The plasma membrane plays a central role in cells response to mechanical stress.

The mechanisms by which lymphocytes recognize and interpret mechanical stimuli and translate these into the triggering of select signaling cascades that are critical for lymphocyte function are still not fully understood. In this work, we investigated the association of mechanical stress (MS)-induced changes in membrane physical properties with changes in cytoskeleton dynamics and cell signaling. In Jurkat T cells, MS was associated with the immediate and transient depolymerization of both beta-tubulin and F-actin. The fluidity of the plasma membrane measured in the hydrophobic region of the bilayer, increased 0.5 min post-MS, recovering the initial value in the following 2 min. This effect was accompanied by the rearrangement of lipids in the lateral phase of the plasma membrane, transient lipid rafts' alteration, and membrane hyperpolarization. The consequent increase in cellular [Ca2+] triggered the activation of the transcription factors NFAT, AP-1, and NF-kappaB. Results indicate that the cytoplasmic membrane, through changes in membrane physical properties, senses MS, and transduces an initial physical stimulus into microtubules rearrangements, Ca2+ mobilization, and the subsequent changes in cell signaling.

The role of zinc in the modulation of neuronal proliferation and apoptosis.

Although a requirement of zinc (Zn) for normal brain development is well documented, the extent to which Zn can modulate neuronal proliferation and apoptosis is not clear. Thus, we investigated the role of Zn in the regulation of these two critical events. A low Zn availability leads to decreased cell viability in human neuroblastoma IMR-32 cells and primary cultures of rat cortical neurons. This occurs in part as a consequence of decreased cell proliferation and increased apoptotic cell death. In IMR-32 cells, Zn deficiency led to the inhibition of cell proliferation through the arrest of the cell cycle at the G0/G1 phase. Zn deficiency induced apoptosis in both proliferating and quiescent neuronal cells via the intrinsic apoptotic pathway. Reductions in cellular Zn triggered a translocation of the pro-apoptotic protein Bad to the mitochondria, cytochrome c release, and caspase-3 activation. Apoptosis is the resultant of the inhibition of the prosurvival extracellular-signal-regulated kinase, the inhibition of nuclear factor-kappa B, and associated decreased expression of antiapoptotic proteins, and to a direct activation of caspase-3. A deficit of Zn during critical developmental periods can have persistent effects on brain function secondary to a deregulation of neuronal proliferation and apoptosis.

(-)-Epicatechin and related procyanidins modulate intracellular calcium and prevent oxidation in Jurkat T cells.

This study investigated the effects of (-)-epicatechin (EC), its oligomers, dimer B2 (B2) and trimer C1 (C1), on calcium-dependent cell oxidation. Jurkat T cells were subjected to a Ca(2+) mobilization challenge by replacing Na(+) with K(+) in the incubation media. A 249+/-38% increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed and that effect was prevented by the presence of inhibitors of Ca(2+) mobilization and entrance. The pre-incubation of the cells in the presence of EC, B2 or C1 prevented K(+)-mediated increase in [Ca(2+)](i). IC(50) were 10, 24 and 196 nMfor EC, B2 and C1, respectively. Cell membrane depolarization was affected by K(+), but neither inhibitors of Ca(2+) mobilization nor EC, B2 or C1 modified the increase in membrane potential. An 84+/-7% increase in cell oxidants was observed after cell exposure to K(+). This increase was prevented by the inhibition of Ca(2+) mobilization, NADPH oxidase and protein kinase C, as well as by 10 nM EC, 10 nM B2 or 100 nM C1. In addition, EC and B2 (100 nM) significantly inhibited the activation of the [Ca(2+)](i)-regulated transcription factor NFAT. These results indicate that EC and related oligomers, assayed at physiologically achievable concentrations, can modulate [Ca(2+)](i) and then prevent cell oxidation and other Ca(2+)-regulated events.

alpha-Lipoic acid and N-acetyl cysteine prevent zinc deficiency-induced activation of NF-kappaB and AP-1 transcription factors in human neuroblastoma IMR-32 cells.

This work investigated the capacity of alpha-lipoic acid (LA) and N-acetyl-L-cysteine (NAC) to reduce zinc deficiency-induced oxidative stress, and prevent the activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), and the cross-talk between both activated cascades through beta-Transducin Repeat-containing Protein (beta-TrCP). IMR-32 cells were incubated in control media or media containing variable concentrations of zinc, without or with 0.5 mM LA or 1 mM NAC. Relative to control and zinc supplemented (15 microM Zn) groups, Hydrogen peroxide (H(2)O(2)) and total oxidant cell concentrations were higher, and total glutathione concentrations were lower in the zinc deficient groups (1.5 and 5 microM Zn). Both, LA and NAC, markedly reduced the increase in cell oxidants and the reduction in glutathione concentrations in the zinc deficient cells. Consistent with this, LA and NAC prevented zinc deficiency-induced activation of the early steps of NF- kappaB (IkappaBalpha phosphorylation) and AP-1 [c-Jun-N-terminal kinase (JNK) and p38 phophorylation] cascades, and the high NF-kappaB- and AP-1-DNA binding activities in total cell extracts. Thus, LA and NAC can reduce the oxidative stress associated with zinc deficiency and the subsequent triggering of NF-kappaB- and AP-1-activation in neuronal cells.

Metals in neurodegeneration: involvement of oxidants and oxidant-sensitive transcription factors.

Oxidant-mediated damage and the triggering of oxidant-sensitive transcription factors could be associated with the neurotoxic actions of aluminum, zinc and lead. Aluminum and lead could induce oxidative stress through their capacity to interact with active oxygen species, increasing their oxidant activity, or by affecting membrane rheology. Aluminum-membrane interactions can also affect signaling cascades. Zinc, at high and low concentrations, increases cell oxidant concentrations, affects AP-1 and NF-kappaB transcription factors and induces neuronal cell death. The capacity of lead to promote oxidative stress, affect cell signals and to induce cell death by apoptosis has been mostly attributed to its effect on different calcium-mediated cellular events. The mentioned mechanisms as well as the contribution of these metals to different neurodegenerative disorders are discussed.

Influence of zinc deficiency on cell-membrane fluidity in Jurkat, 3T3 and IMR-32 cells.

We investigated whether zinc deficiency can affect plasma membrane rheology. Three cell lines, human leukaemia T-cells (Jurkat), rat fibroblasts (3T3) and human neuroblastoma cells (IMR-32), were cultured for 48 h in control medium, in zinc-deficient medium (1.5 microM zinc; 1.5 Zn), or in the zinc-deficient medium supplemented with 15 microM zinc (15 Zn). The number of viable cells was lower in the 1.5 Zn group than in the control and 15 Zn groups. The frequency of apoptosis was higher in the 1.5 Zn group than in the control and 15 Zn groups. Membrane fluidity was evaluated using the 6-(9-anthroyloxy)stearic acid and 16-(9-anthroyloxy)palmitic acid probes. Membrane fluidity was higher in 1.5 Zn cells than in the control cells; no differences were observed between control cells and 15 Zn cells. The effect of zinc deficiency on membrane fluidity at the water/lipid interface was associated with a higher phosphatidylserine externalization. The higher membrane fluidity in the hydrophobic region of the bilayer was correlated with a lower content of arachidonic acid. We suggest that the increased fluidity of the membrane secondary to zinc deficiency is in part due to a decrease in arachidonic acid content and the apoptosis-related changes in phosphatidylserine distribution.

Epicatechin, catechin, and dimeric procyanidins inhibit PMA-induced NF-kappaB activation at multiple steps in Jurkat T cells.

The capacity of the flavan-3-ols [(-)-epicatechin (EC) and (+)-catechin (CT)] and a B dimeric procyanidin (DP-B) to modulate phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB activation in Jurkat T cells was investigated. The classic PMA-triggered increase in cell oxidants was prevented when cells were preincubated for 24 h with EC, CT, or DP-B (1.7-17.2 microM). PMA induced the phosphorylation of IKKbeta and the subsequent degradation of IkappaBalpha. These events were inhibited in cells pretreated with the flavonoids. PMA induced a 4.6-fold increase in NF-kappaB nuclear binding activity in control cells. Pretreatment with EC, CT, or DP-B decreased PMA-induced NF-kappaB binding activity and the transactivation of the NF-kappaB-driven gene IL-2. EC, CT, and DP-B inhibited, in vitro, NF-kappaB binding to its DNA consensus sequence, but they had no effect on the binding activity of CREB or OCT-1. Thus, EC, CT, or DP-B can influence the immune response by modulating NF-kappaB activation. This modulation can occur at early (regulation of oxidant levels, IKK activation) as well as late (binding of NF-kappaB to DNA) stages of the NF-kappaB activation cascade. A model is presented for possible interactions between DP-B and NF-kappaB proteins, which could lead to the inhibition of NF-kappaB binding to kappaB sites.

Zinc status of human IMR-32 neuroblastoma cells influences their susceptibility to iron-induced oxidative stress.

The current work tested the hypothesis that the zinc status of a cell influences its sensitivity to iron-induced oxidative stress. Human IMR-32 neuroblastoma cells were cultured for 24 h in nonchelated control media (5 microM zinc; 4.5 microM iron), or in media that was treated with DTPA to reduce its zinc content (chelated media). Chelated media was supplemented with zinc to achieve concentrations of 1.5-50 microM Zn. The media was then replaced with serum-free complex media (0.9 microM Zn) with either no added iron (0.6 microM Fe), or iron (FeCl(3)) added at concentrations ranging from 15 to 100 microM. Cells were cultured for an additional 3- to 24-hour period. Over the 24-hour period, cells cultured in the control iron media had good viability, and they displayed the gross morphology typical of these cells in culture. With 100 microM iron, cell viability was low in all groups. After 24 h and at iron concentrations between 15-50 microM, cells that had been cultured in the low zinc-chelated media (1.5 microM Zn) showed a concentration-dependent increase in 5 (or 6)-carboxy-2'7'-dichlorodihydrofluorescein diacetate (DCDCDHF) fluorescence (oxidative stress) and decrease in cell viability. A positive correlation between both parameters was observed (r = 0.92). These cells had altered morphology and high level of nucleosomes suggestive of cell death by apoptosis. These results support the concept that the zinc status of IMR-32 neuroblastoma cells modulates their sensitivity to iron overload.

Comparative study on the antioxidant capacity of wines and other plant-derived beverages.

Consistent epidemiological data point to a reduced morbidity and mortality from coronary heart disease and atherosclerosis in people consuming plant-derived beverages such as tea or wine. We studied the antioxidant capacity of three red wines (W) and compared it those of tea and herbal "mate" tea infusions. The antioxidant capacity was evaluated measuring: (1) the inhibition of the luminol-induced chemiluminescence assay (TRAP); (2) the inhibition of 2.2'-thiobarbituric-reactive substances (TBARS) formation in liposomes by fluorescence; (3) the protection of Jurkat cells from AMVN-induced oxidation, measuring the oxidation of 5-(and-6)-carboxy-2'7'-dichlorodihydrofluorescein diacetate to a fluorescent derivative. The polyphenolic content was estimated spectrophotometrically and by HPLC with electrochemical detection. All three beverages provided antioxidant protection in the three assays in a dose-dependent manner. Significant and positive correlations were found between antioxidant capacity and total polyphenol content, especially in the Jurkat cell oxidation assay (r: 0.96, p < 0.01). Results suggest that these dietary components could be a source of antioxidants that protect from oxidative stress. Further studies of absorption and metabolism of the active compounds will judge the physiological relevance of these results for human health.

Low intracellular zinc impairs the translocation of activated NF-kappa B to the nuclei in human neuroblastoma IMR-32 cells.

In the current work, we studied how variations in extracellular zinc concentrations modulate different steps involved in nuclear factor kappaB (NF-kappaB) activation in human neuroblastoma IMR-32 cells. Cells were incubated in media containing varying concentrations of zinc (1.5, 5, 15, and 50 microm). Within 3 h, the intracellular zinc content was lower in cells exposed to 1.5 and 5 microm, compared with the other groups. Low intracellular zinc concentrations were associated with the activation of NF-kappaB, based on high levels of IkappaBalpha phosphorylation, low IkappaBalpha concentrations, and high NF-kappaB binding activity in total cell fractions. However, the active dimer accumulated in the cytosol, as shown by a low ratio of nuclear/cytosolic NF-kappaB binding activity. This altered nuclear translocation was accompanied by a decreased transactivation of an endogenous NF-kappaB-driven gene (ikba) and of a reporter gene (pNF-kappaB-luc). In cells with low intracellular zinc concentrations, a low rate of in vitro tubulin polymerization was measured compared with the other groups. We conclude that low intracellular zinc concentrations induce tubulin depolymerization, which may be one signal for NF-kappaB activation. However, NF-kappaB nuclear translocation is impaired, which inhibits the transactivation of NF-kappaB-driven genes. This could affect cell survival, and be an important factor in certain zinc deficiency-associated pathologies.