Correction of image instability in confocal microscopy using image realignment. Effects on the analysis of intracellular calcium

Using confocal microscopy, we have examined the increases in [Ca(2+)](i) evoked by sodium channel toxins in cells labelled with the fluorescent dye INDO-1. We describe a new image analysis method that improves the detection of region-specific, toxin-induced patterns of change of intracellular calcium. This method is based on correction of global image motion followed by calculation of the strength of correlation between calcium changes in "seed" or reference pixels chosen to represent different regions of cells and those in other regions of the image. When the selected "seed" pixel was chosen to be in either varicosities or neurites, correlations were detected in the same regions of other cells as well as in the soma, indicating specific but spatially distinct patterns of behaviour. Control images (without changes in [Ca(2+)](i)) did not reveal significant interpixel correlations. The ability to recognize correlated patterns of calcium change in different regions of cells was greatly improved by correction for global motion.     

The copper(II) binding properties of the cyclic peptide c(HGHK)

The new cyclic tetrapeptide c(HGHK) was synthesised in the solid phase and its complexes with copper(II) were studied in aqueous solution at various pH values by means of potentiometric and spectroscopic methods (UV, EPR, CD). Six mononuclear coordination species were clearly identified within the pH range 3-11. Spectroscopic data strongly suggest sequential formation of N, 2N, 3N and 4N equatorial donor sets around the copper(II) centre from the lowest to the highest pH, involving both imidazole nitrogens and amide nitrogens. A detailed comparison with the copper(II) binding properties of HGHG and Ac-HGHG ligands is also reported.     

Solid state and solution studies of a vanadium(III)-L-cysteine compound and demonstration of its antimetastatic, antioxidant and inhibition of neutral endopeptidase activities

Reaction of one equivalent of vanadium(III) chloride with three equivalents of l-cysteine(H2Cys) in methyl alcohol affords a VIII-Cys compound that is formulated as [VIII(Hcys)3].2HCl.2.5H2O 1. The solid state characterization of 1 was performed by microanalysis, circular dichroism (CD) and infrared studies as well as room temperature magnetic susceptibility. These studies have shown coordination of each HCys- ligand to the VIII atom through an amine nitrogen and a carboxylate oxygen atoms. Solution studies of 1 were carried out in water and methanol by UV-visible, CD and electron paramagnetic resonance (EPR) spectroscopies. According to these studies, it was evident that despite the progressive oxidation of 1 to oxovanadium(IV) species, some V(III) species were also present in solution after several hours. Compound 1, VIVOSO4.5H2O and l-cysteine were examined for their total antioxidant capacity (TAC) and lag time. Compound 1 exhibited significantly greater total antioxidant capacity and lag time values than l-cysteine. VIVOSO4.5H2O did not show any total antioxidant capacity or lag time. The inhibition of neutral endopeptidase (NEP) activity caused by 1, VIVOSO4.5H2O and thiorphan was also measured. Compound 1, at a concentration of 10(-3) M, showed inhibition of NEP activity as potent as thiorphan at 10(-6) M, while VIVOSO4.5H2O in the same concentration exhibited less than 50% inhibitory activity than that of thiorphan at 10(-6) M. Moreover, the antimetastatic effects of compound 1, l-cysteine and VIVOSO4.5H2O were examined on Wistar rats, treated with 3,4-benzopyrene. The results revealed that 1 prevents significantly lung metastases (only 9.5% of animals treated with 1 showed metastases), whereas 47-52% of the rats of the control group and those treated with l-cysteine and VIVOSO4.5H2O exhibited metastases.     

Ceramide Induces Release of Pro-Apoptotic Proteins from Mitochondria by Either a Ca2+-Dependent or a Ca2+-Independent Mechanism

Several observations have been reported in the last years indicating that ceramide may activate the mitochondrial route of apoptosis. We show here that on addition of either C2- or C16-ceramide to mitochondria isolated from rat heart and suspended in a saline medium, release of cytochrome c and apoptosis-inducing factor (AIF) from the intermembrane space takes place. The release process is Ca2+ -independent and is not inhibited by Cyclosporin A (CsA). For the protein release process to occur, the presence of an oxidizable substrate is required. When mitochondria are suspended in sucrose instead of potassium medium, only short chain C2-ceramide causes cytochrome c release through a Ca2+ -dependent and CsA sensitive mitochondrial permeability transition (MPT) mechanism. The latter effect appears to be related to the membrane potential dissipating ability exhibited by short chain C2-ceramide.     

Semisystematic nomenclature of brassinosteroids

The assignment of the trivial name to new isolated or detected brassinosteroid is based on the trivial names of seven different brassinosteroids, with names assigned according to the plant source from which they were first isolated. To avoid some observed mistakes in assigning trivial names to these compounds and the impractical constant usage of their systematic names, we propose a semisystematic nomenclature of brassinosteroids, in which (22R,23R)-2,3,22,23-tetrahydroxy-5-campestane, the trivial name of which is 6-deoxocastasterone, is considered the functional parent compound and is named brassinostane or brassinane. A set of rules for naming the remaining natural brassinosteroids is presented.     

Changes in membrane cholesterol affect caveolin-1 localization and ICC-pacing in mouse jejunum

Pacing of mouse is dependent on the spontaneous activity of interstitial cells of Cajal in the myenteric plexus (ICC-MP). These ICC, as well as intestinal smooth muscle, contain small membrane invaginations called caveolae. Caveolae are signaling centers formed by insertions of caveolin proteins in the inner aspect of the plasma membrane. Caveolins bind signaling proteins and thereby negatively modulate their signaling. We disrupted caveolae by treating intestinal segments with methyl beta-clodextrin (CD) to remove cholesterol or with water-soluble cholesterol (WSC) to load cholesterol. Both of these treatments reduced pacing frequencies, and these effects were reversed by the other agent. These treatments also inhibited paced contractions, but complete reversal was not observed. To evaluate the specificity of the effects of CD and WSC, additional studies were made of their effects on responses to carbamoyl choline and to stimulation of cholinergic nerves. Neither of these treatments affected these sets of responses compared with their respective time controls. Immunochemical and ultrastructural studies showed that caveolin 1 was present in smooth muscle membranes and ICC-MP. CD depleted both caveolin 1 and caveolae, whereas WSC increased the amount of caveolin 1 immunoreactivity and altered its distribution but failed to increase the number of caveolae. The effects of each agent were reversed in major part by the other. We conclude that signaling through caveolae may play a role in pacing by ICC but does not affect responses to acetylcholine from nerves or when added exogenously.     

Purification and characterization of an<Emphasis Type="Italic"> N</Emphasis>-acetylglucosaminidase produced by a<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> strain which controls<Emphasis Type="Italic"> Crinipellis perniciosa</Emphasis>

Isolate 1051 of Trichoderma harzianum, a mycoparasitic fungus, was found to impair development of the phytopathogen, Crinipellis perniciosa, in the field. This Trichoderma strain growing in liquid medium containing chitin produced substantial amounts of chitinases. The N-acetylglucosaminidase present in the culture-supernatant was purified to homogeneity by gel filtration and hydrophobic interaction chromatography, as demonstrated by SDS-PAGE analysis. The enzyme had a molecular mass of 36 kDa and hydrolyzed the synthetic substrate -nitrophenyl-N-acetylglucosaminide (NGlcNAc) with Michaelis–Menten kinetics. Maximal activities were determined at pH 4.0 and a temperature range of 50–60°C. K _m and V _max values for NGlcNAc hydrolysis were 8.06 moles ml^–1 and 3.36 moles ml^–1 min^–1, respectively, at pH 6.0 and 37°C. The enzyme was very sensitive to Fe³⁺, Mn²⁺ and Co²⁺ ions, but less sensitive to Zn²⁺, Al³⁺, Cu²⁺ and Ca²⁺. Glucose at a final concentration of 1 mM inhibited 65% of the original activity of the purified enzyme. Determination of the product (reducing sugar) of hydrolysis of C. perniciosa mycelium and scanning electron microscopic analysis revealed that the N-acetylglucosaminidase hydrolyses the C. perniciosa cell wall.