Use of the fluorescent dye 10-N-nonyl acridine orange in quantitative and location assays of cardiolipin: a study on different experimental models

The fluorescent dye 10-N-nonyl acridine orange (NAO) is extensively used for location and quantitative assays of cardiolipin in living cells on the assumption of its high specificity for cardiolipin; however, the limits and the mechanism of this specificity are not clear. Moreover, whether factors such as the membrane potential in mitochondria may limit the consistency of the results obtained by this method is open to discussion. The aim of this research was to investigate the effects of some experimental factors on the selective fluorescence of NAO in the presence of cardiolipin in artificial and natural membranes (mitochondria). The results show that the fluorescence of NAO, due to interaction with cardiolipin, is significantly modified by factors that control the spatial arrangement of cardiolipin molecules within the space of the membrane under investigation. Moreover, the present observations suggest that the specific effect of cardiolipin is to facilitate the dimerization of this fluorescent dye, thus confirming that reliable measurements of cardiolipin concentration can be obtained only when the NAO/cardiolipin molar ratio is equal to 2. The finding is also reported that in isolated respiring mitochondria the interaction of NAO with cardiolipin is somewhat related to the respiratory state of mitochondria.     

Involvement of cytochrome P450 2E1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease

Elucidation of the biochemical steps leading to the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of the nigrostriatal dopamine (DA) pathway has provided new clues to the pathophysiology of Parkinson's disease. In line with the enhancement of MPTP toxicity by diethyldithiocarbamate (DDC), here we demonstrate how other cytochrome P450 (CYP) 2E1 inhibitors, such as diallyl sulphide (DAS) and phenylethylisothiocyanate (PIC), also potentiate the selective DA neurone degeneration in C57/bl mice. In addition, we show that CYP 2E1 is present in the brain and in the basal ganglia of this mouse strain, as measured by RT-PCR, western blot analysis and immunohistochemistry. A kinetic analysis of MPTP and its metabolites, by means of the microdialysis technique in the striatum, indicates that no detoxification metabolic pathway is affected by any of these inhibitors. This does not rule out, however, that an undetected detoxification pathway involving CYP 2E1 is operating. In order to provide direct evidence for this isozyme involvement, CYP 2E1 knockout mice were challenged with MPTP or the combined treatment. Here we show that these transgenic mice have a low sensitivity to MPTP alone, similar to their wild-type counterparts, suggesting that it is likely that transgenic mice compensate for the missing enzyme. However, DDC pretreatment completely fails to enhance MPTP toxicity in CYP 2E1 knockout mice, whereas this enhancement is regularly present in wild-type animals. This study indicates that the occurrence of CYP 2E1 in C57/bl mouse brain is relevant to MPTP toxicity, and suggests that this isozyme may have a detoxificant role related to the efflux transporter of the toxin.     

Genotyping of Cryptosporidium isolates from Chamelea gallina clams in Italy

Chamelea gallina clams collected from the mouths of rivers along the Adriatic Sea (central Italy) were found to harbor Cryptosporidium parvum (genotype 2), which is the lineage involved in zoonotic transmission. The clams were collected from the mouths of rivers near whose banks ruminants are brought to graze. This paper reports the environmental spread of C. parvum in Italy and highlights the fact that genotyping of seaborne Cryptosporidium isolates is a powerful tool with which to investigate the transmission patterns and epidemiology of this microorganism.     

Hollow-fiber enzyme reactor operating under nonisothermal conditions

A hollow-fiber enzyme reactor, operating under isothermal and nonisothermal conditions, was built employing a polypropylene hollow fiber onto which beta-galactosidase was immobilized. Hexamethylenediamine and glutaraldehyde were used as spacer and coupling agent, respectively. Glucose production was studied as a function of temperature, substrate concentration, and size of the transmembrane temperature gradient. The actual average temperature differences across the polypropylene fiber, to which reference was done to evaluate the effect of the nonisothermal conditions, were calculated by means of a mathematical approach, which made it possible to know, using computer simulation, the radial and axial temperature profiles inside the bioreactor and across the membrane. Percent activity increases, proportional to the size of the temperature gradients, were found when the enzyme activities under nonisothermal conditions were compared to those measured under comparable isothermal conditions. Percent reductions of the production times, proportional to the applied temperature gradients, were also calculated. The advantage of employing nonisothermal bioreactors in biotechnological industrial process was discussed.