Effects of pulsed electric field on the viscoelastic properties of potato tissue

We have investigated whether transient permeabilization caused by the application of pulsed electric field would give rise to transient changes in the potato tissue viscoelastic properties. Potato tissue was subjected to nominal field strengths (E) ranging from 30 to 500 V/cm, with a single rectangular pulse of 10−5, 10−4, or 10−3 s. The changes on the viscoelastic properties of potato tissue during pulsed electric fields (PEF) were monitored through small amplitude oscillatory dynamic rheological measurements. The elastic (G′) and viscous moduli (G″) were measured every 30 s after the delivery of the pulse and the loss tangent change (tan-δ) was calculated. The results were correlated with measurements of changes on electrical resistance during the delivery of the pulse. Results show a drastic increase of tan-δ in the first 30 s after the application of the pulse, followed by a decrease 1 min after pulsation. This response is strongly influenced by pulsing conditions and is independent of the total permeabilization achieved by the pulse. Our results, supported by similar measurements on osmotically dehydrated control samples, clearly show that PEF causes a rapid change of the viscoelastic properties of the tissue that could be attributed to a partial loss in turgor pressure. This would be an expected consequence of electroporation. The recovery of tan-δ to values similar to those before pulsation strongly suggests recovery of cell membrane properties and turgor, pointing at reversible permeabilization of the cells. A slight increase of stiffness traduced by a negative change of tan-δ after application of certain PEF conditions may also give an indication of events occurring on cell wall structure due to stress responses. This study set the basis for further investigations on the complex cell stress physiology involving both cell membrane functional properties and cell wall structure that would influence tissue physical properties upon PEF application.Fundação para a Ciência e a Tecnologia (FCT

Overexpression, crystallization and preliminary X-­ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1

The C-terminal cytosolic domain of mouse anoctamin 1 (mANO1, also known as TMEM16A) was cloned, overexpressed, purified and crystallized. The crystals belonged to space group P212121 and diffracted to 2.3 Å resolution

Efficient Cultivation Conditions for Human Limbal Epithelial Cells

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding casette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM

HIV infection and HERV expression: a review

The human genome contains multiple copies of retrovirus genomes known as endogenous retroviruses (ERVs) that have entered the germ-line at some point in evolution. Several of these proviruses have retained (partial) coding capacity, so that a number of viral proteins or even virus particles are expressed under various conditions. Human ERVs (HERVs) belong to the beta-, gamma-, or spuma- retrovirus groups. Endogenous delta- and lenti- viruses are notably absent in humans, although endogenous lentivirus genomes have been found in lower primates. Exogenous retroviruses that currently form a health threat to humans intriguingly belong to those absent groups. The best studied of the two infectious human retroviruses is the lentivirus human immunodeficiency virus (HIV) which has an overwhelming influence on its host by infecting cells of the immune system. One HIV-induced change is the induction of HERV transcription, often leading to induced HERV protein expression. This review will discuss the potential HIV-HERV interactions

Sharp boundaries of Dpp signalling trigger local cell death required for Drosophila leg morphogenesis

Article available at http://dx.doi.org/10.1038/ncb1518Morphogens are secreted signalling molecules that govern many developmental processes1. In the Drosophila wing disc, the transforming growth factor (TGF) homologue Decapentaplegic (Dpp) forms a smooth gradient and specifies cell fate by conferring a defined value of morphogen activity. Thus, neighbouring cells have similar amounts of Dpp protein, and if a sharp discontinuity in Dpp activity is generated between these cells, Jun kinase (JNK)-dependent apoptosis is triggered to restore graded positional information2, 3. To date, it has been assumed that this apoptotic process is only activated when normal signalling is distorted. However, we now show that a similar process occurs during normal development: rupture in Dpp activity occurs during normal segmentation of the distal legs of Drosophila. This sharp boundary of Dpp signalling, independently of the absolute level of Dpp activity, induces a JNK—reaper-dependent apoptosis required for the morphogenesis of a particular structure of the leg, the joint. Our results show that Dpp could induce a developmental programme not only in a concentration dependent manner, but also by the creation of a sharp boundary of Dpp activity. Furthermore, the same process could be used either to restore a normal pattern in response to artificial disturbance or to direct a morphogenetic process.This work has been supported by grants from the Dirección General de Investigación Científica y Técnica (BMC 2002-00300), the Comunidad Autónoma de Madrid (08.1/0031/2001.1 and GR/SAL/0147/2004) and an Institutional Grant from the Fundación Ramón Areces. C.M. is a recipient of a Formación del Personal Universitario (F.P.U.) fellowship from the Ministerio de Educación y Ciencia.Peer reviewe

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma