The influence of alkyl pyridinium sponge toxins on membrane properties, cytotoxicity, transfection and protein expression in mammalian cells

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca²⁺ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.     

Resprouting in the Mediterranean‐type shrub Erica australis afffected by soil resource availability

Abstract. Soil resource availability may affect plant regeneration by resprouting in disturbed environments directly, by affecting plant growth rates, or indirectly by determining allocation to storage in the resprouting organs. Allocation to storage may be higher in stressful, low resource‐supply soils, but under such conditions plant growth rates may be lower. These factors could act in opposite directions leading to poorly known effects on resprouting. This paper analyses the role played by soil resources in the production and growth of resprouts after removal of above‐ground plant tissues in the Mediterranean shrub Erica australis. At 13 sites, differing in substrate, we cut the base of the stems of six plants of E. australis and allowed them to resprout and grow for two years. Soils were chemically analysed and plant water potential measured during the summer at all sites to characterize soil resource availability. We used stepwise regression analysis to determine the relationships between the resprouting response [mean site values of the number of resprouts (RN), maximum length (RML) and biomass (RB)] and soil nutrient content and plant water potential at each site. During the first two years of resprouting there were statistically significant differences among sites in the variables characterizing the resprouting response. RML was always different among sites and had little relationship with lignotuber area. RN was less different among sites and was always positively correlated with lignotuber area. RB was different among sites after the two years of growth. During the first months of resprouting, RN and RML were highly and positively related to the water status of the plant during summer. At later dates soil fertility variables came into play, explaining significant amounts of variance of the resprouting variables. Soil extractable cations content was the main variable accounting for RML and RB. Our results indicate that resprout growth of E. australis is positively affected by high water availability at the beginning of the resprouting response and negatively so by high soil extractable cation content at later periods. Some of these factors had previously shown to be related, with an opposite sign, to the development of a relatively larger lignotuber. Indeed, RML and RB measured in the second year of resprouting were significantly and negatively correlated with some indices of biomass allocation to the lignotuber at each site. This indicates that sites favouring allocation to the resprouting organ may not favour resprout growth.     

A new species of water mite <Emphasis Type="Italic">Atractides</Emphasis> (<Emphasis Type="Italic">Atractides</Emphasis>) <Emphasis Type="Italic">turcicus</Emphasis> sp. n. (Acari: Hydrachnidia: Hygrobatidae) from Turkey

In this study, the structural characteristics, unique features, various organ measurements of males and females of the water mite Atractides (Atractides) turcicus sp. n. from Turkey are described. In addition, the study compares their characteristics with related species.     

Effect of retinoic acid on the aggregation of erythrocytes and leukocytes in the blood

Human and animal blood smear staining with PAPh has revealed mononuclear leukocyte-erythrocyte aggregates. Administration of retinoic acid increased concentration and dimensions of these aggregates and was followed by preferential accumulation of PAPh-negative osmotically unstable erythrocytes. Similar changes were detected in the blood samples of women engaged in the production of retinoic acid. Aggregate concentration showed positive correlation with erythrocyte sedimentation rate and no correlation with prothrombin time.     

Phospholipid oxidation catalyzed by cytochrome c in liposomes

Oxidation of liposome phospholipids has been studied in the presence of cytochrome c. Sonicated vesicles of soya bean or egg-yolk lipids, or purified phospholipid preparations, were treated with oxidized cytochrome c at a 10:4 lipid/protein ratio (w/w). Lipid peroxidation was examined by oxygen polarography, gas-liquid chromatography (GLC) and the thiobarbituric acid test. Oxidized, but not reduced, cytochrome effectively catalyzes lipid oxidation under these conditions. Oxygen consumption and disappearance of unsaturated fatty acids follow closely similar patterns, the O2 consumption rate showing a maximum (1.53 mol O2/min per mol heme) shortly before fatty acid loss reaches its peak. GLC and O2 consumption data suggest that monohydroperoxides are the most abundant oxidized species in the system. The thiobarbituric acid reaction, however, appears only to be of qualitative value in peroxidation studies. In order to test the mechanism through which oxidation occurs in our system, the effect of liposome composition and the presence of antioxidants was tested, both on cytochrome c binding to bilayers and on O2 consumption. Oxidized and reduced cytochrome c bind the lipid bilayers with similar affinity, but only the oxidized form is active in autoxidation. Antioxidants do not modify either cytochrome c binding to sonicated liposomes. Lipid composition does influence considerably cytochrome binding, and O2 consumption is correspondingly altered. Studies with various antioxidants and inhibitors suggest that both free radicals and singlet oxygen may be involved in the process under study.     

Laser light-scattering characterization of mitochondrial complex III-Triton X-100-phospholipid mixed micelles

Bovine-heart mitochondrial complex III was purified in the presence of Triton X-100, and the size and shape of the resulting protein-surfactant-phospholipid mixed micelles were investigated by laser light-scattering. The protein appears to be present in the form of a dimer, irrespective of temperature (between 25 and 40 degrees C) and protein concentration (between 0.5 and 5 mg/ml). The molecular weight of the micelle increases with temperature from 600 000 (25 degrees C) to 692 000 (40 degrees C). The variation of the solvent second virial coefficient in this temperature range suggests that, with increasing temperature, some of the free surfactant molecules become integrated in the mixed micelles. The average quadratic radius of gyration of these is of 42 +/- 5 nm, corresponding in our case to an ellipsoidal shape.     

Physiological properties of microbial population as a basis for graphical solution of multi-stage continuous cultivations

A proposal for a graphical solution of the design of a multi-stage continuous culture system is presented. The holding times in individual stages are derived from the reciprocal values of the growth rate and of the product formation rate and plotted against the cell and product concentrations. Characteristic changes of physiologically significant paramaters are then projected on these production curves. Dedicated to Academican Ivan Málek on the occasion of his 60th birthday     

Untersuchung der eiweißspindeln des kakteen-X-Virus mit fluoreszierenden Antikörpern

A?koliv na zá kladě mnoha pokus? se p? edpokládalo, ?e tzv. bÍlkovinná v?etena v buňkách tzn. buně?né inkluse X-viru kaktus? (Ca XV), jsou slo?ena z ?etních prodlou?ených ?ásti Ca XV, p?esto to dosud nebylo proká zá no. Proto jsme se pokusili pomocÍ fluoreskujÍcÍch protilátek doká ?at, ?e bilkovinná v?etena jsou skute?ně agregáty virových ?ástic. V těto práci jsme pouzili tzv. nep?Ímé metody. Nejprve jsme p? sobili na buňky obsahujÍci tato v?etena homologiokým antisé rem proti Ca XV, zÍskanym imunizacÍ králÍk? a teprve potom jsme buňky vlo?ili do roztoku fluoreskujicÍch protilátek proti králicimu γglobulinu. BÍlkovinná v?etena svitila potom ve fluorescen?nÍm mikroskopu silně ?lutozeleně (bylopou?ito fluoresceinisothiocyaná tu). Tato fluorescence ná m uká zala, ?e nastala pozitivnÍ reakce a ?e bÍlkovianá v?etena jsou slo?ena z virových ?ástic. ?etné kontrolnÍ pokusy potvrdily ná? základnÍ pokus.