Protein kinase C isozymes in hypertension and hypertrophy: Insight from SHHF rat hearts

Chronic hypertension results in cardiac hypertrophy and may lead to congestive heart failure. The protein kinase C (PKC) family has been identified as a signaling component promoting cardiac hypertrophy. We hypothesized that PKC activation may play a role mediating hypertrophy in the spontaneously hypertensive heart failure (SHHF) rat heart. Six-month-old SHHF and normotensive control Wistar Furth (WF) rats were used. Hypertension and cardiac hypertrophy were confirmed in SHHF rats. PKC expression and activation were analyzed by Western blots using isozyme-specific antibodies. Compared to WF, untreated SHHF rats had increased phospho-active (10-fold), (4-fold), and (3-fold) isozyme expression. Furthermore, we analyzed the effect of an angiotensin II type 1 receptor blocker (ARB) and hydralazine (Hy) on PKC regulation in SHHF rat left ventricle (LV). Both the ARB and Hy normalized LV blood pressure, but only the ARB reduced heart mass. Neither treatment affected PKC expression or activity. Our data show differential activation of PKC in the hypertensive, hypertrophic SHHF rat heart. Regression of hypertrophy elicited by an ARB in this model occurred independently of changes in the expression and activity of the PKC isoforms examined. (Mol Cell Biochem 270: 63–69, 2005)     

Rare cancer cell analyzer for whole blood applications: microcytometer cell counting and sorting subcircuits

We demonstrate sorting of rare cancer cells from blood using a thin ribbon monolayer of cells within a credit-card sized, microfluidic laboratory-on-a-card ("lab card") structure. This enables higher cell throughput per minute thereby speeding up cell interrogation. In this approach, multiple cells are viewed and sorted, not individually, but as a whole cell row or section of the ribbon at a time. Gated selection of only the cell rows containing a tagged rare cell provides enrichment of the rare cell relative to background blood cells. We also designed the cell injector for laminar flow antibody labeling within 20s. The approach combines rapid laminar flow cell labeling with monolayer cell sorting thereby enabling rare cell target detection at sensitivity levels 1000 to 10,000 times that of existing flow cytometers. Using this method, total cell labeling and data acquisition time on card may be reduced to a few minutes compared to 30-60 min for standard flow methods.     

Alcohol-Related Brain Damage in Humans

Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann’s area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin β II, and α- and β-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in α-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in β-spectrin protein levels, and a significant increase in transmembranous α3 (catalytic) subunit of the Na⁺,K⁺-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of α-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic α- and β-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics.     

Global warming will affect the genetic diversity and uniqueness of Lycaena helle populations

The climate warming of the postglacial has strongly reduced the distribution of cold‐adapted species over most of Central Europe. Such taxa have therefore become extinct over most of the lowlands and shifted to higher altitudes where they have survived to the present day. The lycaenid butterfly Lycaena helle follows this pattern of former widespread distribution and later restriction to mountain areas such as the European middle mountains. We sampled 203 individuals from 10 populations representing six mountain ranges (Pyrenees, Jura, Massif Central, Morvan, Vosges and Ardennes) over the species' western distribution. Allozyme and microsatellite polymorphisms were analysed to study the genetic status of these highly fragmented populations. Both molecular marker systems revealed a strong genetic differentiation among the analysed populations, coinciding with the orographic structure and highly restricted gene flow among them. The large‐scale genetic differentiation is more pronounced in allozymes (F_CT: 0.326) than in microsatellites (R_CT: 0.113), but microsatellites show a higher resolution on the regional scale (R_SC: 0.082) compared with allozymes (F_SC: n.s.). For both analytical tools, we found private alleles occurring exclusively in a single mountain area. The highly fragmented and isolated occurrence of populations is supported by the distribution pattern of potentially suitable climate suggested by species distribution models. Model projections under two climate warming scenarios predict a decline of climatically suitable areas, which will result in the extinction of most of the populations showing unique genetic characteristics.     

Experimental climate effect on seasonal variability of polyphenol/phenoloxidase interplay along a narrow fen–bog ecological gradient in Sphagnum fallax

Extracellular phenoloxidase enzymes play an important role in the stability of soil carbon storage by contributing to the cycling of complex recalcitrant phenolic compounds. Climate warming could affect peatland functioning through an alteration of polyphenol/phenoloxidase interplay, which could lead them to becoming weaker sinks of carbon. Here, we assessed the seasonal variability of total phenolics and phenoloxidases subjected to 2–3 °C increase in air temperature using open‐top chambers. The measurements were performed along a narrow fen–bog ecological gradient over one growing season. Climate warming had a weak effect on phenoloxidases, but reduced phenolics in both fen and bog areas. Multivariate analyses revealed a split between the areas and also showed that climate warming exacerbated the seasonal variability of polyphenols, culminating in a destabilization of the carbon cycle. A negative relationship between polyphenols and phenoloxidases was recorded in controls and climate treatments suggesting an inhibitory effect of phenolics on phenoloxidases. Any significant decrease of phenolics through repeatedly elevated temperature would greatly impact the ecosystem functioning and carbon cycle through an alteration of the interaction of polyphenols with microbial communities and the production of extracellular enzymes. Our climate treatments did not have the same impact along the fen–bog gradient and suggested that not all the peatland habitats would respond similarly to climate forcing.     

ParA ATPases can move and position DNA and subcellular structures

Prokaryotic chromosomes and plasmids can be actively segregated by partitioning (par) loci. The common ParA-encoding par loci segregate plasmids by arranging them in regular arrays over the nucleoid by an unknown mechanism. Recent observations indicate that ParA moves plasmids and chromosomes by a pulling mechanism. Even though ParAs form filaments in vitro it is not known whether similar structures are present in vivo. ParA of P1 forms filaments in vitro at very high concentrations only and filament-like structures have not been observed in vivo. Consequently, a 'diffusion-ratchet' mechanism was suggested to explain plasmid movement by ParA of P1. We compare this mechanism with our previously proposed filament model for plasmid movement by ParA. Remarkably, ParA homologues have been discovered to arrange subcellular structures such as carboxysomes and chemotaxis sensory receptors in a regular manner very similar to those of the plasmid arrays.     

Maintenance of EGFR and EGFRvIII expressions in an in vivo and in vitro model of human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common, and most aggressive primary brain tumor among adults. A vast majority of the tumors express high levels of the epidermal growth factor receptor (EGFR) as a consequence of gene amplification. Furthermore, gene amplification is often associated with mutation of EGFR, and the constitutive activated deletion variant EGFRvIII is the most common EGFR mutation found in GBM. Activated EGFR signaling, through overexpression and/or mutation, is involved in increased tumorigenic potential. As such, EGFR is an attractive target for GBM therapy. However, clinical studies with EGFR inhibitors have shown inconsistent results, and as such, further knowledge regarding the role of EGFR and EGFRvIII in GBM is needed. For this, an appropriate in vivo/in vitro tumor model is required. Here, we report the establishment of an experimental GBM model in which the expressions of EGFR and EGFRvIII are maintained both in xenograft tumors growing subcutaneously on mice and in cell cultures established in stem cell conditions. With this model it will be possible to further study the role of EGFR and EGFRvIII, and response to targeted therapy, in GBM.     

DETERMINATION OF SENSORY OIL-WATER PARTITION COEFFICIENTS OF SINGLE AROMA COMPOUNDS COMBINING PANELIST FREE INTENSITY RATING AND THEORETICAL MODELING OF ODOR INTENSITY

Using an intensity rating with no external calibration, experiments were designed to measure the sensory oil-water partition coefficients of four aroma molecules (benzaldehyde, ethyl butyrate, linalool and acetophenone) as the ratios of concentration producing stimuli of equivalent intensities. Flavored water and oil phases were successively assessed for odor intensity by 24 panelists who were given total freedom regarding scaling strategy. Each session combined five concentration levels of three out of the four studied aromas in a solvent (water or oil). A predominant concentration effect was found for each aroma in both solvents and concentration dependencies of perceived intensity above water and oil were similar. Experimental data were modeled with Fechner, Stevens and Hill equations. Combining results above water and oil solutions to feed a common model led to the evaluation of an overall sensory oil-water partition coefficient for each aroma compound. All three models produced similar partition coefficient values for each aroma that were lower than the related instrumental partition coefficients. Biases previously detected when external calibration had been used were reduced in a large proportion while suggested enhancement of odor intensity by water vapor could not be excluded.