Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related to modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c−cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73, and Lys79 residues play in the cytochrome c−cardiolipin interaction, as these side chains appear to be critical for cytochrome c−cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, ultraviolet−visible, and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination and are less stable than the wild-type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin, and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73, and Lys79 residues stabilize the native axial Met80−Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c−cardiolipin recognition, the simultaneous presence of Lys72, Lys73, and Lys79 is necessary for the peroxidase activity of cardiolipin-bound cytochrome c.

To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5±10 mm) of hydrogen peroxide for 1 h at 37 8C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mm H 2 O 2 ), oxidative stress produced, starting from 1 mm H 2 O 2 , progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H 2 O 2 concentration (4 mm). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMPdeaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H 2 O 2 used; this enzymatic activity increased with increasing H 2 O 2 in the medium, and reached its maximum at 4 mm H 2 O 2 -treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H 2 O 2 , being detectable at the lowest H 2 O 2 concentration and increasing without appreciable haemolysis up to 4 mm H 2 O 2 . Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.Keywords: AMP-deaminase; energy metabolism; human erythrocytes; IMP; oxidative stress.Reactive oxygen species (ROS) are produced in living organisms from the partial reduction of molecular oxygen under physiological and pathological conditions. Because ROS has the capability of reacting nonspecifically with a vast number of compounds, all living organisms contain several enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and nonenzymatic (a-tocopherol, ascorbic and uric acids, glutathione and other thiol protein groups) antioxidants with the specific purpose of protecting the functional and structural integrity of biologically fundamental macromolecules (nucleic acids, proteins, phospholipids). Imbalance between ROS production and antioxidant cell defences has been reported to occur in several physiopathological conditions of both animals and man: for example tissue ischemia and reperfusion Many in vitro and in vivo studies showed that several parameters of red blood cell function and integrity are ne...

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The effect of mild closed head trauma, induced by the weight-drop method (450 g from a 1-m height), on lipid peroxidation and energy metabolism of brain tissue was determined at various times after cerebral injury in spontaneously breathing rats (1, 10, 30 minutes and 2, 6, 15, 24, 48, and 120 hours). Animals were continuously monitored for the evaluation of blood pressure, blood gases, heart rate, and intracranial pressure. Analysis of malondialdehyde (MDA) as an index of lipid peroxidation, ascorbic acid, high-energy phosphates, nicotinic coenzymes, oxypurines, and nucleosides was performed by high-performance liquid chromatography (HPLC) on neutralized perchloric acid extract of the whole brain. Data showed that MDA, undetectable in control, sham-operated rats, was already present within 1 minute of trauma (1.77 nmol/g wet weight; SD = 0.29) and reached maximal values by 2 hours (72.26 nmol/g w.w.; SD = 11.26), showing a progressive slow decrease thereafter. In contrast, ATP, GTP, and nicotinic coenzyme (NAD and NADP) concentrations showed significant reduction only by the second hour postinjury. Maximal decrease of the ATP and GTP concentrations were seen at 6 hours postinjury, whereas NAD and NADP concentrations showed maximum decline by 15 hours. Values recorded in mechanically ventilated rats did not differ significantly from those obtained in spontaneously breathing animals. These findings, supported by the absence of blood gas and blood pressure changes in the spontaneously breathing rats, strongly support the premise that biochemical changes (primarily lipid peroxidation) are not caused by secondary ischemic-hypoxic phenomena but rather are triggered by these forces acting on the brain at the time of impact. In addition, these results suggest that depression of energy metabolism might be caused by peroxidation of the mitochondrial membrane with a consequent alteration of the main mitochondrial function-that is, the energy supply.

Dietary supplementation with EPA significantly increases levels of this fatty acid in colonic mucosa, associated with significantly reduced proliferation and increased mucosal apoptosis. Further studies are needed to assess the potential efficacy of EPA supplementation in preventing polyps in the chemoprevention of colorectal cancer.

The excretion of the compound was principally via urine, giving dose to the kidney and the urinary bladder wall. As SSTR2 is the most frequently expressed somatostatin receptor and (68)Ga-DOTANOC has high affinity to it, this compound might play an important role in PET oncology in the future. The dosimetric evaluation carried out by our team demonstrated that (68)Ga-DOTANOC delivers a dose to organs comparable to, and even lower than, analogous diagnostic compounds.

Over the last twenty years, newly developed chemical sensor systems (socalled "electronic noses") have odour analyses made possible. This paper describes the applications of these systems for microbial detection in different fields such as medicine and the food industry, where fast detection methods are essential for appropriate management of health care. Several groups have employed different electronic noses for classification and quantification of bacteria and fungi to obtain accurate medical diagnosis and food quality control. So far, detection and identification of bacterial and fungal volatiles have been achieved by use of e-noses offering different correct classification percentages. The present review includes examples of bacterial and fungal species producing volatile compounds and correlated to infectious diseases or food deterioration. The results suggest the possibility of using this new technology both in medical diagnostics and in food control management.

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