Correlating Live Cell Viability with Membrane Permeability Disruption Induced by Trivalent Chromium.

Cr(III) is often regarded as a trace essential micronutrient that can be found in many dietary supplements due to its participation in blood glucose regulation. However, increased levels of exposure have been linked to adverse health effects in living organisms. Herein, scanning electrochemical microscopy (SECM) was used to detect variation in membrane permeability of single cells (T24) resulting from exposure to a trivalent Cr-salt, CrCl3. By employing electrochemical mediators, ferrocenemethanol (FcMeOH) and ferrocenecarboxylic acid (FcCOO-), initially semipermeable and impermeable, respectively, complementary information was obtained. Three-dimensional COMSOL finite element analysis simulations were successfully used to quantify the permeability coefficients of each mediator by matching experimental and simulated results. Depending on the concentration of Cr(III) administered, three regions of membrane response were detected. Following exposure to low concentrations (up to 500 µM Cr(III)), their permeability coefficients were comparable to that of control cells, 80 µm/s for FcMeOH and 0 µm/s for FcCOO-. This was confirmed for both mediators. As the incubation concentrations were increased, the ability of FcMeOH to permeate the membrane decreased to a minimum of 17 µm/s at 7500 µM Cr(III), while FcCOO- remained impermeable. At the highest examined concentrations, both mediators were found to demonstrate increased membrane permeability. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability studies were also conducted on Cr(III)-treated T24 cells to correlate the SECM findings with the toxicity effects of the metal. The viability experiments revealed a similar concentration-dependent trend to the SECM cell membrane permeability study.

Identification of metabolites associated with water stress responses in Solanum tuberosum L. clones.

Water deficiency has become a major issue for modern agriculture as its effects on crop yields and tuber quality have become more pronounced. Potato genotypes more tolerant to water shortages have been identified through assessment of yield and dry matter. In the present study, a combination of metabolite profiling and physiological/agronomical measurements has been used to explore complex system level responses to non-lethal water restriction. The metabolites identified were associated with physiological responses in three different plant tissues (leaf, root and tuber) of five different potato genotypes varying in susceptibility/tolerance to drought. This approach explored the potential of metabolite profiling as a tool to unravel sectors of metabolism that react to stress conditions and could mirror the changes in the plant physiology. The metabolite results showed different responses of the three plant tissues to the water deficit, resulting either in different levels of the metabolites detected or different metabolites expressed. The leaf material displayed the most changes to drought as reported in literature. The results highlighted genotype-specific signatures to water restriction over all three plant tissues suggesting that the genetics can predominate over the environmental conditions. This will have important implications for future breeding approaches.

Mutation of conserved aspartates affect maturation of presenilin 1 and presenilin 2 complexes.

Presenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW) complexes. During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments (NTF/CTF). Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of APP and Notch. We show that aspartate-mutant holoprotein presenilins are not incorporated into the high molecular weight, NTF/CTF-containing complexes. Aspartate-mutant presenilin holoproteins also preclude entry of endogenous wild-type PS1/PS2 into the high molecular weight complexes, but do not affect the incorporation of wild-type holoproteins into lower molecular weight holoprotein complexes. These data suggest that the loss-of-function aspartate-mutants cause altered PS complex maturation, and argue that the functional presenilin moieties are contained in the high molecular weight presenilin NTF/CTF-containing complexes.

Fibrillogenesis of Alzheimer Abeta peptides studied by fluorescence energy transfer.

Pathogenesis of Alzheimer's disease is associated with the polymerization of the Abeta peptide into fibrils that accumulate to form plaques. One strategy for therapy is the targeting of inhibitors against fibrillogenesis; however, prior to the formulation of specific tactics, a thorough understanding of the polymerization mechanism is essential. We have applied the principle of fluorescence energy transfer to monitor fibrillogenesis. In theory, this method is capable of measuring fibrillogenesis at physiological concentrations of peptide. Using this assay, we have determined that: fibril formation by Abeta(9-25) is reversible and cooperative, there are two imidazole-carboxylate salt bridges per monomer, monomers are in free exchange with fibrils, and the exchange process displays measurable kinetics.

Bilayer-stabilizing properties of myelin basic protein in dioleoylphosphatidylethanolamine systems.

31P-NMR and X-ray diffraction techniques are used to study the comparative ability of myelin basic protein (MBP) vs. other basic proteins to convert hexagonal (HII) phases to stable lamellar (L alpha) structures. Pure dioleoylphosphatidylethanolamine (DOPE) at pH 9 and 7, and mixtures of DOPE/phosphatidylserine (PS) (95:5 and 80:20% w/w) at pH 7 were employed for this investigation. The polymorphic behavior of the lipid suspensions was evaluated in the presence and absence of several basic proteins (MBP, calf thymus histone, lysozyme, melittin) and the cationic polypeptide, polylysine (PL). Each of the proteins and PL was capable of binding the pure DOPE HII phase at pH 9 but with varying morphological consequences, i.e., lamellar stabilization (MBP, histone, PL), formation of new protein-DOPE HII phases (lysozyme) or lipid disordering/vesiculation (melittin). Reduction to pH 7 resulted in the dissociation of protein from DOPE - with the exception of melittin - and the reformation of a pure lipid HII phase. Additions of PS to DOPE at pH 7 facilitated protein binding, but among the proteins examined, only MBP was capable of converting the lipid suspension into a stable multilamellar form. Differences in the lipid morphology produced by each protein are discussed in terms of protein physicochemical characteristics. In addition, a possible relationship between MBP-lipid interactions and the stability of myelin sheath lipid multilayers is inferred from the significant bilayer-stabilizing capacity of MBP.

Effects of dietary supplementation with marine fish oil on leukocyte lipid mediator generation and function in rheumatoid arthritis.

Twelve patients with active rheumatoid arthritis supplemented their usual diet with 20 gm of Max-EPA fish oil, daily, for 6 weeks. Following this supplementation, the ratio of arachidonic acid to eicosapentaenoic acid in the patients' neutrophil cellular lipids decreased from 81:1 to 2.7:1, and the mean generation of leukotriene B4 (with calcium ionophore stimulation) significantly declined by 33%. The mean neutrophil chemotaxis to both leukotriene B4 and FMLP significantly increased toward the normal range at week 6. The generation of 5-lipoxygenase products by calcium ionophore-stimulated monocytes was not significantly suppressed, but a significant decline (37%) in platelet-activating factor generation was noted at week 6. The modulation of these measures of leukocyte inflammatory potential suggests that fish oil supplementation may have an antiinflammatory effect.

Clinical trial of a new carbenoxolone analogue (BX24), zinc sulphate, and vitamin A in the treatment of gastric ulcer.

The effects of a new carbenoxolone analogue (BX24), zinc sulphate, and vitamin A on the healing of gastric ulcer have been assessed in a multifactorial clinical trial conducted in out-patients treated for four weeks.Forty-eight patients completed the trial. Three groups of eight patients were given respectively 300, 600, and 1 200 mg of BX24 daily and were compared with 24 patients who were given 300 mg of carbenoxolone sodium daily. The size of the ulcer niche was reduced on average by 14.6% in the eight patients given BX24 300 mg daily, by 47.6% in the patients given 600 mg daily, and by 51.0% in the patients given 1 200 mg daily. In the patients given carbenoxolone the size of the niche was reduced by 68.9%. These results were compared with those obtained previously with carbenoxolone and inert tablets and it was concluded that BX24 is without clinically useful effect in the doses used. Eleven of the 24 patients (46%) treated with carbenoxolone sodium developed side effects due to fluid retention and electrolyte disturbances. None of the patients given BX24 experienced such effects. In addition to carbenoxolone or BX24, 24 patients were given zinc sulphate, 660 mg daily, and in 24 patients these tablets were withheld. Among the patients given carbenoxolone the reduction in the size of the niche was much the same irrespective of whether or not the patients received zinc sulphate. Among the 12 patients given BX24 with zinc sulphate the ulcer healed completely in four and, on average, the size of the niche was reduced by 53.5%, compared with 21.9% in the 12 patients given BX24 alone. This difference is not statistically significant but the possibility of a beneficial effect from zinc is not excluded. No side effects attributable to zinc were observed.Twenty-four patients were also given vitamin A, 50 000 units daily, and in 24 patients the vitamin was withheld. No evidence was obtained to suggest that vitamin A had any beneficial effect on the healing of gastric ulcer.