Efficiency of two different transfection reagents for use with human NTERA2 cells

The teratocarcinoma cell line NTERA2 is recently used in a wide range of researches (from developmental biology to toxicology, for their ability to be induced to neural differentiation. In order to study the genetic potential of these cells, it is needed to use methods for gene silencing and/or mRNA interference, allowing cell viability and further differentiation. To check these features, we simultaneously tested the transfection efficiency of NTERA2, A549 and HeLa cells with Metafectene PRO (Biontex, Germany) and another optimal transfection reagent currently used in our Laboratory, using as a reporter gene the DsRed2 vector (Clontech, Mountain View, CA). Under our culture conditions for NTERA2 and HeLa cells, Metafectene PRO transfection method was found to possess high throughput performance, that allows low concentration rate and low exposure time to excitation light source, thus reducing both toxicity and phototoxicity.     

Evidence for aerobic ATP synthesis in isolated myelin vesicles

Even though brain represents only 2–3% of the body weight, it consumes 20% of total body oxygen, and 25% of total body glucose. This sounds surprising, in that mitochondrial density in brain is low, while mitochondria are thought to be the sole site of aerobic energy supply. These data would suggest that structures other than mitochondria are involved in aerobic ATP production. Considering that a sustained aerobic metabolism needs a great surface extension and that the oxygen solubility is higher in neutral lipids, we have focused our attention on myelin sheath, the multilayered membrane produced by oligodendrocytes, hypothesizing it to be an ATP production site. Myelin has long been supposed to augment the speed of conduction, however, there is growing evidence that it exerts an as yet unexplained neuro-trophic role. In this work, by biochemical assays, Western Blot analysis, confocal laser microscopy, we present evidence that isolated myelin vesicles (IMV) are able to consume O₂ and produce ATP through the operation of a proton gradient across their membranes. Living optic nerve sections were exposed to MitoTracker, a classical mitochondrial dye, by a technique that we have developed and it was found that structures closely resembling nerve axons were stained. By immunohystochemistry we show that ATP synthase and myelin basic protein colocalize on both IMV and optic nerves. The complex of data suggests that myelin sheath may be the site of oxygen absorption and aerobic metabolism for the axons.     

The role of the thiol N-acetylcysteine in the prevention of tumor invasion and angiogenesis

We have extensively studied the effects of N-acetylcysteine (NAC), a cytoprotective drug that can prevent in vivo carcinogenesis. Here we review our findings NAC completely inhibits gelatinolytic activity of metalloproteases and chemotactic and invasive activities of tumor cells. In addition, NAC reduces the number of lung metastases when malignant murine melanoma cells are injected into nude mice. NAC treatment decreases the weight of primary tumors and produces a dose-related increase in tumor latency. Moreover, oral administration of NAC reduces the formation of spontaneous metastases. In experimental metastasis assays, we have found a synergistic reduction in the number of lung metastases after treatment with doxorubicin (DOX) and NAC in nude mice. In tumorigenicity and spontaneous metastasis assays, the combined administration of DOX and oral NAC again has shown synergistic effects on the frequency and weight of primary tumors and local recurrences and completely prevented the formation of lung metastases. The addition of NAC to endothelial cells strongly reduces their invasive activity in response to angiogenic stimuli. NAC inhibited the degradation and release of radiolabeled type IV collagen by activated endothelial cells, indicating that NAC blocks gelatinase activity. Oral administration of NAC reduces the angiogenic response induced by KS tumor cell products, confirming the ability of NAC to inhibit the invasive activity of endothelial cells in vivo and thereby blocking angiogenesis.     

Molecular characterization of the human PLC beta1 gene

Inositide-specific phospholipase C (PLC) signaling constitutes a central intermediate in a number of cellular functions among which the control of cell growth raises a particular interest. Indeed, we have previously shown that nuclear phospholipase C beta1 (PLC beta1) is central for the regulation of mitogen-induced cell growth. We have also assigned by fluorescence in situ hybridization (FISH) analysis the PLC beta1 to human chromosome 20p12. In this study, we have carried out a detailed analysis of the human gene, showing the existence of alternative splicing, which gives rise, besides the two forms (1a and 1b) already shown in rodents, to a new 600 bp smaller form coding for a 110 kDa protein. We have also identified a new exon at the 5', showing no homology with the rodent sequence. Here we provide the complete determination of the exon/intron structure of the gene spanning 250 kb of DNA. We found that the exons are quite small, ranging from 49 to 222 bp, while the introns vary between 108 bp and 34,400 bp. The availability of the understanding of the genome organization of this inositide-specific PLC, which represents a key step of the cell cycle related signaling, could actually pave the way for further genetic analysis of p12 region of human chromosome 20 in diseases involving alterations of the control of cell growth such as malignancies.     

Ricerche sull'infrastruttura del coleottile di avena: I. — Il fascio vascolare in corso di differenziazione

Abstract

STUDIES ON THE INFRASTRUCTURE OF THE OAT COLEOPTILE. I. - THE DEVELOPING XYLEM CELLS. — The writers are engaged with a research program dealing with the morphological changes effected in the cell infrastructure by growth substances. The problem, obviously, requires an intimate knowledge of cell infrastructure of all different tissues of the test used, the oat coleoptile in this case. With this first note, cell infrastructures of the xylem region of the bundle (parenchyma and tracheids) are described, limitedly to the portion 1 mm behind the coleoptile tip, where cell divisions are completely suppressed while cell elongation and differentiation are set on.     

Sea urchin development: an alternative model for mechanistic understanding of neurodevelopment and neurotoxicity

Echinoderm early developmental stages might supply a good tool for toxicity testing in different fields, ranging from environment to food contamination, and in full respect of the 3Rs objectives (reduction, refinement, and replacement of animal experiments) that will eventually lead to the replacement of high vertebrate animal testing in toxicology. Sea urchin is one of the few organismic models considered by the European Agency for Alternative models. Actually, sea urchin embryonic development has been studied for over a century, and the complex nets of intercellular communications leading to the different events are well known, as well the possibility for environmental molecules and their residuals to interfere with such communications, causing developmental anomalies. In particular, the main goal of toxicologists since several years has been to establish a correlation between the cell-to-cell communications occurring during different developmental events and the signals occurring during neurogenesis, with the aim to pursue a mechanistic understanding of these processes and their deviations caused by stressors from different sources.     

Postnatal Expression of Glucose-6-Phosphate Dehydrogenase in Different Brain Areas

The activity of glucose-6-phosphate dehydrogenase (G6PD) was studied in five brain areas of rats aged 5 to 90 days. The areas studied were: the olfactory bulb (OB), cortex, hippocampus, striatum and septum. The G6PD activity increased more than 2-fold from 5 to 90 days in the OB, while it was almost constant in the other areas. At every stage of development, the G6PD activity was significantly higher in the OB than in the other areas. The G6PD pattern was compared with 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR); glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) in order to find synergistic interactions among activities of these enzymes during development. Over the considered period, the activity of 6PGD increased significantly in the OB, while no significant difference in activity was detected in the other areas. GR increased significantly and progressively at each developmental stage in all areas. GPX showed a progressive increase in the OB, while in other areas a significant increase was detected at 90 days only. CAT and SOD showed a different and independent pattern which differred from the G6PD pattern. CAT showed the highest level of activity at 5 days then progressively decreased or was constant until 90 days; SOD had the highest value at 5 days, than it decreased at 10 days and increased from 10 to 90 days. In all areas, G6PD activity showed three electrophoretic bands, whose relative activity changed with development. At histochemical level, we found a marked G6PD activity in the periglomerular zone of the OB, which increased with age, while other areas showed a homogeneous staining. The present results demonstrate that G6PD activity increases in the OB during the developmental stages and there is a coordinated simultaneous activation of 6PGD, GPX and GR. It is likely that this enzyme induction increases the antioxidant defense of periglomerular cells that are subject to a rapid renewal and thus much more exposed to oxidant stress.     

Dietary Triacylglycerols with Palmitic Acid in the sn-2 Position Modulate Levels of N-Acylethanolamides in Rat Tissues

Background

Several evidences suggest that the position of palmitic acid (PA) in dietary triacylglycerol (TAG) influences different biological functions. We aimed at evaluating whether dietary fat with highly enriched (87%) PA in sn-2 position (Hsn-2 PA), by increasing PA incorporation into tissue phospholipids (PL), modifies fatty acid profile and biosynthesis of fatty acid—derived bioactive lipids, such as endocannabinoids and their congeners.

Study Design

Rats were fed for 5 weeks diets containing Hsn-2 PA or fat with PA randomly distributed in TAG with 18.8% PA in sn-2 position (Lsn-2 PA), and similar total PA concentration. Fatty acid profile in different lipid fractions, endocannabinoids and congeners were measured in intestine, liver, visceral adipose tissue, muscle and brain.

Results

Rats on Hsn-2 PA diet had lower levels of anandamide with concomitant increase of its congener palmitoylethanolamide and its precursor PA into visceral adipose tissue phospholipids. In addition, we found an increase of oleoylethanolamide, an avid PPAR alpha ligand, in liver, muscle and brain, associated to higher levels of its precursor oleic acid in liver and muscle, probably derived by elongation and further delta 9 desaturation of PA. Changes in endocannabinoids and congeners were associated to a decrease of circulating TNF alpha after LPS challenge, and to an improved feed efficiency.

Conclusions

Dietary Hsn-2 PA, by modifying endocannabinoids and congeners biosynthesis in different tissues may potentially concur in the physiological regulation of energy metabolism, brain function and body fat distribution.