Volatile profiles of human skin cell cultures in different degrees of senescence

It is known that skin releases volatile organic compounds to the environment, and also that its emission pattern changes with aging of the skin. It could be considered, that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, a simple and non-destructive method consisting of SPME sampling and GC/MS analysis was developed to identify volatile organic emanations from cell cultures. This technique, applied to skin cells culture, indicates that the cells or cell metabolism produce several skin emissions. Chemometric analysis was performed in order to explore the relationship between a volatile profile and the senescence of cell cultures. Volatile profiles were different for cell cultures in different degrees of senescence, indicating that volatile compound patterns could be used to provide information about the age of skin cells.     

Separation of plant membranes by electromigration techniques

The review focuses on the multiple separating regimes that offers the free flow electrophoresis technique: free flow zone electrophoresis, isoelectric focusing, isotachophoresis, free flow step electrophoresis. Also, the feasibility to apply either interval or continuous flow electrophoresis is evaluated. The free flow zone electrophoresis regime is generally selected for the separation of cells, organelles and membranes while the other regimes find their largest fields of applications in the purification of proteins and peptides. The latter regimes present the highest resolution efficiency. Therefore, a large part of this review is devoted to the applicabilities of these different regimes to the purification of organelles and membrane vesicles at the preparative scale. Recent developments, both in instrumentation and procedures, are described. The major achievements in plant membrane fractionation obtained with free flow electrophoresis are outlined. The related procedures are both analytical and preparative: they separate tonoplast and plasma membrane simultaneously from the same homogenate, they discriminate for one type of membrane vesicles of opposite orientation, and process large quantities of membrane material by reason of the continuous flow mode. Recent advances using electromigration techniques that permit confirmation of the dynamic state of membranes, characterisation of complex membrane-dependent functions and discovery of new membrane-localised activities are presented.     

Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

DNA Sequences from Formalin-Fixed Nematodes: Integrating Molecular and Morphological Approaches to Taxonomy

To effectively integrate DNA sequence analysis and classical nematode taxonomy, we must be able to obtain DNA sequences from formalin-fixed specimens. Microdissected sections of nematodes were removed from specimens fixed in formalin, using standard protocols and without destroying morphological features. The fixed sections provided sufficient template for multiple polymerase chain reaction-based DNA sequence analyses.     

A predictive model for freshwater bioassessment (Mondego River,Portugal)

We sampled macroinvertebrates at 75 locations in the Mondego river catchment, Central Portugal, and developed a predictive model for water quality assessment of this basin, based on the Reference Condition Approach. Sampling was done from June to September 2001. Fifty-five sites were identified as “Reference sites” and 20 sites were used as “Test sites” to test the model. At each site we also measured 40 habitat variables to characterize water physics and chemistry, habitat type, land use, stream hydrology and geographic location. Macroinvertebrates were generally identified to species or genus level; a total of 207 taxa were found. By Unweighted Pair Group Method with Arithmetic mean (UPGMA) clustering and analysis of species contribution to similarities percentage (SIMPER), two groups of reference sites were established. Using Discriminant Analysis (stepwise forward), four variables correctly predicted 78% of the reference sites to the appropriate group: stream order, pool quality, substrate quality and current velocity. Test sites’ environmental quality was established from their relative distance to reference sites, in MDS ordination space, using a series of bands (BEAST methodology). The model performed well at upstream sites, but at downstream sites it was compromised by the lack of reference sites. As with the English RIVPACS predictive model, the Mondego model should be continually improved with the addition of new reference sites. The adaptation of the Mondego model methodology to the Water Framework Directive is possible and would consist mainly of the integration of the WFD typology and increasing the number of ellipses that define quality bands. Handling editor: K. Martens     

Quantitative histology of the hypertrophied human heart

Myocardial hypertrophy accompanies systemic hypertension and aortic stenosis, i.e., pressure overload. In man cardiac failure only appears after years of pressure overload, during which time cardiac function had been maintained. The structural correlates of cardiac failure have been a subject of much interest for many years. Several hypotheses relating alterations in muscle fiber alignment, capillary density, or collagen content have been offered. The application of morphometric techniques has provided essential quantitative information on the structural components of the normal and diseased heart. These data indicate that muscle fiber alignment remains normal in the pressure overloaded heart despite the presence of hypertrophy or the appearance of clinical failure. On the other hand, capillary density is decreased and collagen content is increased in hypertrophied hearts. Chemical studies on collagen concentration however have yielded inconsistent results. The relative contribution of the microcirculation and collagenous structure of the myocardium on its respective O2 availability, mechanical behavior, and deterioration in pump function will require further investigation.     

Membrane microheterogeneity: Förster resonance energy transfer characterization of lateral membrane domains

Lateral membrane heterogeneity, in the form of lipid rafts and microdomains, is currently implicated in cell processes including signal transduction, endocytosis, and cholesterol trafficking. Various biophysical techniques have been used to detect and characterize lateral membrane domains. Among these, Förster resonance energy transfer (FRET) has the crucial advantage of being sensitive to domain sizes smaller than 50-100 nm, below the resolution of optical microscopy but, apparently, similar to those of rafts in cell membranes. In the last decade, several formalisms for the analysis of FRET in heterogeneous membrane systems have been derived and applied to the study of microdomains. They are critically described and illustrated here.