Two-photon thermal bleaching of single fluorescent molecules

We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations.     

A very rare association of three mutations of the HFE gene for hemochromatosis

In the present paper, we describe a patient who is a compound heterozygote for three mutations in the HFE gene: C282Y, H63D, and E168Q. The patient's mother carries two copies of H63D and one copy of E168Q; the patient's father is heterozygous for C282Y. The family study indicates that the patient, as well as his sister, a maternal uncle, and a first cousin, all have inherited a single HFE allele that contains two mutations H63D and E168Q. The clinical symptoms and laboratory findings of the patient and his relatives are consistent with the conclusion that the E168Q mutation by itself is unlikely to result in hemochromatosis.     

Evaluation of the antioxidant activity of flavonoids by "ferric reducing antioxidant power" assay and cyclic voltammetry

Flavonoids, naturally occurring phenolic compounds, have recently been studied extensively for their antioxidant properties. The structure-antioxidant activity relationships (SAR) of flavonoids have been evaluated against different free radicals, but "ferric reducing antioxidant power" (FRAP) assay, which determines directly the reducing capacity of a compound, has not been used for this purpose. In this study, the antioxidant activities of 18 structurally different flavonoids were evaluated by FRAP assay modified to be used in 96-well microplates. Furthermore, their oxidation potentials were also measured, which were in the range of +0.3 V (myricetin) to +1.2 V (5-hydroxy flavone) and were in good agreement with FRAP assay results. Quercetin, fisetin and myricetin had the lowest oxidation potentials and appeared the most active compounds in FRAP assay and were 3.02, 2.52 and 2.28 times more active than Trolox, respectively. Indications were found that the o-dihydroxy structure in the B ring and the 3-hydroxy group and 2,3-double bond in the C ring give the highest contribution to the antioxidant activity.     

Bioenergetics of mitochondrial diseases associated with mtDNA mutations

This mini-review summarizes our present view of the biochemical alterations associated with mitochondrial DNA (mtDNA) point mutations. Mitochondrial cytopathies caused by mutations of mtDNA are well-known genetic and clinical entities, but the biochemical pathogenic mechanisms are often obscure. Leber's hereditary optic neuropathy (LHON) is due to three main mutations in genes for complex I subunits. Even if the catalytic activity of complex I is maintained except in cells carrying the 3460/ND1 mutation, in all cases there is a change in sensitivity to complex I inhibitors and an impairment of mitochondrial respiration, eliciting the possibility of generation of reactive oxygen species (ROS) by the complex. Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa (NARP), is due to a mutation in the ATPase-6 gene. In NARP patients ATP synthesis is strongly depressed to an extent proportional to the mutation load; nevertheless, ATP hydrolysis and ATP-driven proton translocation are not affected. It is suggested that the NARP mutation affects the ability of the enzyme to couple proton transport to ATP synthesis. A point mutation in subunit III of cytochrome c oxidase is accompanied by a syndrome resembling MELAS: however, no major biochemical defect is found, if we except an enhanced production of ROS. The mechanism of such enhancement is at present unknown. In this review, we draw attention to a few examples in which the overproduction of ROS might represent a common step in the induction of clinical phenotypes and/or in the progression of several human pathologies associated with mtDNA point mutations.     

Influence of conformational flexibility on biological activity in cyclic astin analogues

The astins, a family of natural antitumor cyclopeptides, from the roots of Aster tataricus, consist of a 16-membered ring system containing uncoded amino acid residues. The backbone conformation, with a cis-3,4-dichlorinated proline residue, plays an important role in antineoplastic activity. The acyclic astins, on the other hand, do not show antitumor activity, suggesting that the cyclic nature of astins may be a key role in their biological properties. Although the antineoplastic activity of natural astins has been screened in vitro and in vivo, the mechanism of action has never been investigated. With the aim at elucidating the influence of conformational flexibility on biological activity, we have designed and synthesized several astin analogues containing either Aib and the nonproteinogenic Abu and (S)beta3-hPhe residues, able to modify the peptide backbone structure, or the peptide bond surrogate -SO2-NH-. Tested for their antitumor effect, our astin-related cyclopeptides are able to inhibit the growth of tumor cell lines, while the acyclic astins are inefficacious. The present work reports on the structure-activity study of a selected synthetic cyclotetrapeptide corresponding to the sequence c[Thr-Aib-(S)beta3-hPhePsi(CH2-SO2-NH)-Abu], synthesized by classical methods and characterized conformationally by two-dimensional NMR and molecular dynamics analyses.     

Guanidine-induced dissociation of mitochondrial F1-ATPase

The effect of guanidine hydrochloride on ATPase activity, gel filtration, turbidity, and the fluorescence emission intensity of mitochondrial F1-ATPase was examined. Purified F1 from bovine heart mitochondria was slowly inactivated at low denaturant concentration, and inactivation was associated with delta and epsilon subunit dissociation. delta and epsilon subunits were bound together to form a stable and soluble heterodimer. In parallel, appearance of turbidity was observed. This was caused by the formation of alpha3beta3gamma non-covalent aggregates, as analyzed by SDS-PAGE. Short periods of exposition of the F1 complex to high concentrations of guanidine hydrochloride (0.8-3 M) again induced deltaepsilon dissociation as a heterodimer and the formation of an inactive alpha3beta3gamma subcomplex. This eventually dissociated progressively into single subunits caused by partial unfolding, as evidenced through changes of the protein intrinsic fluorescence emission. Our results suggest that the delta and epsilon subunits are loosely bound to alpha3beta3gamma , and play an important role in determining structural stability to isolated mitochondrial F1-ATPase.     

Function of the hepatic melanogenesis in the newt, Triturus carnifex

Like the majority of lower vertebrates, the newt Triturus carnifex holds varying quantities of melanin and hemosiderin in the Kupffer cells of the liver. Following hypoxic treatment, the amount of these two pigments can increase to such an extent that they can occupy nearly a quarter of the surface of histological sections. A group of six specimens, anesthetised with chlorbutol, were subjected to hypoxic treatment by keeping them in a respiratory chamber containing degassed water under vacuum, with only 1.1 ppm of residual oxygen, until they had consumed the oxygen completely (4 hours, at a temperature of 18 degrees C). Using hematological and histochemical techniques and computerised image analysis, it has been shown that hypoxic animals not only increase the extent of the melanic areas of the liver from about 5-7% to almost 24% compared to control groups kept under two different respiratory conditions (6 anesthetised specimens exposed to the air and 6 submerged in normoxic water), they also went through a remarkable hemolytic process to justify a parallel increase in hemosiderin deposits. Melanin was extracted from the liver by keeping fragments of the organ for one hour at 37 degrees C in an oxidising solution (20 mL of benzyl alcohol, 10 mL of acetone, 5 mL of 10% hydrogen peroxide, and 4 drops of concentrated ammonia solution), then quickly rinsing them in 50% acetone and subsequently letting them stand for 6 hours in 10 mL of distilled water alkalised to pH 12 with a drop of ammonia solution. The extract was then left to sediment at pH 2.5 and the black precipitate washed and dried under vacuum. Elemental and spectrophotometric analyses revealed a significant presence of purines in the melanic pigment. This phenomenon can be explained by the animals' need under hypoxic crisis to rapidly neutralise purines resulting from lysis of the nucleated red blood cells by introducing them into an inert molecular complex. A partial model of structure is proposed here. Synthesis of the mixed polymer is possible through the well-known capacity of ferrous iron to activate tyrosinase (the enzyme responsible for melanogenesis) even in the absence of DOPA.     

Nitric oxide release and distribution following oral and intraperitoneal administration of nitroaspirin (NCX 4016) in the rat

The metabolic fate of nitric oxide (NO) released from nitroaspirin, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX 4016), the lead compound of a new class of NO-releasing non steroidal anti-inflammatory drugs (NO-NSAIDs), has been studied in the rat following p.o. and i.p. administration of 100 mg/kg, by monitoring in plasma the bioactive storage forms of NO (S-nitrosothiols, RS-NO) and its oxidation products (nitrites/nitrates, NOx) by a chemiluminescent assay. In parallel, plasma was analyzed for unchanged drug and metabolites by reverse-phase HPLC. In orally treated rats, no unchanged drug is observed in the 0-24 h interval post-dosing, but only salicylic acid (SA), NOx and RS-NO. The time-course of SA formation parallels that of plasma NOx (plateau after 6 h). Nitrosothiols in plasma are detectable at 1 h, peak at 4 h post-administration, and decline thereafter. The results relative to i.p. administration show a more pronounced and rapid NO delivery (peak of both NOx and RS-NO at 1 h and plateau between 1 and 2 h), still coincident with the peak of SA, and the presence in plasma of NCX 4015 (a metabolite of NCX 4016 which still bears the nitrate function). In myocardial tissue from p.o. treated rats, no drug or metabolites were ever detected, and the NOx levels were always in the range of the controls. Conversely, following i.p. treatment, we observed a rapid compartmentalization within the heart of the unchanged drug, which rapidly disappears in favour of its breakdown products NCX 4015 and SA, with a concomitant rise in myocardial NOx levels up to 2 h. To check the stability of NCX 4016 in the acidic gastric milieu and to explain the different distribution of the drug following p.o. or i.p. administration, the gastric content of the orally-treated animals at different post-dosing times was analysed by HPLC. The unchanged drug was detected up to 8 h post-dosing (levels slowly decreased with time), and the only metabolite to be detected was the O-deacetylated derivative (NCX 4023), which was present in low concentrations up to 4 h post-dosing. This indicates that NCX 4016 does not undergo biotransformation in the upper part of gastrointestinal tract (no direct release of NO in this district) and that the stomach acts as a reservoir for the drug.     

Overexpression of the ped/pea-15 gene causes diabetes by impairing glucose-stimulated insulin secretion in addition to insulin action

Overexpression of the ped/pea-15 gene is a common feature of type 2 diabetes. In the present work, we show that transgenic mice ubiquitously overexpressing ped/pea-15 exhibited mildly elevated random-fed blood glucose levels and decreased glucose tolerance. Treatment with a 60% fat diet led ped/pea-15 transgenic mice to develop diabetes. Consistent with insulin resistance in these mice, insulin administration reduced glucose levels by only 35% after 45 min, compared to 70% in control mice. In vivo, insulin-stimulated glucose uptake was decreased by almost 50% in fat and muscle tissues of the ped/pea-15 transgenic mice, accompanied by protein kinase Calpha activation and block of insulin induction of protein kinase Czeta. These changes persisted in isolated adipocytes from the transgenic mice and were rescued by the protein kinase C inhibitor bisindolylmaleimide. In addition to insulin resistance, ped/pea-15 transgenic mice showed a 70% reduction in insulin response to glucose loading. Stable overexpression of ped/pea-15 in the glucose-responsive MIN6 beta-cell line also caused protein kinase Calpha activation and a marked decline in glucose-stimulated insulin secretion. Antisense block of endogenous ped/pea-15 increased glucose sensitivity by 2.5-fold in these cells. Thus, in vivo, overexpression of ped/pea-15 may lead to diabetes by impairing insulin secretion in addition to insulin action.     

Fluorescence study on rat epithelial cells and liposomes exposed to aromatic nitroxides

This study was performed to evaluate the effects, if any, of aromatic nitroxides, namely, indolinic nitroxides, on membrane fluidity of rat epithelial cells using steady-state fluorescence. These nitroxides are being increasingly considered as new and versatile compounds to reduce oxidative stress in biological systems. Hence, the results obtained in this study will give more insights on the interaction of these compounds with biological structures which at present is lacking, especially in view of their possible application as antioxidant therapeutic agents. The probes DPH and Laurdan which give information on the hydrophobic and hydrophilic-hydrophobic regions of the membrane bilayer, respectively, showed that nitroxide 1 (1,2-dihydro-2-methyl-3H-indole-3-one-1-oxyl) significantly increases membrane fluidity, whereas the corresponding phenylimino nitroxide derivative 2 (1,2-dihydro-2-methyl-3H-indole-3-phenylimino-1-oxyl) leads to membrane rigidification. The aliphatic nitroxide TEMPO included in this study for comparison produced no modifications. Consequently, it appears that the structure of the heterocyclic rings (aromatic or aliphatic) and the substituents may affect membrane fluidity differently.