Veronica Carnicelli

Isomerization of an Antimicrobial Peptide Broadens Antimicrobial Spectrum to Gram-Positive Bacterial Pathogens

The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.

Lactoferrin Derived Peptides: Mechanisms of Action and their Perspectives as Antimicrobial and Antitumoral Agents

Antimicrobial peptides, AMPs, exert their function acting mainly at the membrane level. In the wide AMPs panorama a particular position is occupied by lactoferrin derived peptides. They also possess antiviral, antifungal and antitumor activities and their parent molecules are available in several mammalian fluids and in dairy industries waste.

Regional differences in signalling transduction pathways among smooth muscle cells from rabbit colon

Smooth muscle cells (SMC) from the circular muscle layer of rabbit colon, taken from the proximal and distal regions that are known to have different physiological and motor activities, were used to highlight distinct regional intrinsic myogenic properties and to investigate the correlations between receptor and signalling transduction pathways. Contractile agonists were shown to be more potent on proximal than on distal SMC in inducing contraction and intracellular Ca(2+) increase. Concentration-response curves of agonists-induced Ca(2+) increase were constantly shifted to the right, though remaining parallel, with respect to contraction curves, independently of the region analysed. Using agents activating different steps of cAMP-or cGMP-mediated intracellular cascades, main regional differences were revealed as far as relaxation was concerned. Relaxation of proximal SMC was found to be essentially cGMP mediated, while that of distal SMC was cAMP mediated. In conclusion, the motor patterns of the two regions appear to be influenced by distinct regional biochemical characteristics that are intrinsic to colonic SMC.

An EPR study of lipid vesicles as paramagnetic agent vectors

Paramagnetic nitroxides have been proposed as probes in electron paramagnetic resonance (EPR) imaging and in clinical diagnosis. However, nitroxides are rapidly reducedin vivo to hydroxylamines, diamagnetic EPR-inactive species. Reduction occurs in blood via soluble agents such as ascorbic acid, as well as in the cells via enzymatic and non-enzymatic endocellular systems. To prevent the reduction, a water soluble nitroxide, i.e., potassium peroxylamine disulfonate, is entrapped in reverse phase evaporation vesicles. The loaded liposomes have a high entrapment capacity, and vesicles with the encapsulated agent are stable for days, even at room temperature. The vesiclesin vitro can almost completely prevent the reduction of the entrapped nitroxide by ascorbic acid. In blood of a rat, enriched with a homogenate of rat liver proteins, the vesicles are able to greatly prolong the life time of the nitroxide. In particular, the encapsulated nitroxide has a half-life of more than one hour, compared to two minutes for free nitroxide under the same conditions. Due to these protective effects, the lipid vesicles might be useful as a delivery system for paramagnetic agents.

Apoptotic effects of bovine apo‐lactoferrin on HeLa tumor cells

Lactoferrin (Lf), a cationic iron‐binding glycoprotein of 80 kDa present in body secretions, is known as a compound with marked antimicrobial activity. In the present study, the apoptotic effect of iron‐free bovine lactoferrin (apo‐bLf) on human epithelial cancer (HeLa) cells was examined in association with reactive oxygen species and glutathione (GSH) levels. Apoptotic effect of iron‐free bovine lactoferrin inhibited the growth of HeLa cells after 48 hours of treatment while the diferric‐bLf was ineffective in the concentration range tested (from 1 to 12.5 μM). Western blot analysis showed that key apoptotic regulators including Bax, Bcl‐2, Sirt1, Mcl‐1, and PARP‐1 were modulated by 1.25 μM of apo‐bLf. In the same cell line, apo‐bLf induced apoptosis together with poly (ADP‐ribose) polymerase cleavage, caspase activation, and a significant drop of NAD+. In addition, apo‐bLf–treated HeLa cells showed a marked increase of reactive oxygen species level and a significant GSH depletion. On the whole, apo‐bLf triggered apoptosis of HeLa cells upon oxygen radicals burst and GSH decrease.

EPR study of Fremy’s salt nitroxide roduction by ascorbic acid; influence of the bulk pH values

EPR and UV spectroscopy were used to investigate the efficiency of ascorbic acid in reducing Fremy’s salt. Our data indicates that the first proton electron transfer from ascorbate occurs within the mixing time. Even after the disappearance of the UV signal of the ascorbate, EPR measurements showed that the reaction goes forward, indicating a biphasic redox process. The slower time-course of this second phase was related to the initial concentrations of the reductant. Experiments performed at four different pH values demonstrated that the reduction was a function of the bulk solution pH. At the lower pH, after a fast initial reduction, the Fremy’s salt EPR signal remained constant, while at physiological or higher pH a further reduction was found.The reaction rates demonstrate that the reducing power of ascorbic acid towards Fremy’s salt strongly depends on its dissociation state.

Static magnetic field effect on the Fremy's salt-ascorbic acid chemical reaction studied by continuous-wave electron paramagnetic resonance.

Static magnetic field effect in the framework of the radial pair mechanism (RPM) theory was studied on the biologically significant chemical reaction between ascorbic acid and Fremys salt. The data indicate that the reaction rate depends on the applied magnetic field strength. The time scale of the studied reaction and the improved continuous-wave electron paramagnetic resonance system allowed for the first time the direct comparison of the amplitude differences between exposed and control samples in the strictly same boundary conditions. Until now the RPM was studied in a different time scale, focusing only on faster reactions by time-resolved techniques or by spectrophotometer measurement. The magnetic field effects presently measured can not be extended tout court to living systems; however the understanding of magnetic field sensitivity in basic chemical reaction in vitro could help clarifying the underlying basic step of interaction between magnetic fields and biological systems.

EPR study of the effect, induced by zidovudine (AZT), on the membrane lipid dynamics in leukemic cell

Zidovudine (AZT) was the first drug approved by the FDA for treatment of HIV infections. To investigate the AZT effects on the physical properties of K562 cell membranes, experiments were performed by measuring an order parameter value in these cells, previously labelled with 16-DSA (16-doxyl-stearic acid) or 5-DSA (5-doxyl-stearic acid). The EPR spectra of the labelled fatty acid were used to detect the alteration in their freedom of motion and to provide indications of membrane fluidity. Two different parameters were calculated from EPR spectra for the 16-DSA and 5-DSA. Preliminary data indicate that, for both the probes used, these parameters were not significantly different in the control cells with respect to the AZT-treated ones. Control measurements, performed to test the sensitivity of the technique using the DMSO agent, showed a significant increase in the K562 membrane fluidity.

Effects of azidothymidine on protein kinase C activity and expression in erythroleukemic cell K562 and acute lymphoblastic leukemia cell HSB-2

Azidothymidine (AZT) is one of the anti-retroviral drugs currently used for the treatment of HIV-infected patients. Several other effects of the drug have been studied in vitro, such as the alterations of some enzymes, the inhibition of cell proliferation, and the increase of transferrin receptor expression. In this study, we investigated the alterations of protein kinase C (PKC) activity, PKCα and PKCβII expressions and plasmatic membrane fluidity induced by AZT in two cancer cell lines, human chronic myeloid (K562) and human acute lymphoblastic (HSB-2) leukemia cells, respectively. The results showed that both PKC activity and membrane fluidity in HSB-2 cells increased after 24 h of drug incubation. PKCα expression in HSB-2 cells decreased after 48 h of AZT exposure, when the cell growth also decreased. However, in K562 cells, the PKCα and PKCβII expressions enhanced in the presence of the drug when the growth was inhibited. The results indicate that AZT is less effective in inhibiting the growth of acute lymphoblastic HSB-2 leukemia cells than inhibiting that of chronic myeloid K562 cells. In fact, after 24 h exposure, the HSB-2 cell growth decreased less than K562 cell growth.