Argante Bozzi

Resveratrol: A Natural Polyphenol with Multiple Chemopreventive Properties (Review)

Resveratrol, a naturally occurring polyphenol, shows pleiotropic health beneficial effects, including anti-oxidant, anti-inflammatory, anti-aging, cardioprotective and neuroprotective activities. Due to the several protective effects and since this compound is widely distributed in the plant kingdom, resveratrol can be envisaged as a chemo-preventive/curative agent introduced almost daily with the diet. Currently, a number of preclinical findings suggest resveratrol as a promising natures weapon for cancer prevention and treatment. A remarkable progress in elucidating the molecular mechanisms underlying anti-cancer properties of resveratrol has been achieved in the last years. Concerning the resveratrol mechanism of action as a protective (vs. normal cells and tissues) and toxic (vs. cancer cells) compound, many studies focus on its antioxidant capacity as well as on its ability to trigger and favor the apoptotic cascade in malignant cells. However, a generalized mechanism of action able to explain this dual effect of resveratrol has not yet been clearly established. In addition to these important functions, resveratrol is reported to exhibit several other biological/biochemical protective effects on heart, circulation, brain and age-related diseases which are summarized in this Review.

Antimicrobial peptides: natural templates for synthetic membrane-active compounds

Abstract.The innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (AMPs) to resist microbial invasion. Crafted by evolution into an extremely diversified array of sequences and folds, AMPs do share a common amphiphilic 3-D arrangement. This feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. This minireview reports on current understanding of the modes of interaction of AMPs with biological and model membranes, especially focusing on recent insights into the folding and oligomerization requirements of peptides to bind and insert into lipid membranes and exert their antibiotic effects. Given the potential of AMPs to be developed into a new class of anti-infective agents, emphasis is placed on how the information on peptide-membrane interactions could direct the design and selection of improved biomimetic synthetic peptides with antibiotic properties.

Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles

The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), isolated from the skin of the European red frog Rana temporaria. The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains. At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram-negative strains such as Pseudomonas aeruginosa A.T.C.C. 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria. In addition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested. A study of the interaction of temporin L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition. In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L. We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that perturbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings.

Cytotoxic activity and antioxidant capacity of purified lichen metabolites: an in vitro study.

The purpose of this study was to investigate the effects of six lichen metabolites (diffractaic acid, lobaric acid, usnic acid, vicanicin, variolaric acid, protolichesterinic acid) on proliferation, viability and reactive oxygen species (ROS) level towards three human cancer cell lines, MCF‐7 (breast adenocarcinoma), HeLa (cervix adenocarcinoma) and HCT‐116 (colon carcinoma). Cells were treated with different concentrations (2.5–100 μM) of these compounds for 48 h. In this comparative study, our lichen metabolites showed various cytotoxic effects in a concentration‐dependent manner, and usnic acid was the most potent cytotoxic agent, while variolaric acid did not inhibit the proliferation of any of the three cell lines used. All tested lichen compounds did not exhibit free radical scavenging activity using the 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) assay. The lichen metabolites did not significantly increase the intracellular ROS level and did not prevent oxidative injury induced by t‐butylhydroperoxide in HeLa cells. To better clarify the mechanism(s) of cytotoxic effect induced by protolichesterinic acid in HeLa cells, we investigated apoptotic markers such as condensation and fragmentation of nuclear chromatin and activation of caspase‐3, 8 and 9. Our results revealed that the antiproliferative activity of 40 μM protolichesterinic acid in HeLa cells is related to its ability to induce programmed cell death involving caspase‐3, 8 and 9 activation. Copyright

The biochemical mechanism of selective heat sensitivity of cancer cells—IV. Inhibition of RNA synthesis

Abstract The mechanism by which exposure of ascites tumour cells to supranormal temperatures causes an irreversible inhibition of uridine incorporation into RNA was investigated. Heat-treated cells were still able to incorporate labeled nucleotides, and even nucleosides, into RNA, if these precursors were added at sufficiently high concentrations. The passive permeability of the cell membrane increased exponentially with temperature, but this increase was fully reversible. At variance with the results obtained with agents which increase cell permeability, or which inhibit nucleoside permeation, heat treatment of Ehrlich ascites cells did not modify the ability of labeled uridine to be metabolized by these cells. The inhibition of uridine incorporation following heat treatment cannot therefore be attributed to a damage at the cell membrane level; the experimental data can be tentatively accounted for by the hypothesis of a block in the maturation of pre-rRNA into rRNA, with reutilization of the pre-rRNA degradation products.

Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria

ABSTRACT Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptides affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used β-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and β-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-α levels, resulting in the highest survival rates.

Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis

The cytosolic and mitochondrial alcohol dehydrogenases from Kluyveromyces lactis (KlADHs) were purified and characterised. Both the N-terminally blocked cytosolic isozymes, KlADH I and KlADH II, were strictly NAD-dependent and exhibited catalytic properties similar to those previously reported for other yeast ADHs. Conversely, the mitochondrial isozymes, KlADH III and KlADH IV, displayed Ala and Asn, respectively, as N-termini and were able to oxidise at an increased rate primary alcohols with aliphatic chains longer than ethanol, such as propanol, butanol, pentanol and hexanol. Interestingly, the mitochondrial KlADHs, at variance with cytosolic isozymes and the majority of ADHs from other sources, were capable of accepting as a cofactor, and in some case almost equally well, either NAD or NADP. Since Asp-223 of horse liver ADH, thought to be responsible for the selection of NAD as coenzyme, is strictly conserved in all the KlADH isozymes, this amino-acid residue should not be considered critical for the coenzyme discrimination with respect to the other residues lining the coenzyme binding pocket of the mitochondrial isozymes. The relatively low specificity of the mitochondrial KlADHs both toward the alcohols and the cofactor could be explained on the basis of an enhanced flexibility of the corresponding catalytic pockets. An involvement of the mitochondrial KlADH isozymes in the physiological reoxidation of the cytosolic NADPH was also hypothesized. Moreover, both cytosolic and KlADH IV isozymes have an additional cysteine, not involved in zinc binding, that could be responsible for the increased activity in the presence of 2-mercaptoethanol.

AZT: An Old Drug With New Perspectives

The science of antiviral research was well advanced when HIV/AIDS appeared as a major new virus disease in the early 1980s. The first effective antiviral compound (AZT, azidothymidine, zidovudine) was already among the library of compounds screened and was promptly reported to be a specific inhibitor of retroviruses, including HIV. Due to the pivotal role of AZT in HIV treatment, this review summarizes the most known effects -some of which are toxic side effects- induced by AZT a drug which is still used in the combined therapy of HIV-infected patients. Among the toxic side effects, a severe bone marrow toxicity manifested as anemia, neutropenia and siderosis, and caused by inhibition of heme and globin synthesis together with a general derangement of iron supply, have been reported. In this regard, we proved that while AZT and its monophosphorylated derivative AZTMP were unable to chelate iron, the triphosphate form AZTTP displayed a significant capacity to remove iron from transferrin. Moreover, we have previously demonstrated that AZT-exposed K562 cells showed an increase of transferrin receptors located on the cell membrane without affecting their biosynthesis, but slowing down their endocytotic pathway. Interestingly, literature data report the impairement of glycosylation reactions by AZT. Indeed, we have shown that AZT-treated K562 cells exhibited a reduced sialylation of proteins and lipids, and a strong inhibition of alpha,(2-->8) sialyltransferase activity while beta,(1-->4)galactosyltransferase and beta-galactosidase activities were significantly increased. These latter observations could be of clinical relevance since alterations of intracellular and cell surface carbohydrate expression and composition, often are associated with several diseases. However, contrarily to previous reports by other authors on AZT as an inhibitor of plant and bacterial toxins activity, we have demonstrated that AZT not only did not inhibit saporin toxicity, but even increased the cytotoxic activity of this plant toxin on K562 cells. Furthermore, the review enlightens the potential utilization of AZT as a tool in proteomics since in the recent years several genes responding to this drug have been identified in different cell lines. We have shown, for the first time, an over-expression of two proteins (PDI-A3 and sthatmin), and a full repression of two others (HSP-60 and SOD1) in AZT-exposed K562 cells. At present, we are investigating if the above reported alterations are a general feature of AZT-treatment of cultured cells, or they represent a peculiar characteristic of a specific cell line. Finally, the paper reviews a number of novel methodologies aimed at enhancing the AZT plasma levels and its bioavailability in all human organs in order to improve its therapeutic efficacy against HIV infection. These new possibilities, namely the AZT prodrug strategy, the AZT transdermal delivery and the targeted brain delivery, are yet not in use for humans but they are under experimental studies.

pH-dependent disruption of Escherichia coli ATCC 25922 and model membranes by the human antimicrobial peptides hepcidin 20 and 25

The human hepcidin 25 (hep‐25) and its isoform hepcidin 20 (hep‐20) are histidine‐containing, cystein rich, β‐sheet structured peptides endowed with antimicrobial activity. We previously reported that, similar to other histidine‐containing peptides, the microbicidal effects of hep‐25 and hep‐20 are highly enhanced at acidic pH. In the present study, we investigated whether pH influences the mode of action of hep‐25 and hep‐20 on Escherichia coli American Type Culture Collection 25922 and model membranes. A striking release of β‐galactosidase by hepcidin‐treated E. coli was observed at pH 5.0, whereas no inner membrane permeabilization capacity was seen at pH 7.4, even at bactericidal concentrations. Similar results were obtained by flow cytometry when assessing the internalization of propidium iodide by hepcidin‐treated E. coli. Scanning electron microscope imaging revealed that both peptides induced the formation of numerous blebs on the surface of bacterial cells at acidic pH but not at neutral pH. Moreover, a phospholipid/polydiacetylene colourimetric vesicle assay revealed a more evident membrane damaging effect at pH 5.0 than at pH 7.4. The leakage of entrapped dextrans of increasing molecular size from liposomes was also assessed at pH 7.4. Consistent with the lack of β‐galactosidase release from whole E. coli observed at such a pH value, evident leakage of only the smallest 4‐kDa dextran (and not of dextrans of 20 or 70 kDa) was observed, indicating a poor ability of hepcidin peptides to permeabilize liposome vesicles at pH 7.4. Altogether, the data obtained in the present study using different approaches strongly suggest that the ability of hepcidins to perturb bacterial membranes is markedly pH‐dependent.

Membrane interaction and antibacterial properties of two mildly cationic peptide diastereomers, bombinins H2 and H4, isolated from Bombina skin

Bombinins H are mildly cationic antimicrobial peptides isolated from the skin of the anuran genus Bombina, the fire-bellied toad. Some members of this peptide family coexist in skin secretions as diastereomers in which a single d-amino acid (alloisoleucine or leucine) is incorporated as a result of the post-translational modification of the respective gene-encoded l-amino acid. Here we report on the antimicrobial properties and membrane interactions of bombinins H2 and H4. The latter differs from H2 by the presence of a d-alloisoleucine at the second N-terminal position. Specifically, we have evaluated the antimicrobial activity of H2 and H4 against a large panel of reference and clinical isolates of Gram-negative and Gram-positive bacteria; performed membrane permeation assays on both intact cells and model membranes (lipid monolayers and liposomes) mimicking the composition of the plasma membrane of Gram-negative/positive bacteria; used biochemical tools, such as trypsin-encapsulated liposomes and capillary electrophoresis, to monitor the peptides’ ability to translocate through the membrane of liposomes mimicking Escherichia coli inner membrane. The results revealed interesting relationships between the presence of a single d-amino acid in the sequence of an antimicrobial peptide and its target microbial cell selectivity/membrane-perturbing activity.