyzed through biochemical assays and cDNA microarray hybridization and compared with Asiatic citrus canker infection caused by Xanthomonas citri subsp. citri. Citrus leaves exposed to the non-host bacterium Xcv showed hypersensitive response (HR) symptoms (cell death), a defense mechanism common in plants but poorly understood in citrus. The HR response was accompanied by differentially expressed genes that are associated with biotic stress and cell death. Moreover, 58 transcription factors (TFs) were differentially regulated by Xcv in citrus leaves, including 26 TFs from the stress-associated families AP2-EREBP, bZip, Myb and WRKY. Remarkably, in silico analysis of the distribution of expressed sequence tags revealed that 10 of the 58 TFs, belonging to C2C2-GATA, C2H2, CCAAT, HSF, NAC and WRKY gene families, were specifically over-represented in citrus stress cDNA libraries. This study identified candidate TF genes for the regulation of key steps during the citrus non-host HR. Furthermore, these TFs might be useful in future strategies of molecular breeding for citrus disease resistance. Fil: Daurelio, Lucas Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Romero, Maria S.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Petrocelli, Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Merelo, Paz. Institut Valencià d’Investigacions Agràries. Centre de Genómica; España Fil: Cortadi, Adriana Amalia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Talon, Manuel. Institut Valencià d’Investigacions Agràries. Centre de Genómica; España Fil: Tadeo, Francisco R.. Institut Valencià d’Investigacions Agràries. Centre de Genómica; España Fil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo₂Hex₆GalA₃Fuc3NAcRha₄ and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.

Plants are continuously exposed to pathogen challenge. The most common defense response to pathogenic microorganisms is the nonhost response, which is usually accompanied by transcriptional changes. In order to identify genes involved in nonhost resistance, we evaluated the tobacco transcriptome profile after infection with Xanthomonas axonopodis pv. citri (Xac), a nonhost phytopathogenic bacterium. cDNA-amplified fragment length polymorphism was used to identify differentially expressed transcripts in tobacco leaves infected with Xac at 2, 8 and 24h post-inoculation. From a total of 2087 transcript-derived fragments (TDFs) screened (approximately 20% of the tobacco transcriptome), 316 TDFs showed differential expression. Based on sequence similarities, 82 differential TDFs were identified and assigned to different functional categories: 56 displayed homology to genes with known functions, 12 to proteins with unknown functions and 14 did not have a match. Real-time PCR was carried out with selected transcripts to confirm the expression pattern obtained. The results reveal novel genes associated with nonhost resistance in plant-pathogen interaction in tobacco. These novel genes could be included in future strategies of molecular breeding for nonhost disease resistance.

Background Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase). Methodology/principal findings Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation. Conclusions Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

We have used hepatocyte suspensions to study how hypothermic storage in a modified University of Wisconsin (mUW) solution affects liver cell metabolism and cell membrane properties. At present the UW solution is the gold standard of organ preservation. However it contains several ingredients which either are expensive or cannot easily be obtained worldwide. The aim of the present study was the development of a novel preservation solution for rat hepatocytes effective and comparable with the mUW, with enhanced buffer capacity and less expensive. In particular, we investigated the effects of the buffering agent BES, a derivate of aminosulfonic acid, on liver cells metabolism during cold storage and rewarming. In the development of this novel preservation solution we have included three key components: gluconate as impermeant anion, sucrose to give additional osmotic support and the aminosulfonic acid BES for its buffer capacity. Our results shown that BGS solution was equally effective to mUW to protect rat hepatocytes against cold preservation injury due to ischemia and reoxigenation. Also, BGS solution is a good alternative with its high buffer capacity, best indices of respiration activity and it is considerably less expensive than the mUW solution. The use of this solution for the storage of isolated hepatocytes may facilitate hepatocyte research in situations in which the more complex and expensive mUW solution is not available since the cost of one liter of BGS is about 1/3 that an equal volume of mUW solution.

Xanthomonas axonopodis pv. citri (Xac) is the phytopathogen responsible for citrus canker, one of the most devastating citrus diseases in the world. A broad range of pathogens is recognized by plants through so-called pathogen-associated molecular patterns (PAMPs), which are highly conserved fragments of pathogenic molecules. In plant pathogenic bacteria, lipopolisaccharyde (LPS) is considered a virulence factor and it is being recognized as a PAMP. The study of the participation of Xac LPS in citrus canker establishment could help to understand the molecular bases of this disease. In the present work we investigated the role of Xac LPS in bacterial virulence and in basal defense during the interaction with host and non host plants. We analyzed physiological features of Xac mutants in LPS biosynthesis genes (wzt and rfb303) and the effect of these mutations on the interaction with orange and tobacco plants. Xac mutants showed an increased sensitivity to external stresses and differences in bacterial motilities, in vivo and in vitro adhesion and biofilm formation. Changes in the expression levels of the LPS biosynthesis genes were observed in a medium that mimics the plant environment. Xacwzt exhibited reduced virulence in host plants compared to Xac wild-type and Xacrfb303. However, both mutant strains produced a lower increase in the expression levels of host plant defense-related genes respect to the parental strain. In addition, Xac LPS mutants were not able to generate HR during the incompatible interaction with tobacco plants. Our findings indicate that the structural modifications of Xac LPS impinge on other physiological attributes and lead to a reduction in bacterial virulence. On the other hand, Xac LPS has a role in the activation of basal defense in host and non host plants.

Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.

ID: mamprin Abstract No.: 158 Volume Regulation and mRNA Expression of Aaquaporine 8 in Hepatocytes Cold Stored at 0◦C in Preservation Solutions Maŕia Mamprin, Cristina Bellarosa, Silvana Petrocelli, Edgardo Guibert, Claudio Tiribelli, Joaquin Rodŕiguez a Ciencias Fisiologicas, Facultad de Ciencias Bioquimicas y Farmaceuticas. Universidad Nacional de Rosario. UNESCO Chair in Cryobiology, S2002LRK Rosario, Argentina b Ciencias Biologicas, Facultad de Ciencias Bioquimicas y Farmaceuticas. Universidad Nacional de Rosario. UNESCO Chair in Cryobiology, S2002LRK Rosario, Argentina d BBCM, Centro Studi Fegato, AREA Science Park, Basovizza SS 14 Km 136.5. University of Trieste, 34012 Trieste, Italy Different storage solutions, such as the University of Wisconsin solutions (UW) have been successfully used for the temporal maintenance of the liver of animals and humans. Many studies have demonstrated the effectiveness of cold storage of hepatocytes and successful application on Bioartificial liver devices and hepatocellular transplantation. The aim of the present study was to investigate the effect of cold storage time at 0◦C with two different preservation solutions: UW and a novel BES-based solution (BGS), on gene expression (at mRNA level for Aquaporine 8 (AQP8)) and water movements of isolated rat hepatocytes. Hepatocytes were isolated from male Wistar rats by collagenase perfusion and cold stored for 72 h at 0◦C in either UW or a 70 mM BES solution containing 50mM sucrose and 100mM potassium gluconate. Total RNA was isolated from hepatocytes taken at different time intervals. Real time RT-PCR was used to quantify the AQP8 mRNA in freshly isolated hepatocytes and after 72 h of cold storage and during a subsequent 2 h rewarming period; s-actin was used as housekeeping gene to normalize the expression of target genes. For the AQP8 (accession bank: NM 019158) we used Upper primer: 5-TTG GGG CTC ATC ATT GCT AC-3, position 333; and Lower primer: 5-AAG GCC TCC AAC CAG TGT GA-3, position 403. To evaluate the water content of cells during preservation and rewarming, the [wet weight dry weight] / dry weight of hepatocytes collected at different time intervals was determinate after 48 h desiccation at 90◦C. To monitor the evolution of hepatocyte water content (Vend / Vinitial), each volume (Vend) was normalized to the volume of hepatocyte at the corresponding time of control cells at 37◦C (Vinitial). Cold storage induced a significant hepatocyte shrinkage at 72 h: UW 0.56 ± 0.03; BCG: 0.87 ± 0.13 vs. Control 1.00 (n=10). On the contrary, no changes in expression of AQP8 at mRNA level was found During the rewarming, cells preserved in both solutions recover a volume similar to the control group (control: 1.15 ± 0.06; UW: 1.03 ± 0.09, BCG: 1.21 ± 0.12, n=10). From these data we conclude that BES-based solution is as effective as UW solution for hepatocyte preservation. The change in cell volume observed both during cold storage and after rewarming is not related to change in the mRNA levels of AQP8. Irrespective of the cell shrinkage observed at 0◦C, hepatocytes recover a normal cellular volume during the rewarming pointing to the possibility to safety preserve the cells in both solutions for further use. Conflict of interest: None declared Source of funding: Ministero degli Affari Esteri, Italy. Progetti di Grande Rilevanza Prot. 269/P/0114337. ANPCyT, Argentina, PICT 03-14492. UNESCO Chair in Cryobiology. Cryo 2006 www.cryo2006.org sputtek@uke.uni-hamburg.de