AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC₅₀ value of 15 μM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na⁺ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.     

Interaction of an artificial antimicrobial peptide with lipid membranes

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.     

Isolation, purification and quantification of BRCA1 protein from tumour cells by affinity perfusion chromatography

A new procedure for the isolation, purification and quantification of the product of the oncosuppressor gene brca1 in breast tissues, was carried out. It involves internal cell protein [³⁵S]methionine labelling followed by two perfusion chromatographies. The first one is heparin affinity chromatography, to purify all of the cell DNA-binding proteins. A subsequent specific immunoprecipitation of BRCA1 protein was performed with an antibody raised against BRCA1. The immune complex was isolated using the second chromatographic step, Protein A affinity chromatography. The amount of BRCA1 expressed by cells was expressed as a ratio, in percent, calculated as follows: 100× amount of labelled DNA-binding proteins (dpm) that bound specifically to the anti-BRCA1 polyclonal antibodies (K-18)/amount of whole labelled DNA-binding protein (dpm) purified on a heparin column. Applications to MCF7 and T-47D human breast tumour cell lines, which were treated or not using 2 mM sodium butyrate demonstrated an increase in BRCA1 protein expression.     

Mutually Independent Cyclic AMP and Sodium Responses to Nerve Growth Factor in Embryonic Chick Dorsal Root Ganglia

Abstract: Chick embryo dorsal root ganglia display a rapid and transient rise in their cyclic AMP content when presented with nerve growth factor. These ganglia also depend on nerve growth factor for control of their intracellular Na⁺ and K⁺ levels. A sequential relationship between the cyclic AMP and Na⁺ responses is not readily apparent. Incubation of chick sensory ganglia in a sodium-free medium does not prevent the cyclic AMP response to nerve growth factor from occurring. When ganglia are first incubated with ouabain for 6 h, presentation of nerve growth factor elicits a cyclic AMP response, but no Na⁺ response. The cyclic AMP response therefore does not depend on the Na⁺ environment. An initial presentation of nerve growth factor to the ganglia for 30 min, followed by its withdrawal and subsequent re-administration at different intervals over several hours failed to result in a second cyclic AMP response. Nevertheless, the expected Na⁺ behaviors were still observed. Dibutyryl cyclic AMP is capable of eliciting a cyclic AMP response in chick sensory ganglia after 6 h of nerve growth factor deprivation. When both agents were presented simultaneously to the ganglia, only a single cyclic AMP response was obtained, corresponding in time to the response elicited by dibutyryl cyclic AMP alone-indicating that this drug acts on the NGF-sensitive cells. At the same time dibutyryl cyclic AMP alone failed to result in a Na⁺ response, leading one to conclude that the cyclic AMP response to nerve growth factor is truly not mediating the Na⁺ response. Additional support for the mutual independence of these two short-latency responses is provided by the apparent inability of nerve growth factor to cause a cyclic AMP response in chick embryo sympathetic ganglia, another traditional target for the factor, which is capable of displaying a Na⁺ response.     

The clastogenic effects of chronic exposure to particulate and soluble Cr(VI) in human lung cells

Hexavalent chromium (Cr(VI)) is a well-designated human lung carcinogen, with solubility playing an important role in its carcinogenic potential. Although it is known that particulate or water-insoluble Cr(VI) compounds are more potent than the soluble species of this metal, the mechanisms of action are not fully elucidated. In this study, we investigated the hypothesis that the difference in potency between particulate and soluble Cr(VI) is due to more chronic exposures with particulate chromate because it can deposit and persist in the lungs while soluble chromate is rapidly cleared. Chronic exposure to both insoluble lead chromate and soluble sodium chromate induced a concentration and time-dependent increase in intracellular Cr ion concentrations in cultured human lung fibroblasts. Intracellular Pb levels after chronic exposure to lead chromate increased in a concentration-dependent manner but did not increase with longer exposure times up to 72 h. We also investigated the effects of chronic exposure to Cr(VI) on clastogenicity and found that chronic exposure to lead chromate induces persistent or increasing chromosome damage. Specifically, exposure to 0.5 μg/cm² lead chromate for 24, 48 and 72 h induced 23, 23 and 27% damaged metaphases, respectively. Contrary to lead chromate, the amount of chromosome damage after chronic exposure to sodium chromate decreased with time. For example, cells exposed to 1 μM sodium chromate for 24, 48 and 72 h induced 23, 13 and 17% damaged metaphases, respectively. Our data suggest a possible mechanism for the observed potency difference between soluble and insoluble Cr(VI) compounds is that chronic exposure to particulate Cr(VI) induces persistent chromosome damage and chromosome instability while chromosome damage is repaired with chronic exposure to soluble Cr(VI).     

Effects of Selenium Supplementation on Antioxidant Defense and Glucose Homeostasis in Experimental Diabetes Mellitus

The objective of this study was to investigate the effects of different forms of Se supplementation on the antioxidant defense and glucose homeostasis in experimental diabetes. Sodium selenate (SS) or selenomethionine (SM) were administered (2 μmol Se kg⁻¹ day⁻¹) via orogastric route to streptozotocine (STZ)-induced diabetic rats in addition to basal diet for 12 weeks. Glucose levels in whole blood, glutathione peroxidase (GSH-Px) activity in erythrocytes, Se and fructosamine levels in plasma were evaluated monthly. Plasma Se levels increased significantly in all diabetic groups compared to basal measurements, being more prominent in SM group [p(SM₃/SM₀) = 0.018]. The increase in GSH-Px activities was significant at the end of the second month in SS [p(SS₂/SS₀) = 0.028], whereas at the end of the third month in SM the value was lower [p(SM₃/SM₀) = 0.018] and the unsupplemented diabetic control (DC) groups, p(DC₃/DC₀) = 0.012. Glucose increased significantly only in DC group. Fructosamine increased gradually in all diabetic groups, being significant in DC and SS groups. At the end of the third month, highest fructosamine levels were observed in SS group, which were significantly higher than the SM group [p(SM/SS) = 0.010]. In conclusion, Se augmented the antioxidant defense by increasing GSH-Px activity and this effect was more prominent when Se was supplemented as SM, which exerted positive effects also on glucose homeostasis.     

Gender-dependent effects of selenite on the perfused rat heart

Gender differences are related to the manner in which the heart responds to chronic and acute stress conditions of physiological and pathological nature. Depending on dose, sodium selenite acts as an antioxidant proven to have beneficial effects in several pathological conditions G. Drasch, J. Schopfer, and G. N. Schrauzer, Selenium/cadmium ratios in human prostates: indicators of prostate cancer risk of smokers and non-smokers, and relevance to the cancer protective effects of selenium,Biol. Trace Element Res. 103(2), 103–107 (2005); R. G. Kasseroller and G. N. Schrauzer, Treatment of secondary lymphedema of the arm with physical decongestive therapy and sodium selenite: a review,Am. J. Ther. 7(4), 273–279 (2000); G. N. Schrauzer, Anticarcinogenic effects of selenium,Cell. Mol. Life Sci. 57(13–14), 1864–1873 (2000); I. S. Palmer and O. E. Olson, Relative toxicities of selenite and selenate in the drinking water of rats,J. Nutr. 104(3), 306–314 (1974). To date, little is known about the gender-dependent direct effects of toxic doses of selenite on electrophysiology of the cardiovascular system H. A. Schroeder and M. Mitchener, Selenium and tellurium in rats: effect on growth, survival and tumors,J. Nutr. 101(11), 1531–1540 (1971); G. N. Schrauzer, The nutritional significance, metabolism and toxicology of selenomethionine,Adv. Food Nutr. Res. 47, 73–112 (2003). In the present study, the effects of in vitro toxic concentrations of sodium selenite ranging from 10^-6 M to 10^-3 M were tested on both male and female rat heart preparations. The toxic effects seen in an electrocardiogram and left ventricular pressure were dose and sex dependent at most of the tested concentrations. The present study clearly shows that at toxic doses, stress conditions are induced by selenite, resulting in gender-dependent modifications of the heart function. This modification is more pronounced in the contraction cascade of female rats. Males, on the other hand, had been much more affected in excitation-related parameters.     

Comparison of proteomes in various human plasma preparations by two-dimensional gel electrophoresis

Serum or plasma can be utilized in a variety of studies targeted toward the discovery of disease biomarkers. In this study, the proteome profiles of plasma samples prepared using various anticoagulants (EDTA, heparin or citrate), were compared with those of serum using two-dimensional electrophoresis (2-DE). Proteins which evidenced different levels in the plasma and serum were screened and identified using ESI-Q-TOF MS/MS. The proteins which became detectable after the removal of fibrinogen from serum were identified as pigment epithelial differentiating factor (four spots), fetuin-like protein, and the hemopexin precursor. In particular, three proteins, pre-serum amyloid P component, plasma glutathione peroxidase precursor, and tetranectin, evidenced increased volume intensity only in the plasma samples prepared with EDTA.     

Density fractionation of forest soils: methodological questions and interpretation of incubation results and turnover time in an ecosystem context

Soil organic matter (SOM) is often separated by physical means to simplify a complex matrix into discrete fractions. A frequent approach to isolating two or more fractions is based on differing particle densities and uses a high density liquid such as sodium polytungstate (SPT). Soil density fractions are often interpreted as organic matter pools with different carbon (C) turnover times, ranging from years to decades or centuries, and with different functional roles for C and nutrient dynamics. In this paper, we discuss the development and mechanistic basis of common density-based methods for dividing soil into distinct organic matter fractions. Further, we directly address the potential effects of dispersing soil in a high density salt solution on the recovered fractions and implications for data interpretation. Soil collected from forested sites at H. J. Andrews Experimental Forest, Oregon and Bousson Experimental Forest, Pennsylvania was separated into light and heavy fractions by floatation in a 1.6 g cm⁻³ solution of SPT. Mass balance calculations revealed that between 17% and 26% of the original bulk soil C and N content was mobilized and subsequently discarded during density fractionation for both soils. In some cases, the light isotope was preferentially mobilized during density fractionation. During a year-long incubation, mathematically recombined density fractions respired ∼40% less than the bulk soil at both sites and light fraction (LF) did not always decompose more than the heavy fraction (HF). Residual amounts of tungsten (W) present even in well-rinsed fractions were enough to reduce microbial respiration by 27% compared to the control in a 90-day incubation of O_a material. However, residual W was nearly eliminated by repeated leaching over the year-long incubation, and is not likely the primary cause of the difference in respiration between summed fractions and bulk soil. Light fraction at Bousson, a deciduous site developed on Alfisols, had a radiocarbon-based mean residence time (MRT) of 2.7 or 89 years, depending on the interpretation of the radiocarbon model, while HF was 317 years. In contrast, both density fractions from H. J. Andrews, a coniferous site developed on andic soils, had approximately the same MRT (117 years and 93 years for LF and HF). At H. J. Andrews the organic matter lost during density separation had a short MRT (19 years) and can account for the difference in respired CO₂ between the summed fractions and the bulk soil. Recognition and consideration of the effects of the density separation procedure on the recovered fractions will help prevent misinterpretation and deepen our understanding of the specific role of the recovered organic matter fractions in the ecological context of the soil studied.     

Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na⁺/H⁺ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na⁺/H⁺ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na⁺/H⁺ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.