The effect of exposure to radiofrequency LTE signal and coexposure to mitomycin-C in Chinese hamster lung fibroblast V79 cells

This study aims to investigate the cellular effects of radiofrequency exposure, 1950 MHz, long-term evolution (LTE) signal, administered alone and in combination with mitomycin-C (MMC), a well-known cytotoxic agent. Chinese hamster lung fibroblast (V79) cells were exposed/sham exposed in a waveguide-based system under strictly controlled conditions of both electromagnetic and environmental parameters, at specific absorption rate (SAR) of 0.3 and 1.25 W/kg. Chromosomal damage (micronuclei formation), oxidative stress (reactive oxygen species [ROS] formation), and cell cycle progression were analyzed after exposure and coexposure. No differences between exposed samples and sham-controls were detected following radiofrequency exposure alone, for all the experimental conditions tested and biological endpoints investigated. When radiofrequency exposure was followed by MMC treatment, 3 h pre-exposure did not modify MMC-induced micronuclei. Pre-exposure of 20 h at 0.3 W/kg did not modify the number of micronuclei induced by MMC, while 1.25 W/kg resulted in a significant reduction of MMC-induced damage. Absence of effects was also detected when CW was used, at both SAR levels. MMC-induced ROS formation resulted significantly decreased at both SAR levels investigated, while cell proliferation and cell cycle progression were not affected by coexposures. The results here reported provide no evidence of direct effects of 1950 MHz, LTE signal. Moreover, they further support our previous findings on the capability of radiofrequency pre-exposure to induce protection from a subsequent toxic treatment, and the key role of the modulated signals and the experimental conditions adopted in eliciting the effect.     

Purification and characterization of a novel high molecular weight exotoxin produced by red tide phytoplankton,Alexandrium tamarense

Our recent studies have demonstrated that the aqueous extract prepared from Alexandrium tamarense, a harmful red tide phytoplankton, showed cytotoxicity on Vero cells. In this study, the toxic substance was purified from the culture supernatant of A. tamarense. Based on the gel‐filtration profile, the molecular mass of a purified toxin was estimated to be about 1,000 kDa. On sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis, a main band with molecular mass of 1,000 kDa was detected with periodic acid‐Schiff (PAS) staining, but no protein bands were detected by Coomassie brilliant blue (CBB) protein staining. Sugar composition analysis of the toxin suggested that the toxin contains galactose, fucose, mannose, N‐acetylglucosamine, xylose, and other minor saccharides, whereas no significant levels of amino acids were detected by amino acid analysis. These results suggest that the toxin is a polysaccharide‐based compound. The toxin showed cytotoxic effects on various cell lines in a concentration‐dependent manner. Among the cell lines tested, U937 cells were the most susceptible to the toxin. In U937 cells treated with the toxin, a typical apoptotic nuclear morphological change and DNA fragmentation were observed. This is the first report demonstrating that a polysaccharide‐based toxin isolated from red tide phytoplankton can induce apoptotic cell death. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:405–415, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20253     

AQP1 expression analysis in human diseases: implications for proteomic characterization

Aquaporin (AQP)1 belongs to a ubiquitous family of water channel proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs existing in almost all organs and tissues. Currently, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules, such as AQP1, or also glycerol and other small solutes. The genomic, structural and functional aspects of AQP1 are briefly described. An in-depth discussion is devoted to proteomic approaches that are useful for identifying and characterizing AQP1, mainly through electrophoretic techniques combined with different extraction procedures followed by mass spectrometry analysis. Moreover, the relevance of AQP1 in human diseases is also explained. Its role in human tumors and, in particular, those of the kidney (e.g., clear cell renal carcinoma) is discussed.     

Changes of the hepatic proteome in hepatitis B-infected mouse model at early stages of fibrosis

Liver fibrosis (LF) is the accumulation of extracellular matrix (ECM) proteins due to chronic liver injury. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) to perform a comparative analysis of cytosolic and nuclear protein patterns of nontransgenic (NTg) and HBV transgenic (Tg) mice livers at early stages of fibrosis. We identified several candidate proteins, involved in a variety of pathways, which could be used as putative biomarkers for LF early detection.     

Harpooning the Cvt complex to the phagophore assembly site

Autophagy is a catabolic process employed by eukaryotes to degrade and recycle intracellular components. When this pathway is induced by starvation conditions, part of the cytoplasm and organelles are sequestered into double-membrane vesicles called autophagosomes, and delivered into the lysosome/vacuole for degradation. In addition to the random bulk elimination of cytoplasmic contents, the selective removal of specific cargo molecules has also been described. These selective types of autophagy are characterized by the recruitment of the cargo destined for degradation in close proximity to the forming double-membrane vesicle that results in an exclusive incorporation (that is, without bulk cytoplasm). A number of factors required for selective types of autophagy have been identified. In particular, we have recently shown that actin and the actin-binding Arp2/3 protein complex are involved in the cytoplasm to vacuole targeting (Cvt) pathway, a yeast selective type of autophagy. The contribution at a molecular level of these factors, however, remains unknown. In this addendum, we present mechanistic models that take into account possible roles of actin and the Arp2/3 complex in the Cvt pathway.     

Mutation of Y179 on phospholipase D2 (PLD2) upregulates DNA synthesis in a PI3K-and Akt-dependent manner

Phospholipase D2 (PLD2), one of the two mammalian members of the PLD family, has been implicated in cell proliferation, transformation, tumor progression and survival. However, as precise mechanistic details are still unknown, we investigated here if the PLD2 isoform would signal through the PI3K/AKT pathway. Transient expression of PLD2 in COS7 cells with either the WT or with a Y179F mutant, resulted in an increased basal phosphorylation of AKT in residues T308 and S473, in a PI3K-dependent manner. Transfection of PLD2-Y179F (but not the wild type) caused an increased (>2-fold) DNA synthesis even in the absence of extracellular stimuli. Other signaling mechanisms downstream such PLD/PI3K dependence (that might lead to DNA synthesis regulation) were further studied. PLD2-Y179F caused an increase in phosphorylation of p42/p44 ERK and in the expression of G0/G1 phase transition markers (p21 CIP, PCNA), and these effects, too, were dependent on PI3K. Interestingly, Akt, once activated induced the phosphorylation of PLD2 on residue T175, an effect that was inhibited by LY296004. Lastly, if PLD2-Y179F is further mutated in residue K758 (PLD2 Y179F-K758R), which renders inactive a catalytic site, DNA synthesis is then abrogated, indicating that the activity of the enzyme (i.e. synthesis of PA) is necessary for the observed effects. In conclusion, the unavailability of residue Y179 on PLD2 to become phosphorylated leads to an augmentation of DNA synthesis concomitantly with MEK and AKT phosphorylation, in a process that is dependent on PI3K and independent of any extracellular stimuli. This might be critical for the maintenance of the PLD2-regulated proliferative status.     

Ultrastructural Analysis of Nanogold-labeled Endocytic Compartments of Yeast Saccharomyces cerevisiae Using a Cryosectioning Procedure

Yeast Saccharomyces cerevisiae has been a valuable model organism for the study of the endosomal system of eukaryotic cells. Morphological analyses, however, have been limited because of the lack of specific protein markers and of procedures that lead to a satisfactory ultrastructural resolution. We have recently developed an immunoelectron microscopy (IEM) protocol adapted from the Tokuyasu method to prepare cryosections from mildly fixed yeast. This novel approach allows excellent cell preservation and a unique resolution of the yeast morphology. Here, we present a protocol that combines this procedure with the specific labeling of the various endosomal compartments with positively charged Nanogold. In particular, we show that this new protocol generates excellent results when applied for the examination of early and late endosomes, and of mutants with an endosomal trafficking defect. Importantly, this method is compatible with immunogold labeling of protein markers, and it is consequently appropriate for localization studies of both resident and cargo proteins. This new IEM protocol will be a valuable tool for the large community of scientists using yeast as a model system to investigate the membrane transport and the biogenesis of the endosomal system. (J Histochem Cytochem 57:801–809, 2009)