Assessment of the effect of vessel curvature on Doppler measurements in steady flow

Blood vessel curvature is responsible for the appearance of nonaxial velocity components and for minor changes in the pattern of the axial flow. All the velocity components are expected to contribute to the Doppler signal produced by the ultrasound (US) backscattered by the insonated blood cells, the axial velocity, contributing to the actual volumetric blood flow, and the transverse velocity, causing the recirculating vortices. A detailed, separate analysis of the velocity components is, therefore, mandatory to quantify how vessel curvature can affect results and clinical diagnosis. Both experimental in vitro measures and numerical simulations were performed on a curved tube and the Doppler power spectra so obtained were compared. The satisfactorily agreement of the above spectra shows that the nonaxial velocity components are easily detectable with clinical equipment and that their amplitude, as expected, is not negligible and can bias Doppler measurements and resulting clinical diagnosis.     

Oxidative stress in the animal model: the possible protective role of milk serum protein

In the field of biology, free radicals which are derived from the incomplete reduction of oxygen take on great importance; they belong to the so called reactive oxygen species, whose production in the organism is an inevitable consequence of various external or internal factors to which it is exposed. Once free radicals are generated they are often capable of giving rise to chain reactions. A lot of biological molecules are susceptible to the attack by free radicals including lipids, proteins, carbohydrates and nucleic acids. Molecular alterations caused by the radical reactions have been frequently studied and are considered as pathogenetically main passages in the development of many diseases and ageing. In order to face a radical attack, living organisms have developed several biological defensive systems against it: the main ones are represented by anti oxidizing molecules and by enzymatic anti oxidizing systems. Among the various defence systems, glutathione stands out as the principal guarantor of homoeostatic intra-cellular oxidation–reduction. One of glutathione’s most important functions is to act as cysteine “tank”; this amino acid is extremely unstable in the extra-cellular environment and it rapidly auto-oxidates. Whey proteins (WP) are particularly rich in cysteine (cys) and in glutamine (glu) and therefore potentially capable of increasing the organism’s antioxidant defences. It is thought that the principal mechanism which allows WPs to exert their properties is through the contribution of cys and glu, which is rich in these proteins and is used intra-cellularly for the synthesis of glutathione. A diet based on milk serum proteins which supplies a superior quantity of cys, allows for a greater synthesis of hepatic glutathione in oxidative stress conditions. The use of ultra-filtrated WP could represent a useful tool in the control of oxidative stress in numerous pathological situations.     

Compartmentalization and insulin-induced translocations of insulin receptor substrates, phosphatidylinositol 3-kinase, and protein kinase B in rat liver

Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling proteins between subcellular compartments in rat liver. In plasma membranes (PM) and microsomes, insulin induced a rapid decrease in insulin receptor substrate-1/2 (IRS1/2) within 30 sec and an increase in these proteins in endosomes (EN) and cytosol. The level of p85 in PM increased 2.3-fold at 30 sec after insulin stimulation followed by a decrease at 2 min. In this interval, 60-85% and 10-20% of p85 in PM was associated with IRS1 and IRS2, respectively. Thus, in PM, IRS1/2 accounts for almost all of the protein involved in phosphatidylinositol 3-kinase activation. In ENs insulin induced a maximal increase of 40% in p85 recruitment. As in PM, almost all p85 was associated with IRS1/2. The greater level of p85 recruitment to PM was associated with a higher level of insulin-induced recruitment of Akt1 to this compartment (4.0-fold in PM vs. 2.4-fold in EN). There was a close correlation between Akt1 activity and Akt1 phosphorylation at Thr308 and Ser473 in PM and cytosol. However, in ENs the level of Akt1 activity per unit of phosphorylated Akt1 was significantly greater than in PM, indicating that in addition to phosphorylation, another factor(s) modulates Akt1 activation by insulin in rat liver. Our results demonstrate that activation of the insulin receptor kinase and modulation of key components of the insulin signaling cascade occur at the cell surface and within the endosomal system. These data provide further support for the role of the endocytic process in cell signaling.     

Doppler indices in the umbilical arteries: influence of vessel curvature induced by bladder filling

Doppler indices are widely used to assess normal versus pathologic haemodynamics. In obstetrics, the assessment of abnormal values in some critical compartments, such as the umbilical arteries (UA), may be crucial in the clinical management of growth-restricted foetuses. It was recently proposed that the UA should be sampled in their perivesical portion (PVC), i.e., where they surround the foetal urinary bladder. However, measurements at this site could be biased by the degree of curvature of the vessel due to bladder filling. We investigated this possibility in vivo and in vitro, i.e., measurements on rubber tubes at different radii of curvature R_c. There was significant dependence of the Doppler indices A/B and PI on the vessel curvature and insonation angle; in fact, we recorded errors of about 25% when R_c was 10 times larger than the radius of the vessel and about 100% when R_c was five times larger than the radius of the vessel. Therefore, measurements of the UA at the PVC site should only be performed when the foetal bladder is empty. (E-mail: caterina.guiot@unito.it)     

Compartmentalization of signaling-competent epidermal growth factor receptors in endosomes

In this study, the preparation of detergent-resistant membranes (DRMs) and the immunoisolation of intracellular vesicles enriched in raft markers were used to investigate the effect of physiological doses of epidermal growth factor (EGF) in vivo on the compartmentalization and activation of EGF receptor (EGFR) in rat liver endosomes. Both of these techniques show that after EGF administration, a distinctive population of intracellular EGFR, which was characterized by a high level of tyrosine phosphorylation, accumulated in endosomes. EGFR recruited to early endosomes were more tyrosine phosphorylated than those from late endosomes. However, the level of tyrosine phosphorylation of EGFR in DRMs isolated from early and late endosomes was comparable, suggesting that EGFR in endosomal DRMs are more resistant to tyrosine dephosphorylation. In accordance with the higher level of Tyr phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc to endosomal DRMs compared with whole endosomes. Furthermore, a proteomic analysis identified a selective increase of many alpha-subunits of heterotrimeric G proteins in endosomal DRMs in response to EGF. These observations suggest that a distinctive pool of endocytic EGFR, potentially competent for signaling, is actively trafficking through intracellular compartments with the characteristic of lipid rafts.     

GSH loss per se does not affect neuroblastoma survival and is not genotoxic

Depletion of glutathione (GSH) by buthionine sulfoximine (BSO) has been reported to be toxic against some cancer cells and to sensitize many tumours including neuroblastoma (NB) to anticancer drugs. The balance between the production rate of reactive oxygen species (ROS) and the function of GSH affects the intracellular reduction-oxidation status, which is crucial for the regulation of several cellular physiological functions. To assess the role of glutathione in neuroblastoma therapy, the effect of sublethal concentrations of BSO was studied in a panel of neuroblastoma cell lines characterized by different MYCN status. We found that GSH depletion per se not accompanied by ROS overproduction, does not affect cell survival, and is not genotoxic but induces HO-1 expression in GI-ME-N cell line, a representative example of MYCN non-amplified NB cells, having the highest basal levels of GSH among the tested NB lines. These observations might open a novel therapeutic window based on the possibility of modulating the cellular 'activity' of GSH.     

Anti malondialdehyde-adduct immunological response as a possible marker of successful aging

Contrasting results have been obtained by various researchers about oxidative markers of aging. In this study, a healthy over-90-year-old population was examined for various plasma oxidative biomarkers and compared with a healthy population of blood donors (age range 23-66). Plasma malondialdehyde (MDA), evaluated by means of the thiobarbituric acid test, was significantly higher in the over-90-year-old population, confirming the presence of increased lipoperoxidation in old age. The antibody titre against MDA-protein adducts, considered a marker of lipoperoxidative protein damage in vivo, was evaluated in an ELISA test, completely home made and calibrated versus a concentrated pool of human plasma; this antibody titre was significantly higher in the over-90-year-old population. Plasma vitamin E, evaluated in RP-HPLC, was not significantly different between the two groups. Plasma protein-bound carbonyls, a marker of oxidative protein damage, were measured with the 2,4-dinitrophenylhydrazine assay; their level in the over-90-year-old population was lower than in the blood donors. The higher antibody titre against MDA-adducts may result in protection against accumulation of oxidatively damaged proteins by enhancing their removal, and, together with the preserved plasma vitamin E level, it may endow over-90-year-olds with an especially efficient antioxidant profile. The low level of protein carbonyl might reflect the more efficient removal of damaged proteins.     

Curvature affects Doppler investigation of vessels: implications for clinical practice

In clinical practice, blood velocity estimations from Doppler examination of curved vascular segments are normally different from those of nearby straight segments. The observed "accelerations," sometimes considered as a sort of stochastic disturbances, can actually be related to very specific physical effects due to vessel curvature (i.e., the development of nonaxial velocity [NAV] components) and the spreading of the axial velocity direction in the Doppler sample volume with respect to the insonation axis. The relevant phenomena and their dependence on the radius of curvature of the vessels and on the insonation angle are investigated with a beam-vessel geometry as close as possible to clinical setting, with the simplifying assumptions of steady flow, mild vessel curvature, uniform ultrasonic beam and complete vessel insonation. The insonation angles that minimize the errors are provided on the basis of the study results.     

Whey protein, as exclusively nitrogen source, controls food intake and promotes glutathione antioxidant protection in Sprague-Dawley rats

The inclusion of whey protein concentrates (WPC) in the diet can lead to a decrease in food intake. Considering that excessive food intake and weight gain are correlated with increased oxidative stress and other risk factors, the anorectic action of WPC may have important clinical implications. The aims of the current study were to verify the effects of WPC in comparison with those of casein on food intake, weight, and oxidized glutathione (GSSG) and total glutathione (GSH) concentrations in the blood and liver with or without oxidative stress induced by oral carbon tetrachloride intoxication. Male Sprague-Dawley rats were fed a balanced liquid diet for 3 weeks. Half of the rats received WPC (group P), while the control group received casein (group C). Group P rats ate significantly less than group C rats (p < 0.0001), and their weights decreased significantly. After carbon tetrachloride intoxication, there was a significant increase in GSH in rats of group P compared with the levels in rats of group C both in the liver (GSH group P 4,994 ± 652.6, group C 2,196 ± 323.2 nmol/mg, p < 0.01) and in the blood (GSH group P 1,368 ± 69.56, group C 1,088 ± 48.35 nmol/ml, p < 0.05). These findings indicate that WPC is effective in reducing food intake and preventing weight gain, and it may also play a protective role against oxidative stress by increasing glutathione synthesis in the liver.