Phospholipid base exchange enzyme activity in sarcolemmal membranes from the heart of cardiomyopathic hamsters

The activity of phospholipid base exchange enzymes has been evaluated in cardiac sarcolemmal membranes from Syrian Golden hamsters and from a hamster strain (UM-X7.1) characterized by a genetic form of hypertrophic cardiomyopathy. No choline base exchange activity and only a little serine base exchange activity were detected, whereas the ethanolamine base exchange enzyme was found highly active in membranes from both strains. For this reason, the present study is focussed on the ethanolamine base exchange enzyme. The apparent Km for ethanolamine of ethanolamine base exchange enzyme from Syrian Golden membranes and from UM-X7.1 strain membranes are 18 and 32 μM, respectively. The specific activity of the sarcolemmal ethanolamine base exchange enzyme is lower in the UM-X7.1 strain than in Syrian Golden hamsters. The calcium-dependence of the enzyme appears different when the membranes from the two strains are compared. Indeed, after removal of the membrane-bound divalent cations, comparable activities are found in both membrane preparations, whereas, upon addition of Ca²⁺ to the incubation mixtures, the activity of the enzyme is enhanced in the membranes from Syrian Golden strain more than in those from UM-X7.1 strain. The cholesterol content of sarcolemmal membranes is higher in the cardiomyopathic strain than in the Syrian Golden hamsters. A possible relation between changes of the membrane lipid composition and of the ethanolamine base exchange activity is discussed.     

Effect of glucose starvation on germ-tube production byCandida albicans

By incubating starved and unstarved yeast cells in synthetic media with a pH of 4.5 or 6.7 at 37°C the effect of a 3 hours' glucose starvation on germ-tube production byCandida albicans was evaluated. In addition the endocellular content of total carbohydrates, glycogen, trehalose and proteins after and before the starvation were dosed. The most interesting result was the overcoming of the pH-regulated dimorphism, thanks to the starvation treatment. Infact the starved cultures produced germtubes indifferently in neutral or acid media, whereas the filamentation of the unstarved cultures was more copious in pH 6.7 medium. The endocellular content of trehalose and protein was unchanged, whereas total carbohydrates and glycogen showed a shortage after the 3 hours' glucose starvation. The possible involvements of these metabolic changes in the regulation of dimorphic transition are discussed.     

Serum levels of anti-heat shock protein 27 antibodies in patients with chronic liver disease

Heat shock protein 27 (HSP27), an intracellular molecular chaperone, is involved in the pathogenesis of cancer by promoting both tumor cell proliferation and resistance to therapy. HSP27 is also present in the circulation and circulating HSP27 (sHSP27) can elicit an autoimmune response with production of antibodies. Levels of sHSP27 are enhanced in patients with hepatocellular carcinoma (HCC); it is, however, unknown whether changes in HSP27 antibody levels occur in patients with HCC and can be exploited as a circulating biomarker of HCC. Our aim was to assess the potential association between newly diagnosed HCC and serum anti-HSP27 antibody levels. In this cross-sectional study, anti-HSP27 antibody levels were measured in serum samples from 71 HCC patients, 80 subjects with chronic liver disease, and 38 control subjects by immunoenzymatic assay. Anti-HSP27 antibody levels did not differ significantly among groups. However, in patients with chronic active hepatitis/cirrhosis, anti-HSP27 levels were significantly higher in subjects with a positive history of alcoholism (p = 0.03). Our data do not support the hypothesis that anti-HSP27 antibody levels may help identify patients with HCC among subjects with chronic liver disease. However, our finding that alcohol-related liver disease is associated with higher anti-HSP27 levels is novel and deserves further investigations.     

Ubiquinol and a Coenzyme Q Reducing System Protect Platelet Mitochondrial Function of Transfusional Buffy Coats from Oxidative Stress

The conditions under which Coenzyme Q (CoQ) may protect platelet mitochondrial function of transfusional buffy coats from aging and from induced oxidative stress were investigated. The Pasteur effect, i.e. the enhancement of lactate production after inhibition of mitochondrial respiratory chain, was exploited as a marker of mitochondrial function as it allows to calculate the ratio of mitochondrial ATP to glycolytic ATP. Reduced CoQ 10 improves platelet mitochondrial function of transfusional buffy coats and protects the cells from induced oxidative stress. Oxidized CoQ is usually less effective, despite the presence, shown for the first time in this study, of quinone reductase activities in the platelet plasma membranes. The addition of a CoQ reducing system to platelets is effective in enhancing the protection of platelet mitochondrial function from the oxidative stress. The results support on one hand a possibility of protection of mitochondrial function in aging by exogenous CoQ intake, on the other a possible application in protection of transfusional buffy coats from storage conditions and oxidative deterioration.     

Leaflet morphometric variation of service tree (Sorbus domestica L.) in the Balkan Peninsula

Abstract

In most European countries, the service tree (Sorbus domestica L.) is a rare and threatened species and its conservation has been recognised as a priority. The aim of this study was to asses its morphologic variation in the western and central part of the Balkan Peninsula and in southern Central Europe. Three populations were analysed: one in Serbia, one in Bosnia and Herzegovina and one in Slovenia. In each population 30 trees were selected, and from each tree 30 leaves were collected for morphometric analysis based on nine leaflet morphological traits. Univariate (ANOVA) and multivariate (MANOVA) analysis of variance were used to estimate the variation within- and between populations and a discriminant analysis was performed to examine the structure of the between-population differences. The values of particular morphological traits found in our study did not differ considerably from the values reported elsewhere. The results revealed significant within- and between population variation. Variation within populations was highly significant for all the scored leaf morphological traits, while variation between populations was significant for all the studied traits except for the leaflet length. The discrimination between the three populations was significant. High percentages of correctly classified samples demonstrate good discriminating employability of the analysed leaf morphological traits and indicate differentiation of the analysed populations.     

BAFF,APRIL, TWEAK,BCMA, TACI and Fn14 Proteins Are Related to Human Glioma Tumor Grade: Immunohistochemistry and Public Microarray Data Meta-Analysis

Gliomas are common and lethal tumors of the central nervous system (CNS). Genetic alterations, inflammatory and angiogenic processes have been identified throughout tumor progression; however, treatment still remains palliative for most cases. Biological research on parameters influencing cell survival, invasion and tumor heterogeneity identified several cytokines interfering in CNS inflammation, oxidative stress and malignant transformation, including TNF-superfamily (TNFSF) members. In this report we performed a meta-analysis of public gene-array data on the expression of a group of TNFSF ligands (BAFF, APRIL, TWEAK) and their receptors (BAFF-R, TACI, BCMA, Fn14) in gliomas. In addition, we investigated by immunohistochemistry (IHC) the tumor cells'' expression of these ligands and receptors in a series of 56 gliomas of different grade. We show that in IHC, BAFF and APRIL as well as their cognate receptors (BCMA, TACI) and Fn14 expression correlate with tumor grade. This result was not evidenced in micro-arrays meta-analysis. Finally, we detected for the first time Fn14, BAFF, BCMA and TACI in glioma-related vascular endothelium. Our data, combined with our previous report in glioma cell lines, suggest a role for these receptors and ligands in glioma biology and advance these molecules as potential markers for the classification of these tumors to the proliferative, angiogenic or stem-like molecular subtype.     

Monitoring and manipulation of the pH of single cells using infrared spectromicroscopy and a molecular switch

Background

The pH of a biological system is a crucial determinant of the structures and reactivity of its components and cellular homeostasis of H⁺ is critical for cell viability. Control and monitoring of cellular acidity are highly desirable for the purpose of studying biochemical processes in vivo.

Methods

The effect of photolysis of a caged strong acid, the ester 1-(2-nitrophenyl)-ethylhexadecyl sulfonate (HDNS) is used to cause a controlled drop in pH in single cells. An isolated cell is selected under the IR microscope, irradiated with near-UV light and monitored by FTIR.

Results

We demonstrate the use of FTIR spectromicroscopy to monitor light-induced acidification of the cellular medium by measuring the increased concentration of CO₂ and corresponding decrease of HCO₃⁻ in the cell and in the surrounding medium.

Conclusions

We have demonstrated a method to control and accurately monitor the changes in pH of a cellular system by coupling a caged proton-releasing agent with FTIR spectromicroscopy detection. The overall implementation of photolysis and spectroscopic detection in a microscope optical configuration ensures single cell selectivity in both acidification and monitoring. We show the viability of monitoring of pH changes by FTIR spectromicroscopy with sensitivity comparable to that of glass electrodes, better than the existing methods for determining cell pH.

General significance

Reporting the effect of small variations of cellular acidity provides a major improvement in the understanding of the interplay between molecular properties as assessed in vitro and cell physiology.     

Barth syndrome: Cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation

Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I + III₂ + IV_n but also decreased amounts of individual complexes I and IV and supercomplexes I + III and III + IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.