Simonetta Croci

Elastic incoherent neutron scattering as a probe of high pressure induced changes in protein flexibility.

We report here the results of elastic incoherent neutron scattering experiments on three globular proteins (trypsin, lysozyme and beta-lactoglobulin) in different pressure intervals ranging from 1 bar to 5.5 kbar. A decrease of the mean square hydrogen fluctuations, u(2), has been observed upon increasing pressure. Trypsin and beta-lactoglobulin behave similarly while lysozyme shows much larger changes in u(2). This can be related to different steps in the denaturing processes and to the high propensity of lysozyme to form amyloids. Elastic incoherent neutron scattering has proven to be an effective microscopic technique for the investigation of pressure induced changes in protein flexibility.

Acetylphenylhydrazine induced haemoglobin oxidation in erythrocytes studied by Mössbauer spectroscopy

The oxidative action of acetylphenylhydrazine (APH) on red blood cells obtained from healthy donors and from patients with breast cancer has been investigated by Mössbauer spectroscopy. Whole blood was incubated with APH for different time periods and the Mössbauer spectra of the packed red cells were recorded and compared. The evolution with time of the oxidation products has been followed. The largest difference in red cells analysis between healthy persons and patients was found after about 50 min of treatment where Mössbauer spectra of patient samples show a much broader spectral pattern due to an advanced haemoglobin oxidation.

Potassium bicarbonate and D-ribose effects on A72 canine and HTB-126 human cancer cell line proliferation in vitro.

BackgroundThe synergic action of KHCO3 and D-ribose is tested on A72 and HTB-126 cell lines proliferation using K:D-Rib solution. Altered Na+/K+ ATPase expression and activity were shown in patients with cancer. Studies in human epithelial-derived malignancies indicate that K+ depletion also occurs, contributing to the increased intracellular Na+/K+ ratio [1]. D-ribose transformed to piruvate, enters into the Krebss cycle and has a key role on energetic metabolism. The up-regulation of glycolysis in tumor cells is already well known and it is the rationale of F18-FDG PET diagnostic technique. D-ribose is synthesized by the non-oxidative transketolase PPP reaction.ResultsResults with different K:D-Rib concentrations show that MTT salt interferes with K:D-Rib solution and therefore this method is not reliable. The UV/VIS measurements show that K:D-Rib solutions reduce MTT salt to formazan in absence of cells. Cell proliferation has then been evaluated analysing the digital photos of the Giemsa stained cells with MCID™ software. At 5 mM K:D-Rib concentration, the cell growth arrests between 48 h and 72 h; in fact the cell number after 48 h is around the same with respect to the control after 72 h. In case of HTB-126 human cancer cells, the growth rate was valuated counting the splitting times during 48 days: control cells were split sixteen times while 5 mM treated cells eleven times. Most relevant, the clonogenic assay shows that nine colonies are formed in the control cells while only one is formed in the 5 mM and none in 10 mM treated cells.ConclusionsThe K:D-Rib solution has an antioxidant behaviour also at low concentrations. Incubation with 5 mM K:D-Rib solution on A72 cells shows a cytostatic effect at 5 mM, but it needs more than 24 h of incubation time to evidence this effect on cell proliferation. At the same concentration on human HTB-126 cells, K:D-Rib solution shows a clear replication slowing but the cytostatic effect at 10 mM K:D-Rib solution only. Results on A72 cells indicate the K+ uptake could be determinant either to arrest or to slow down cell growth.

Potassium Ascorbate as Protective Agent in the Oxidation of the Red Blood Cells

Free radicals and oxidative substances are involved in many degenerative diseases modifying cell physiological properties. Many mechanisms can be disrupted, including the K-Na channels, with a consequent potassium loss. Free radicals are also able to modify erythrocytes stability leading to peroxidation of the lipid membrane, oxidation of haemoglobin (Hb) and finally to the formation of Heinz Bodies. Such a process can also be induced or enhanced by strong oxidants like acetylphenylhydrazine (APH). In the present work the antioxidant properties of potassium ascorbate acting on the red cells treated with APH are investigated.

Hemoglobin oxidative stress

Venous blood obtained from healthy donors and from patients suffering from breast cancer have been treated with acetylphenylhydrazine (APH) for different time. Mössbauer spectra of the packed red cells have been recorded and compared. The largest difference occurs after 50 min of treatment with APH where the patient samples show a broad spectral pattern indicating an advanced hemoglobin oxidation. These results may have some relevance in early cancer diagnosis.

APH Induced Red Cell Oxidation in Healthy Persons

In a previous work we have shown that the red blood cells of patients suffering from breast cancer (P) seem more sensitive to the denaturing action of acetylphenylhydrazine (APH) than those from healthy donors (H). The amount and the formation time of the hemin, one of the last oxidation products, are significantly different in the patients. Therefore, the spectral contribution of hemin after the same incubation time could be a factor of discrimination between the samples from the patients and healthy population. The aim of the present work was to gain a deeper understanding of the oxidation processes and establish a standard setup for the measurements.