Effects of dietary iodine supplement on sheep milk and cheese.

The work reported in this paper addresses the iodine nutritional deficiency that still affects a large number of people. For this purpose, we analyzed the possibility to use, as iodine vehicle, a hard typical ewe cheese, called Canestrato d'Abruzzo, derived from milk of ewes fed with an iodine-fortified diet. Both in the milk and the cheese of these animals, the iodine level was higher than that measured in sheep with a normal diet. An increase in the lactoferrin and iron content was evident in the whey derived from milk of the iodine group. Furthermore, in derived cheese, the caseins seemed more efficiently transformed in small peptides making the product more digestible and, for this reason, particularly suitable for feeding the elderly. In conclusion, the dairy products obtained from ewes fed with iodine diet contain more bioactive compounds so that they represent a useful food to prevent iodine and iron deficiency in lamb and humans.

Antibacterial and Anti-Inflammatory Activity of an Antimicrobial Peptide Synthesized with D Amino Acids.

The peptide SET-M33 is a molecule synthesized in tetra-branched form which is being developed as a new antibiotic against Gram-negative bacteria. Its isomeric form with D amino acids instead of the L version (SET-M33D) is also able to kill Gram-positive bacteria because of its higher resistance to bacterial proteases (Falciani et al., PLoS ONE, 2012, 7, e46259). Here we report the strong in vitro activity of SET-M33D (MIC range 0.7-6.0 µM) against multiresistant pathogens of clinical interest, including Gram-positives Staphylococcus aureus, Staphylococcus saprophyticus, and Enterococcus faecalis, and various Gram-negative enterobacteriaceae. SET-M33D antibacterial activity is also confirmed in vivo against a MRSA strain of S. aureus with doses perfectly compatible with clinical use (5 and 2.5 mg/Kg). Moreover, SET-M33D strongly neutralized lipopolysaccharide (LPS) and lipoteichoic acid (LTA), thus exerting a strong anti-inflammatory effect, reducing expression of cytokines, enzymes, and transcription factors (TNF-α, IL6, COX-2, KC, MIP-1, IP10, iNOS, NF-κB) involved in the onset and evolution of the inflammatory process. These results, along with in vitro and in vivo toxicity data and the low frequency of resistance selection reported here, make SET-M33D a strong candidate for the development of a new broad spectrum antibiotic.

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.

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 PKCßII 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 PKCßII 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.

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.

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.

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.

Globin interactions with lipids and membranes.

Many bacterial globins have been demonstrated to interact with membrane lipids, and several hypotheses in support of a functional role for membrane localization have been set forth. Bacterial globins have been suggested to facilitate oxygen diffusion to terminal oxidases, to protect oxidases from nitric oxide or eventually to preserve the integrity of the membrane lipids through peroxide-reducing activities as a response to oxidative/nitrosative stress. In this framework, methodological approaches to the study of globin-membrane interactions need to be analyzed in depth in order to single out the relevant features of these interactions and to clearly distinguish the specific membrane and lipid binding process from trivial effects related to the possible partitioning of the lipid side chains to the hydrophobic heme pocket or to the presence of partially folded, insoluble protein aggregates within membranous pellets. Methods for qualitative lipid analysis, liposome-protein binding studies, and analysis of protein insertion into lipid monolayer are thus described with the aim of providing rapid and efficient screening of specific globin-membrane interactions.

Zidovudine inhibits protein kinase C activity in human chronic myeloid (K562) cells.

In this paper we show that human erythroleukaemia (K562) cells exhibited a significant inhibition of protein kinase C activity when cells were exposed to 40 micro M zidovudine in a time interval of 5-180 min., whereas prolonged treatment (24 hr) was uneffective. The addition of an excess of thymidine (125:1, mol:mol), in the cell suspension with or without zidovudine fully restored the protein kinase C activity. Interestingly, either in cell homogenates and in commercially purified rat brain protein kinase C, both zidovudine and its monophosphate derivative, caused inhibition that was higher than in intact cells. This inhibition reached a maximal value of 45% when zidovudine or zidovudine monophosphate were incubated with the pure commercial enzyme and in this case the addition of thymidine did not prevent the enzyme inhibition. The conclusions from these data are that either zidovudine or zidovudine monophosphate interact directly with the pure enzyme, causing inhibition, while in intact cells exposed to the drug, zidovudine monophosphate appears to be the main metabolite responsible for protein kinase C inhibition.

Interaction of antimicrobial peptide temporin L with lipopolysaccharide in vitro and in experimental rat models of septic shock caused by gram-negative bacteria.

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 peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used beta-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-alpha) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and beta-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-alpha levels, resulting in the highest survival rates.

Interaction of Vitreoscilla hemoglobin with membrane lipids.

The interaction of the recombinant hemoglobin from Vitreoscilla sp. (VHb) with the bacterial membrane of Escherichia coli cells has been investigated by measuring the propensity of VHb to interact with monolayers formed by natural bacterial phosholipids. The measurements showed that the protein is capable of penetrating the monolayers, possibly establishing interactions with the hydrophobic acyl chains. VHb is also capable of binding reversibly phospholipids and free fatty acids in solution with a strong selectivity toward cyclopropanated acyl chain species. Lipid binding occurs within the distal heme pocket as demonstrated by a sharp UV-vis spectral change corresponding to a five-coordinate to six-coordinate transition of the heme-iron ferric derivative. Oxygen binding properties are affected by the presence of the lipid component within the active site. In particular, the oxygen affinity is decreased by more than 20-fold in the presence of cyclopropanated phospholipids. The kinetic counterpart of the decrease in oxygen affinity is manifest in a 10-fold decrease in the ligand combination kinetics. Accordingly, the CO and NO combination kinetics were also significantly affected by the presence of the bound lipid within the active site. These studies indicate that the current functional hypotheses about VHb should take into account the association of the protein within the cytoplasmic membrane as well as the presence of a phospholipid within the active site. These data suggest a possible lipid-induced regulation of oxygen affinity as the basis of VHb functioning.

Improvements in technical assessment and protocol for EPR evaluation of magnetic fields effects on a radical pair reaction.

The effects of either static or pulsed magnetic fields on the reaction rate of Fremy's salt-ascorbic acid were studied directly by EPR spectroscopy. Radical pair mechanism (RPM) accounts for the magnetic field effects, but the expected amounts are so small that they need to be observed with particular care with EPR technique. The method is based on the resolution of a pair of EPR signals by the addition of a stationary field gradient, where the signals are coming from the exposed and control capillary sample. To this purpose, a suitable device for the gradient generation was used. Others improvements were the strictly keeping of the same boundary temperature condition in the capillary pairs, obtained by a refrigerating system controlled by a thermocouple, and the use of a pair of Helmholtz coils to generate an external high homogeneous magnetic field. By this experimental set up, we found that the magnetic field induce the decrease of the studied radical reaction rate. This EPR approach is a significant alternative to the spectrophotometric one. Moreover, it offers the advantage to detect both the radicals and/or intermediates involved in the reaction.

Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli.

Antimicrobial peptides are produced by all organisms in response to microbial invasion and are considered as promising candidates for future antibiotics. There is a wealth of evidence that many of them interact and increase the permeability of bacterial membranes as part of their killing mechanism. However, it is not clear whether this is the lethal step. To address this issue, we studied the interaction of the antimicrobial peptide temporin L with Escherichia coli by using fluorescence, confocal and electron microscopy. The peptide previously isolated from skin secretions of the frog Rana temporaria has the sequence FVQWFSKFLGRIL-NH2. With regard to fluorescence microscopy, we applied, for the first time, a triple-staining method based on the fluorochromes 5-cyano-2,3-ditolyl tetrazolium chloride, 4',6-diamidino-2-phenylindole and FITC. This technique enabled us to identify, in the same sample, both living and total cells, as well as bacteria with altered membrane permeability. These results reveal that temporin L increases the permeability of the bacterial inner membrane in a dose-dependent manner without destroying the cell's integrity. At low peptide concentrations, the inner membrane becomes permeable to small molecules but does not allow the killing of bacteria. However, at high peptide concentrations, larger molecules, but not DNA, leak out, which results in cell death. Very interestingly, in contrast with many antimicrobial peptides, temporin L does not lyse E. coli cells but rather forms ghost-like bacteria, as observed by scanning and transmission electron microscopy. Besides shedding light on the mode of action of temporin L and possibly that of other antimicrobial peptides, the present study demonstrates the advantage of using the triple-fluorescence approach combined with microscopical techniques to explore the mechanism of membrane-active peptides in general.

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.

Interactions of the antimicrobial peptides temporins with model biomembranes. Comparison of temporins B and L.

Temporins are short (10-13 amino acids) and linear antimicrobial peptides first isolated from the skin of the European red frog, Rana temporaria, and are effective against Gram-positive bacteria and Candida albicans. To get insight into their mechanism(s) of action, we compared the effects on model membranes exerted by two members of this family, viz., temporin B (LLPIVGNLLKSLL-NH(2)) and temporin L (FVQWFSKFLGRIL-NH(2)). More specifically, we measured their insertion into lipid monolayers as well as their effects on the structural dynamics of liposomal bilayers as revealed by diphenylhexatriene (DPH)- and pyrene-labeled phospholipids. We also observed the impact of these peptides on the topology of giant vesicles. Both temporins readily penetrate into lipid monolayers, their intercalation being enhanced in the presence of the common bacterial negatively charged phospholipid phosphatidylglycerol. Instead, the eukaryotic lipid cholesterol did to some extent counteract their penetration into the lipid films. Both temporin B and temporin L caused an enrichment of phospholipids in the bilayers, and in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), these peptides increased acyl chain order. Temporin B had practically no effect on giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), whereas rapid vesiculation was observed when POPG was present. In contrast, temporin L induced vesiculation of both SOPC and SOPC/POPG giant vesicles while the presence of cholesterol in SOPC giant vesicles attenuated this effect.