Anandamide increases swelling and reduces calcium sensitivity of mitochondria

The endocannabinoid anandamide alters mitochondria-dependent signal transduction, thus controlling key cellular events like energy homeostasis and induction of apoptosis. Here, the ability of anandamide to directly affect the integrity of mitochondria was investigated on isolated organelles. We found that anandamide dose-dependently increases mitochondrial swelling, and reduces cytochrome c release induced by calcium ions. The effects of anandamide were independent of its target receptors (e.g., cannabinoid or vanilloid receptors), and were paralleled by decreased membrane potential and increased membrane fluidity. Overall, our data suggest that anandamide can impact mitochondrial physiology, by reducing calcium sensitivity and perturbing membrane properties of these organelles.     

A silver staining reaction for chromosomes and nuclei

Summary A staining reaction for the nucleus and chromosomes, using silver-hexamethylentetramine, buffered with borax, after hydrolisis with warm IN HC1, has been proposed. Its specificity has been assayed by means of enzymatic digestions (DNase, pepsin, trypsin), and through the comparison of the results with those obtained by the Feulgen reaction.This staining method bears the advantage, compared to the usual methods, of a noteworthy photographic contrast.Moreover this method is probably specific for the DNA containing structures, because of the lack of stainability of nuclear structures, after DNase digestion.

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Riassunto E'stata proposta, per il nucleo ed i cromosomi, una reazione con argentoesa-metilentetrammina, tamponata con borace, previa idrolisi acida con HC1 1N, a caldo, e ne è stata saggiata la specificità mediante digestioni enzimatiche (DNAasi, pepsina, tripsina) e confronto dei risultati ottenuti con la reazione di Peulgen.Questa colorazione presenta il vantaggio, rispetto alle usuali colorazioni, di un notevole contrasto fotografico.Inoltre la non colorabilità delle strutture nucleari, dopo digestione con DNAasi, lascia supporre una specificità del metodo per le strutture contenenti DNA.

     

Demonstration of phosphates in calcium deposits: A modification of von Kossa's reaction

Summary It has been suggested that in von Kóss'as technic silver cations replace calcium bound to phosphate or carbonate groups and are then reduced to black metallic silver during exposure to light. However, in test tube experiments silver phosphate retains its yellow color for days. These differences between reactions of pure calcium phosphates and calcium deposits in tissues were emphasized already by von Kóssa; he regarded only the initial yellow coloration of calcium diagnostic for calcium phosphates and deplored the subsequent blackening caused by organic compounds. Von Kóssa's experiments were easily reproducible. A review of the literature showed that reduction of silver nitrate by organic compounds was well known in the 19th century. For histochemical studies of phosphates it was deemed desirable to avoid the formation of black by-products. Sections of paraffin-embedded human tissues were exposed to solutions of silver nitrate in subdued light or darkness and then treated with sodium thiosulfate. Silver phosphate was yellow to yellowish brown; other tissue structures remained colorless. No darkening was observed in sections stored for eight years. Other compounds which form yellow silver salts, e.g. iodides and periodates, are unlikely to occur in paraffin sections of human tissues.     

Oxidative stress increases internal calcium stores and reduces a key mitochondrial enzyme

Fibroblasts from patients with genetic and non-genetic forms of Alzheimer's disease (AD) show many abnormalities including increased bombesin-releasable calcium stores (BRCS), diminished activities of the mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC), and an altered ability to handle oxidative stress. The link between genetic mutations (and the unknown primary event in non-genetic forms) and these other cellular abnormalities is unknown. To determine whether oxidative stress could be a convergence point that produces the other AD-related changes, these experiments tested in fibroblasts the effects of H(2)O(2), in the presence or absence of select antioxidants, on BRCS and KGDHC. H(2)O(2) concentrations that elevated carboxy-dichlorofluorescein (c-H(2)DCF)-detectable ROS increased BRCS and decreased KGDHC activity. These changes are in the same direction as those in fibroblasts from AD patients. Acute treatments with the antioxidants Trolox, or DMSO decreased c-H(2)DCF-detectable ROS by about 90%, but exaggerated the H(2)O(2)-induced increases in BRCS by about 4-fold and did not alter the reduction in KGDHC. Chronic pretreatments with Trolox more than doubled the BRCS, tripled KGDHC activities, and reduced the effects of H(2)O(2). Pretreatment with DMSO or N-acetyl cysteine diminished the BRCS and either had no effect, or exaggerated the H(2)O(2)-induced changes in these variables. The results demonstrate that BRCS and KGDHC are more sensitive to H(2)O(2) derived species than c-H(2)DCF, and that oxidized derivatives of the antioxidants exaggerate the actions of H(2)O(2). The findings support the hypothesis that select abnormalities in oxidative processes are a critical part of a cascade that leads to the cellular abnormalities in cells from AD patients.     

A method for combined C-banding and silver staining

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.     

Fixation increases sensitivity of India ink staining of proteins and peptides on nitrocellulose paper

The detectability by India ink staining of proteins and peptides dot-blotted on nitrocellulose paper was assessed before and after fixation. Fixation considerably increased the detectability of proteins and peptides. Denaturation by KOH treatment or baking at 100 degrees C for 15 min gave the best results. Precipitation by isopropanol/acetic acid gave intermediate results, whereas crosslinking with glutaraldehyde improved the detectability of small peptides, but not of proteins. Ferridye and Aurodye were also tested after baking. Both dyes were more sensitive and stained more proteins and peptides than India ink. In all cases the detectability of peptides smaller than Mr 1500 was poor.     

Oxalate pretreatment and use of a physical developer render the Kossa method selective and sensitive for calcium

Summary Based on experiments on agarose gels and tissue, a procedure has been developed which greatly improves the sensitivity and the specifity of the Kossa method for demonstrating calcium in tissue. Tissue calcium is immobilized by acetonic oxalic acid, which simultaneously removes the other sorts of anions capable of precipitating silver ions (e.g. phosphate, carbonate). The resulting submicroscopic grains of calcium oxalate are converted first into silver oxalate then into metallic silver by a treatment with silver nitrate followed by an ultra-violet irradiation (Kossa reaction). These submicroscopic metallic silver grains are enlarged up to microscopic visibility by means of physical development, which makes the staining highly sensitive. Costaining of the argyrophil sites in the tissue is totally suppressed by various tricks, which render the silver staining selective for calcium.     

Oxalate pretreatment and use of a physical developer render the Kossa method selective and sensitive for calcium

Based on experiments on agarose gels and tissue, a procedure has been developed which greatly improves the sensitivity and the specifity of the Kossa method for demonstrating calcium in tissue. Tissue calcium is immobilized by acetonic oxalic acid, which simultaneously removes the other sorts of anions capable of precipitating silver ions (e.g. phosphate, carbonate). The resulting submicroscopic grains of calcium oxalate are converted first into silver oxalate then into metallic silver by a treatment with silver nitrate followed by an ultra-violet irradiation (Kossa reaction). These submicroscopic metallic silver grains are enlarged up to microscopic visibility by means of physical development, which makes the staining highly sensitive. Co-staining of the argyrophil sites in the tissue is totally suppressed by various tricks, which render the silver staining selective for calcium.     

Ethanol increases sensitivity of oxalate oxidase assays and facilitates direct activity staining in SDS gels

Oxalate oxidase, and H₂O₂-generating enzyme, has been characterized from several plants, and is widely used for clinical detection of oxalate. Using a germin-like oxalate oxidase from barley leaves, we have developed and optimized novel methods for measuring oxalate oxidase activity. As oxalate oxidase is SDS-tolerant, its activity can be detected directly in SDS-PAGE gels in the presence of ethanol. This ethanol-dependent method is a hundred times more sensitive than the current methods. Furthermore, ethanol also improves the sensitivity of oxalate oxidase assays performed in solution. We found at least a 10-fold increase in sensitivity in comparison to a current method. The assay in solution is, in addition, useful for detection of oxalate. This elevation in sensitivity may be due to the immobilization of the enzyme in protein precipitates as a result of the treatment with ethanol.     

An improved method for increasing the resolution and sensitivity of silver staining of nucleic acid bands in polyacrylamide gels

Staining of polyacrylamide gels with methylene blue prior to silver staining increases band resolution and sensitivity. This method permits resolution of multiple bands less than 1 mm apart, and is able to detect bands containing only 100 pg of RNA.     

Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.     

Serotonin decreases a background current and increases calcium and calcium-activated current in pedal neurons ofHermissenda

The effects of serotonin (5-HT) on membrane potential, membrane resistance, and select ionic currents were examined in large pedal neurons (LP1, LP3) of the mollusk Hermissenda. Calcium (Ca) action potentials were evoked in sodium-free artificial seawater containing tetramethylammonium, tetraethylammonium, and 4-aminopyridine (0-Na, 4-AP, TEA ASW). They failed at stimulation rates greater than 0.5/sec and were blocked by cadmium (Cd). Under voltage clamp the calcium current (ICa) responsible for them also failed with repeated stimulation. Thus, ICa inactivation accounts for refractoriness of the Ca action potential. The addition of 10 microM 5-HT to 0-Na, 4-AP, TEA ASW produced a slight depolarization and increased excitability and input resistance. Under voltage clamp the background current decreased. The voltage-dependent inward, late outward, and outward tail currents, sensitive to Cd, increased. ICa inactivation persisted. Under voltage clamp with Ca influx blocked by Cd, the addition of 10 microM 5-HT decreased the remaining current uniformly over membrane potentials of -10 to -100 mV. Thus, 5-HT reduces a background current that is active within the physiological range of the membrane potential, voltage insensitive, independent of Ca influx, noninactivating, and not blocked by 4-AP or TEA.     

Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis

In proteomics it is essential to be able to detect proteins separated by gel electrophoresis at high sensitivity. Silver staining is currently the most popular method. Here we present silver staining protocols that are optimized for staining sensitivity, peptide recovery and compatibility with digestion and mass spectrometry.     

Combination of silver and fluorescent staining for metaphase chromosomes.

A convenient and reliable method for simulatneous visualization of silver staining (Ag-NOR) of the nucleolus organizers and fluorescent bandings in metaphase chromosomes is described. Studies employing this combined procedure on human chromosomes revealed that the Ag-NOR patterns may be characteristic for each chromosome of each individual.     

Inhibition of SIRT6 in prostate cancer reduces cell viability and increases sensitivity to chemotherapeutics

SI RT6 is an important histone modifying protein that regulates DNA repair, telomere maintenance, energy metabolism, and target gene expression. Recently SIRT6 has been identifi ed as a tumor suppressor and is downregulated in certain cancer types, but not in other cancers. From deposited gene profi ling studies we found that SIRT6 was overexpressed in prostate tumors, compared with normal or paratumor prostate tissues. Tissue microarray studies confi rmed the higher levels of SIRT6 in both prostate tumor tissues and prostate cancer cells than in their normal counterparts. Knockdown of SIRT6 in human prostate cancer cells led to sub-G1 phase arrest of cell cycle, increased apoptosis, elevated DNA damage level and decrease in BCL2 gene expression. Moreover, SIRT6-deficiency reduced cell viability and enhanced chemotherapeutics sensitivity. Taken together, this study provides the fi rst evidence of SIRT6 overexpression in human prostate cancer, and SIRT6 regulation could be exploited for prostate cancer therapy.     

Transgenic expression of a fungal endo-polygalacturonase increases plant resistance to pathogens and reduces auxin sensitivity

Polygalacturonases (PGs), enzymes that hydrolyze the homogalacturonan of the plant cell wall, are virulence factors of several phytopathogenic fungi and bacteria. On the other hand, PGs may activate defense responses by releasing oligogalacturonides (OGs) perceived by the plant cell as host-associated molecular patterns. Tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) plants expressing a fungal PG (PG plants) have a reduced content of homogalacturonan. Here, we show that PG plants are more resistant to microbial pathogens and have constitutively activated defense responses. Interestingly, either in tobacco PG or wild-type plants treated with OGs, resistance to fungal infection is suppressed by exogenous auxin, whereas sensitivity to auxin of PG plants is reduced in different bioassays. The altered plant defense responses and auxin sensitivity in PG plants may reflect an increased accumulation of OGs and subsequent antagonism of auxin action. Alternatively, it may be a consequence of perturbations of cellular physiology and elevated defense status as a result of altered cell wall architecture.