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Epidemiological and experimental evidence indicated that hyperhomocysteinemia is associated with neurodegeneration. However, homocysteine neurotoxic effects have been so far investigated mostly by employing homocysteine concentrations (≥100 µM) much higher than homocysteine mean plasma levels (20 µM) observed in patients with neurodegenerative disorders. While evaluating the effects of a prolonged exposure to ~20 µM homocysteine in neuronal-like differentiated SH-SY5Y cells, we observed a 35 % loss of cell viability and a four-fold increase in reactive oxygen species levels in cells incubated with homocysteine for five days compared with controls. Moreover, homocysteine increased by 30 % and around two-fold, respectively, the Comet-positive cell number and DNA damage indexes (tail length, T-DNA, olive tail moment) compared with controls. Cell response to homocysteine-induced DNA damage involved the up-regulation of Bax and, at a greater extent, Bcl-2, but not caspase-3, in association with a p53-independent increase of p21 levels; concomitantly, also p16 levels were increased. When looking at time-dependent changes in cyclin expression, we found that a significant up-regulation of cyclins D1, A1, E1, but not B1, concomitant with p21 down-regulation, occurred in cells incubated with homocysteine for three days. However, in line with the observed increase of p21 and p16 levels, a five days incubation with homocysteine induced cyclin down-regulation accompanied by a strong reduction of phosphorylated pRB amounts. These results suggest that, when prolonged, the exposure of neuronal-like cells to mildly elevated homocysteine concentrations triggers oxidative and genotoxic stress involving an early induction of cyclins, that is late repressed by G1-S check-point regulators.

The expression of the protein crosslinking enzyme tissue transglutaminase (TG2, tTG), the ubiquitous member of transglutaminase family, can be regulated by multiple factors. Although it has been suggested that TG2 can be involved in apoptotic cell death, high levels of enzyme have also been associated with cell survival in response to different stimuli. Furthermore, evidence indicates that increases in TG2 production cause enzyme translocation to cell membrane. Cell stress can also lead to TG2 accumulation on the cell surface and in the extracellular matrix resulting in changes in cell-matrix interactions.Here, we discuss the underlying mechanisms of TG2 up-regulation induced by various stimuli including glutamate exposure, calcium influx, oxidative stress, UV, and inflammatory cytokines.These findings agree with a postulated role for transglutaminases in molecular mechanisms involved in several diseases suggesting that cross-linking reactions could be a relevant part of the biochemical changes observed in pathological conditions.

Neuroprotection is conceived as one of the potential tool to prevent or slow neuronal death and hence a therapeutic hope to treat neurodegenerative diseases, like Parkinson’s and Alzheimer’s diseases. Increase of oxidative stress, mitochondrial dysfunction, excitotoxicity, inflammatory changes, iron accumulation, and protein aggregation have been identified as main causes of neuronal death and adopted as targets to test experimentally the putative neuroprotective effects of various classes of drugs. Among these agents, antiepileptic drugs (AEDs), both the old and the newer generations, have shown to exert protective effects in different experimental models. Their mechanism of action is mediated mainly by modulating the activity of sodium, calcium and potassium channels as well as the glutamatergic and GABAergic (gamma-aminobutyric acid) synapses. Neurological pathologies in which a neuroprotective action of AEDs has been demonstrated in specific experimental models include: cerebral ischemia, Parkinson’s disease, and Alzheimer’s disease. Although the whole of experimental data indicating that neuroprotection can be achieved is remarkable and encouraging, no firm data have been produced in humans so far and, at the present time, neuroprotection still remains a challenge for the future.

A systematic evaluation of mechanical and biocompatibility properties of different volume fractions of hydroxyapatite whiskers in comparison with three commercial dental composites filled with micro- and nanosilica particles was carried out. Six groups with different hydroxyapatite whiskers mass fractions were taken into account in order to be compared with the performances of silica particles based composites group. Flexural properties were evaluated via a universal testing machine (2.5 kN Zwick Line) with a 2 kN load-cell (sensitivity 0.001 N). The test was replicated 10 times for the seven experimental groups to better identify statically the significance of the mechanical performances data. MTT quantitative colorimetric assay was performed in order to evaluate the mitochondrial activity of living cells exposed to different resin composites. Data obtained show better interfacial interaction with filler/matrix until 20 wt% of hydroxyapatite whiskers partially replaced silica particles filler. After this threshold, the mechanical performances decrease dramatically due to both the hydroxyapatite agglomerates formation and the low degree of resin conversion. In addition, biocompatibility test showed less cytotoxic effect with the addition of 20 wt% of hydroxyapatite in comparison with higher rates.

Heme oxygenase-1 (HO-1) plays a crucial role in oxidative stress processes, apoptosis and cell differentiation. Further, some proteins related to cell cycle including cyclins and p21 are important markers of astrocyte cultures. Aim of investigation was to study the effects of cholinergic precursors (choline, CDP-choline, Acetylcholine and α-Glyceril-Phosphorylcholine) on HO-1 and p21 expression during astroglial cell proliferation and differentiation in primary cultures at 14 and 35 days in vitro (DIV) treated for 24 h with choline metabolites. Our results showed a slight reduction of HO-1 expression (data not statistical significant) in astroglial cell cultures treated with CDP-choline at 14 DIV and 35 DIV. On the contrary, ACh and choline induced a significant increase of HO-1 expression in 14 DIV astrocyte cultures. Surprisingly, choline and ACh dramatically reduced HO-1 expression at 35 DIV. A slight decrease not statistical significant was detectable for α-GPC at 14 DIV and particularly significant at 35 DIV. Data concerning p21 expression, a well known protein inhibiting cell cycle, evidenced a significant increase at 14 and 35 DIV after α-GPC treatment. CDP-choline treatment caused a high increase of p21 expression in 14 DIV astrocyte cultures, but no modification at 35 DIV. Instead, ACh treatment induced a marked increment of p21 expression at 35 DIV. Our data suggest that cholinergic precursors modulate HO-1 and p21 expression during astroglial cell proliferation and differentiation in culture and could be considered a tool to study the induced effects of ischemia and hypoxia diseases in some in vitro models to prevent and reduce its effects after treatment with cholinergic drugs.

In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 DIV astrocyte cultures pretreated or not with 0.5 mM glutamate for 24 h and than maintained under chronic or acute treatment with 50 μM R(+)enantiomer or raceme alpha-lipoic acid (ALA). GFAP expression significantly increased after (R+)enantiomer acute-treatment and also in glutamate-pretreated ones. Vimentin expression increased after R(+)enantiomer acute-treatment, but it decreased after raceme acute-treatment. Nestin expression drastically increased after acute raceme-treatment in glutamate-pretreated or not cultures, but significantly decreased after (R+)enantiomer acute and chronic-treatments. Cyclin D1 expression increased in raceme acute-treated cultures pretreated with glutamate. MAP-kinase expression slightly increased after (R+)enantiomer acute treatment in glutamate-pretreated or unpretreated ones. These preliminary findings may better clarify antioxidant and metabolic role played by ALA in proliferating and differentiating astrocyte cultures suggesting an interactive cross-talk between glial and neuronal cells, after brain lesions or damages.

High levels of homocysteine promote cell damage mainly through induction of oxidative stress, endoplasmic reticulum (ER) stress, and activation of pro-inflammatory factors. The effects of homocysteine were here examined in the continuously dividing neuroblastoma cell line Neuro2a. Cell treatment with homocysteine (100–500 μM) for 4 h increased ROS production while reducing cell viability in a dose-dependent manner. Cell exposure to 250 μM homocysteine was able to induce transglutaminase 2 up-regulation and increased in situ transglutaminase activity. These effects were prevented by the incubation with the transglutaminase activity inhibitor cystamine. Homocysteine also induced NF-κB activation that seemed associated with transglutaminase 2 up-regulation since the specific NF-κB inhibition by SN50 was able to reduce transglutaminase expression and activity levels. In the light of these observations, it may be postulated that TG2 up-regulation is involved in cell response to homocysteine-induced stress, in which NF-κB activation plays also a pivotal role.

OBJECTIVE: To investigate the effects of leptin on platelet aggregation and platelet free calcium (Ca2+) concentrations, and the role of the long form of leptin receptor (ObRb) and the phospholipase C (PLC) in mediating leptin effects on platelet function. DESIGN: Cross-sectional, clinical study. SETTING: Outpatient's Service for Prevention and Treatment of Obesity at the University Hospital of Messina, Italy. SUBJECTS: A total of 19 healthy, 14 overweight, and 16 obese male subjects. MEASUREMENTS: ADP-induced platelet aggregation and platelet Ca2+ were measured after incubation of platelet-rich plasma with leptin alone 5-200 ng/ml, leptin 200 ng/ml and anti-human leptin receptor long-form antibody (ObRb-Ab) 5-10 μl, or leptin 200 ng/ml and PLC inhibitor U73122 0.5-1 nmol/l. RESULTS: Platelet stimulation with leptin lead to a significant and dose-dependent increase in platelet aggregation in healthy subjects. This effect was blunted in overweight, and strongly reduced in obese subjects. Similarly, the incubation with leptin induced a significant and dose-dependent increase in platelet free calcium, which was blunted in overweight and obese patients. The effect of leptin on platelet aggregation and platelet Ca2+ was completely abated by the anti-ObRb-Ab and the PLC inhibitor U73122. CONCLUSIONS: Leptin produces a dose-dependent enhancement of ADP-induced platelet aggregation in humans. Platelet aggregation response to leptin is blunted, but not completely abolished in overweight/obese subjects, thus suggesting that platelet may represent a site of leptin resistance in human obesity. Leptin increases platelet free calcium in a dose-dependent manner. The inhibition of PLC completely abates the effect of leptin on both platelet aggregation and Ca2+ levels. These findings suggest that signaling pathway other than JAK–STAT tyrosine phosphorylation (ie PLC and calcium) may be involved in mediating the prothrombotic action of leptin.

Effects of acetylcholine and of the cholinergic precursors choline, cytidine 5′-diphosphocholine (CDP-choline) and α-glyceril-phosphorylcholine (α-GPC) on transglutaminase (TG) and cyclin D1 expression were studied in primary astrocyte cultures by confocal laser microscopy (CLSM) with monodansyl-cadaverine uptake as a marker of enzyme activity and by immunochemistry (Western blotting). CLSM analysis showed an increased cytofluorescence in 0.1 μM choline-treated astrocytes. Treatment with CDP-choline dose-dependently increased TG. A total of 1 μM CDP-choline exposure in 14 days in vitro (DIV) astrocyte cultures increased cytofluorescence. A total of 1 μM α-GPC 24 h-treated cultures revealed increased cytofluorescence both in cytosol and nuclei. Western blot analysis showed an increased TG expression in cultures exposed for 24 h to 1 μM choline or α-GPC, whereas in 24 h 1 μM CDP-choline and acetylcholine-treated astrocytes TG expression was unaffected. Treatment with 1 μM acetylcholine reduced TG expression at 21 DIV. In cultures at 14 and 35 DIV cholinergic precursor treatment for 24 h induced a marked down-regulation of cyclin D1 expression, with reduced cyclin D1 expression in 1 μM α-GPC treated astrocytes. Our data suggest a role of cholinergic precursors investigated independent from acetylcholine on maturation and differentiation of astroglial cells in vitro, rather than on their growth, proliferation and development in culture.

Neurodegeneration induced by excitotoxicity is a common feature in various neurological disorders. This pathological condition is caused by prolonged stimulation of glutamate receptor subtypes, followed by both intracellular Ca2+ overload and activation of specific genes, resulting in synthesis of enzymes involved in cell stress response. Using experimental in vitro models of excitotoxicity, we demonstrated that glutamate exposure up-regulated tissue transglutaminase in primary cultures of both cerebellar granule cells and astrocytes. These changes were consequent to receptor-mediated Ca2+ influx, as demonstrated by the inhibition with selective antagonists, MK-801 and GYKI 52466. Early increases in different transglutaminase isoforms were also observed in global cerebral ischemia, which closely resembles neuronal damage caused by NMDA receptor activation. These findings agree with a postulated role for transglutaminases in molecular mechanisms of several neurodegenerative diseases. Indeed, increased cross-linking reactions could be of pathologic relevance, as part of biochemical changes observed in neurological disorders.