Ca-mediated regulation of VDAC1 expression levels is associated with cell death induction

VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca^2 + across the OMM and because Ca^2 + has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca^2 + levels ([Ca^2 +]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca^2 + and induce VDAC1 over-expression. Direct elevation of [Ca^2 +]i by the Ca^2 +-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca^2 +]i using the cell-permeable Ca^2 +-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca^2 +]i levels and apoptosis induction, as induced by H₂O₂ or As₂O₃, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca^2 +-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca^2 +]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca^2 + promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau     

The interaction of local anesthetics with the ryanodine receptor of the sarcoplasmic reticulum

The effects of various local anesthetics (LAs) on the skeletal muscle ryanodine receptor were tested. The LAs were divided into three categories according to their effects on the binding of ryanodine to the junctional sarcoplasmic reticulum membranes. Ryanodine binding was assayed in the presence of 0.2 m NaCl and 10 m CaCl₂. Tetracaine and dibucaine inhibit the binding with half-maximal inhibition (CI₅₀) of 0.12 and 0.25 mm, respectively, while inhibition by benzocaine and procaine occurs with CI₅₀ of about 10-fold higher. Lidocaine, its analogue QX-314, and prilocaine, on the other hand, stimulate the binding up to fourfold with half-maximal stimulation occurring with about 2 mm of the drugs. Lidocaine increases both the receptor affinity for ryanodine by about fivefold and the rate of ryanodine association with its binding site by about 10-fold.Tetracaine interacts with the ryanodine receptor in a non-competitive fashion with respect to ryanodine but it competes with lidocaine for its binding site, suggesting the existence of a single site for the inhibitory and stimulatory LA.     

Light‐independent thermoluminescence from thylakoids of greening barley leaves. Evidence for involvement of oxygen radicals and free chlorophyll

A study was conducted to identify biophysical markers which change in response to changing chlorophyll organization during plant development. When heated to around 70°C in the dark, barley thylakoids emit red thermoluminescence (TL). This is a pure chemiluminescence signal and distinct from the lower-temperature TL bands of thylakoids that are seen only with preillumination. The development of the light-independent, 70°C TL band was investigated following transfer of dark-grown barley leaves to the light. Because of the rapidly increasing chlorophyll content of the plastid membrane, the TL signal was normalized against either chlorophyll or tissue mass of the starting material. In either case, the extent of the TL signal reached a maximum in the early hours of greening and then declined. The drop in signal over 20 h was approximately 50% for TL per unit tissue mass, and well over 90% for TL per unit chlorophyll. Exposure of plastid membrane samples to hydrogen peroxide for several minutes caused a large increase in light-independent TL, while addition of ascorbate caused substantial quenching. The fluorescence profiles of dark-grown barley leaves were recorded following transfer to the light. Basal fluorescence (F₀) reaches a substantial level after just seconds of illumination. Over the next few hours, F₀ increases only slightly and then starts to decline. The decline in F₀ is correlated with an increase in variable fluorescence (F_v) which indicates the appearance of active photosystem II. It is concluded that the early peak in F₀ reflects a state in which the leaves contain a maximum amount of disorganized chlorophyll. Considering the TL and fluorescence data together, we propose the following: When chlorophyll first appears in the system, it is not properly assembled into the complexes that offer photochemical or non-photochemical quenching of the excited state. Thus, fluorescence and parallel chlorophyll triplet formation are prevalent. The triplets cause generation of active oxygen resulting in lipid peroxidation and/or other radical-generating processes. When the membranes are heated, increased interaction of the radicals with chlorophyll generates chemiluminescence. We thus conclude that light-independent thermoluminescence is a marker for actual damage arising from poor chlorophyll organization and propose that this parameter might be usefully applied for assessing stress effects.     

Monophenolase activity of avocado polyphenol oxidase

Polyphenol oxidase of avocado mesocarp catalyses (a) the orthohydroxylation of monophenols like l-tyrosine, d-tyrosine, tyramine and p-cresol, and (b) the oxidation of the corresponding o-dihydroxyphenols to quinones. The rate of step b is much greater than that of step a. The hydroxylation of monophenols occurs after a lag period. DOPA or ascorbate effectively eliminate the lag but not dl-6-methyltetrahydropteridine or tetrahydrofolic acid. At 1.66 × 10^?4 M, α,α-dipyridyl has no effect, while diethyldithiocarbamate at this concentration inhibits the hydroxylation reaction by 90%. The tyrosinase activity of avocado polyphenol oxidase is inactivated in the course of the reaction; this inactivation occurs faster and is more pronounced in the presence of exogenously added DOPA. This inactivation is partially prevented by a large excess of ascorbate. The K_m values indicate that tyramine, dopamine, p-cresol and 4-methyl catechol are better substrates for avocado polyphenol oxidase than tyrosine or DOPA.     

Polyphenol oxidase isoenzymes in avocado

Avocado polyphenol oxidase (PPO) was precipitated mainly in the 30–90% saturated ammonium sulfate fraction. The 40–75% saturated ammonium sulfate fraction (the partially purified enzyme) had the highest specific activity in the cultivars Lerman, Horeshim and Fuerte. The PPO was active towards o-dihydroxyphenols. Six active enzymes (a–f) were detected with D,L-DOPA, 4-methylcatechol, catechol, caffeic acid or chlorogenic acid. Band e was the most active in all cases. More isoenzyme bands (fast-moving) were observed with caffeic acid than with 4-methylcatechol. Furthermore, the isoenzyme patterns of the partially purified extracts of the cultivars could be distinguished with respect to caffeic acid.     

Sugar accumulation in chemically debudded potato tubers during cold storage

Potato tuber buds may be excised by immersion of the tubers in a mixture of EtOH-Me₂CO (1:1) for 4 hr. This enabled the study of the effect of tuber aging (at 17°) on the starch-to-sugar conversion during storage at 4°, in the absence of complications due to sprouting. Sugar accumulation during a two-week period of storage at 4° decreased with increasing time of prior storage at 17°.     

Formation and characterization of stable human serum albumin-tris-malonic acid [C60]fullerene complex

The preparation and characterization of the stable human serum albumin (HSA)-C3 isomer of tris-malonic acid [C60]fullerene complex is reported. Other than the anti-fullerene antibody, a stable protein-fullerene complex with a native protein has never been observed. This study may provide valuable answers to the growing concern regarding the effects of carbonaceous nanomaterials on human health on one hand and, on the other, may lead to the development of novel antioxidant therapeutic agents, radiopharmaceuticals, and components for bioelectronic devices.     

Subcellular localization of VDAC in mitochondria and ER in the cerebellum

The voltage-dependent anion channel (VDAC) provides passage for adenine nucleotides, Ca2+ and other metabolites into and from mitochondria. Here, the intracellular localization and oligomeric organization of VDAC in brain mitochondria and ER are demonstrated. Immunohistochemical staining of VDAC in rat cerebellum showed high labeling of the Purkinje neurons. Immunogold labeling and EM analysis of the cerebellar molecular layer showed specific VDAC immunostaining of the mitochondrial outer membrane, highly enhanced in contact sites between mitochondria or between mitochondria and associated ER. Purified ER membranes contain VDAC, but not other mitochondrial proteins. Chemical cross-linking of isolated mitochondria, ER or purified VDAC demonstrated the existence of VDAC in oligomeric form. Based on the enrichment of VDAC in the junctional face of closely associated mitochondrial and ER membranes and the existence of VDAC oligomers, we propose an involvement of VDAC in specialized intermembrane communication between mitochondria or between ER and mitochondria, serving to complement the tight structural and functional coupling observed between these organelles.     

Delineation of Cohen syndrome following a large-scale genotype-phenotype screen

Cohen syndrome is an autosomal recessive condition associated with developmental delay, facial dysmorphism, pigmentary retinopathy, and neutropenia. The pleiotropic phenotype, combined with insufficient clinical data, often leads to an erroneous diagnosis and has led to confusion in the literature. Here, we report the results of a comprehensive genotype-phenotype study on the largest cohort of patients with Cohen syndrome assembled to date. We found 22 different COH1 mutations, of which 19 are novel, in probands identified by our diagnostic criteria. In addition, we identified another three novel mutations in patients with incomplete clinical data. By contrast, no COH1 mutations were found in patients with a provisional diagnosis of Cohen syndrome who did not fulfill the diagnostic criteria ("Cohen-like" syndrome). This study provides a molecular confirmation of the clinical phenotype associated with Cohen syndrome and provides a basis for laboratory screening that will be valuable in its diagnosis.