Acetylcholine synthesis and possible functions during sea urchin development

Cholinergic neurotransmitter system molecules were found to play a role during fertilisation and early cell cycles of a large number of invertebrate and vertebrate organisms. In this study, we investigated the presence and possible function of choline acetyltransferase (ChAT, the biosynthetic enzyme of acetylcholine) in gametes of the sea urchin, Paracentrotus lividus, through localisation and functional studies. ChAT-like molecules were detected in oocytes, mature eggs and zygotes with indirect immunofluorescence methods. Positive immunoreactivity was found in the ovarian egg cytoplasm and surface as well as at the zygote surface. This suggests the eggs' capacity to autonomously synthesise acetylcholine (ACh), the signal molecule of the cholinergic system. Acetylcholinesterase (AChE, the lytic enzyme of acetylcholine) was also found in ovarian eggs, with a similar distribution; however, it disappeared after fertilisation. Ultrastructural ChAT localisation in sperms, which was carried out with the immuno-gold method, showed immunoreactivity in the acrosome of unreacted sperms and at the head surface of reacted sperms. In order to verify a functional role of ACh during fertilization and sea urchin development, in vivo experiments were performed. Exposure of the eggs before fertilisation to 1 mM ACh + 1 microM eserine caused an incomplete membrane depolarisation and consequently enhanced polyspermy, while lower concentrations of ACh caused developmental anomalies. The exposure of zygotes to 0,045 AChE Units/mL of sea water caused developmental anomalies as well, in 50% of the embryos. Altogether, these findings and other previously obtained results, suggest that the cholinergic system may subserve two different tasks during development, according to which particular type of ACh receptor is active during each temporal window. The first function, taking place in the course of fertilisation is a result of autonomously synthesised ACh in sperms, while the second function, taking place after fertilisation, is due to maternal ChAT molecules, assembled on the oolemma along with egg maturation and fertilisation processes.     

One step purification of glucose-6-phosphate dehydrogenase from brain areas by immunoaffinity chromatography

Glucose-6-phosphate dehydrogenase was purified from rabbit brain cortex using a single immunoaffinity chromatographic step and was contaminated only by a 50 kDa protein. The proteins, separated by SDS-PAGE, were sequenced: the glucose-6-phosphate dehydrogenase was blocked at the N-terminal, the co-eluted protein was similar to -tubulin. Our technique can be applied to purification and sequencing of the enzyme from brain areas or to measure its turnover rate in cultured cells.     

Pre-adipocytes commitment to neurogenesis 1: Preliminary localisation of cholinergic molecules

A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimer's disease and motor neuron disease). A stem cell population (i.e. pre-adipocytes) is present in the adipose stromal compartment. Pre-adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre-adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre-adipocytes, in parallel with neural morphology. The passage of the choline-acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic-like phenotype from pre-adipocytes.     

Evidence for Ectopic Aerobic ATP Production on C6 Glioma Cell Plasma Membrane

Extracellular ATP plays a pivotal role as a signaling molecule in physiological and pathological conditions in the CNS. In several glioma cell lines, ATP is a positive factor for one or more characteristics important for the abnormal growth and survival of these cells. This work presents immunofluorescence and biochemical analyses suggesting that an aerobic metabolism, besides mitochondria, is located also on the plasma membrane of C6 glioma cells. An ATP synthesis coupled to oxygen consumption was measured in plasma membrane isolated from C6 cells, sensitive to common inhibitors of respiratory chain complexes, suggesting the involvement of a putative surface ATP synthase complex. Immunofluorescence imaging showed that Cytochrome c oxydase colocalized with WGA, a typical plasma membrane protein, on the plasma membrane of glioma cells. Cytochrome c oxydase staining pattern appeared punctuate, suggesting the intriguing possibility that the redox chains may be expressed in discrete sites on C6 glioma cell membrane. Data suggest that the whole respiratory chain is localized on C6 glioma cell surface. Moreover, when resveratrol, an ATP synthase inhibitor, was added to culture medium, a cytostatic effect was observed, suggesting a correlation among the ectopic ATP synthesis and the tumor growth. So, a potential direction for the design of new targets for future therapies may arise.     

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.     

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.