Carlos Minahk

Enhancement of the Enterocin CRL35 Activity by a Synthetic Peptide Derived from the NH2-Terminal Sequence

ABSTRACT The enterocin CRL35 biosynthetic gene cluster was cloned and sequenced. The sequence was revealed to be highly identical to that of the mundticin KS gene cluster (S. Kawamoto, J. Shima, R. Sato, T. Eguchi, S. Ohmomo, J. Shibato, N. Horikoshi, K. Takeshita, and T. Sameshima, Appl. Environ. Microbiol. 68:3830-3840, 2002). Short synthetic peptides were designed based on the bacteriocin sequence and were evaluated in antimicrobial competitive assays. The peptide KYYGNGVSCNKKGCS produced an enhancement of enterocin CRL35 antimicrobial activity in a buffer system.

The role of oxidative stress in alterations of hematological parameters and inflammatory markers induced by early hypercholesterolemia.

AIMS The investigation of the effects of a high cholesterol diet (HD) for a short-time period on hematological parameters and the potential role of oxidative stress and inflammation markers. MAIN METHODS Rabbits were fed either a control diet or a diet containing 1% cholesterol (HD) for 5-6 weeks. The plasma lipid levels, C reactive protein (CRP), total red blood cells (RBC), total white blood cells (WBC), platelet count, packed cell volume (PCV) and leukocyte formula were determined. Oxidative stress was evaluated by the thiobarbituric acid reactive substances (TBARS), total glutathione and GSH serum level measurements. The osmotic fragility and the membrane fluidity of erythrocytes were determined. The levels of total cholesterol and TBARS were also measured in the erythrocyte membrane suspension. KEY FINDINGS A decrease in the RBC and PCV was observed in rabbits fed on HD. The membrane rigidity and osmotic fragility were increased, and the morphological changes caused by the HD and TBARS levels in the erythrocyte membrane may account for this phenomenon. The inflammatory markers as the CRP levels, the platelet count, the WBC and the neutrophils were increased. The TBARS and GSH levels in the serum were increased and decreased, respectively. SIGNIFICANCE This study shows that feeding rabbits an HD for a short time induces hematological alterations, disturbances in the oxidant-antioxidant balance and an increase of inflammatory markers. These findings support the importance of the early correction or prevention of high cholesterol levels to disrupt the process leading to the development of cardiovascular diseases.

Microcin J25 induces the opening of the mitochondrial transition pore and cytochrome c release through superoxide generation

Microcin J25, an antimicrobial lasso‐structure peptide, induces the opening of mitochondrial permeability transition pores and the subsequent loss of cytochrome c. The microcin J25 effect is mediated by the stimulation of superoxide anion overproduction. An increased uptake of calcium is also involved in this process. Additional studies with superoxide dismutase, ascorbic acid and different specific inhibitors, such as ruthenium red, cyclosporin A and Mn2+, allowed us to establish a time sequence of events starting with the binding of microcin J25, followed by superoxide anion overproduction, opening of mitochondrial permeability transition pores, mitochondrial swelling and the concomitant leakage of cytochrome c.

Microcin J25 triggers cytochrome c release through irreversible damage of mitochondrial proteins and lipids

We previously showed that the antimicrobial peptide microcin J25 induced the over-production of reactive oxygen species with the concomitant release of cytochrome c from rat heart mitochondria via the opening of the mitochondrial permeability transition pore. Here, we were able to demonstrate that indeed, as a consequence of the oxidative burst, MccJ25 induces carbonylation of mitochondrial proteins, which may explain the irreversible inhibition of complex III and the partial inhibition of superoxide dismutase and catalase. Moreover, the peptide raised the levels of oxidized membrane lipids, which triggers the release of cytochrome c. From in silico analysis, we hypothesize that microcin would elicit these effects through interaction with heme c1 at mitochondrial complex III. On the other hand, under an excess of l-arginine, MccJ25 caused nitric oxide overproduction with no oxidative damage and a marked inhibition in oxygen consumption. Therefore, a beneficial anti-oxidative activity could be favored by the addition of l-arginine. Conversely, MccJ25 pro-oxidative-apoptotic effect can be unleashed in either an arginine-free medium or by suppressing the nitric oxide synthase activity.

Resveratrol modulates ATPase activity of liposome-reconstituted ABCG1

ABCG1 is a half‐sized transporter with an unquestionable importance in cholesterol homeostasis. So far, its expression and thus its activity was suggested to be regulated at transcriptional level by LXR and PPAR agonists including polyphenols. However, it is unknown whether there are other mechanisms of up‐regulation of ABCG1 activity. In the present work resveratrol was shown to induce a nearly twofold increase in ATPase activity of reconstituted ABCG1. Evidence is presented for the first time suggesting that resveratrol is able to activate ABCG1 activity by an alternative mechanism that involves an indirect interaction.

Cholesterol induces surface localization of polyphenols in model membranes thus enhancing vesicle stability against lysozyme, but reduces protection of distant double bonds from reactive-oxygen species

The main scope of the present study was to analyze the membrane interaction of members of different classes of polyphenols, i.e. resveratrol, naringenin, epigallocatechin gallate and enterodiol, in model systems of different compositions and phase states. In addition, the possible association between membrane affinity and membrane protection against both lipid oxidation and bilayer-disruptive compounds was studied. Gibbs monolayer experiments indicated that even though polyphenols showed poor surface activity, it readily interacted with lipid films. Actually, a preferential interaction with expanded monolayers was observed, while condensed and cholesterol-containing monolayers decreased the affinity of these phenolic compounds. On the other hand, fluorescence anisotropy studies showed that polyphenols were able to modulate membrane order degree, but again this effect was dependent on the cholesterol concentration and membrane phase state. In fact, cholesterol induced a surface rather than deep into the hydrophobic core localization of phenolic compounds in the membranes. In general, the polyphenolic molecules tested had a better antioxidant activity when they were allowed to get inserted into the bilayers, i.e. in cholesterol-free membranes. On the other hand, a membrane-protective effect against bilayer permeabilizing activity of lysozyme, particularly in the presence of cholesterol, could be assessed. It can be hypothesized that phenolic compounds may protect membrane integrity by loosely covering the surface of lipid vesicles, once cholesterol push them off from the membrane hydrophobic core. However, this cholesterol-driven distribution may lead to a reduced antioxidant activity of linoleic acid double bonds.

The Key Role of Membranes in Amyloid Formation from a Biophysical Perspective

Even though our knowledge of how proteins misfold and aggregate is deeper nowadays, the mechanisms driving this process are still poorly understood. Among the factors involved, membranes should be taken into account. Indeed, convincing evidence suggests that membranes may influence protein folding, misfolding and aggregation. In fact, membrane lipid composition of different cellular types may attenuate or intensify the environmental pressure over protein folding equilibrium. In the present review the aim is to make an up-to-date analysis of the membrane influence on protein aggregation from a biophysical point of view in order to provide useful tools for researchers from other fields. In particular, we discuss how membranes can alter protein environment, e.g. increasing local protein concentration, lowering pH and dielectric constant, allowing accessibility to the hydrophobic milieu and promoting surface crowding, all of which will lead to protein aggregation. In addition, we review the role that specific lipids may exert on protein aggregation and finally we analyse the possible implication of membrane-related oxidative stress on amyloidogenesis.

Differential inhibition of human erythrocyte acetylcholinesterase by polyphenols epigallocatechin‐3‐gallate and resveratrol. Relevance of the membrane‐bound form

The activity of acetylcholinesterase (AChE) from human erythrocytes was tested in the presence of the phenolic compounds resveratrol and epigallocatechin-3-gallate (EGCG). Even though the stilbene barely changed this enzymatic activity, EGCG did inhibit AChE. Importantly, it preferentially acted on the membrane-bound enzyme rather than on its soluble form. Actually, it was shown that this flavonoid may bind to the red blood cell membrane surface, which may improve the interaction between EGCG and AChE. Therefore, caution should be taken when screening AChE inhibitors. In fact, testing compounds with the soluble form of the enzyme may underestimate the activity of some of these potential inhibitors, hence it would be advisable not to use them as a sole model system for screening. Moreover, erythrocyte AChE is proposed as a good model for these enzymatic assays.

Microcin J25-Ga induces apoptosis in mammalian cells by inhibiting mitochondrial RNA-polymerase.

MccJ25, an antimicrobial peptide, was unable to cause apoptosis of COS-7 cells in spite of inducing reactive-oxygen species overproduction as well as cytochrome c release from isolated mitochondria. Surprisingly, MccJ25-Ga, an amidated variant of MccJ25 that displays similar anti-mitochondrial effects, did induce apoptosis in COS-7. The only difference found between the activities of these peptides was the unpredicted inhibition of mitochondrial RNA synthesis by MccJ25-Ga. These results led us to hypothesize that both mitochondrial RNA polymerase and mitochondrial membrane might be the molecular targets of MccJ25-Ga in mitochondria and this combined effect may lead to apoptosis.

Impairment of the class IIa bacteriocin receptor function and membrane structural changes are associated to enterocin CRL35 high resistance in Listeria monocytogenes

BACKGROUND Enterocin CRL35 is a class IIa bacteriocin with anti-Listeria activity. Resistance to these peptides has been associated with either the downregulation of the receptor expression or changes in the membrane and cell walls. The scope of the present work was to characterize enterocin CRL35 resistant Listeria strains with MICs more than 10,000 times higher than the MIC of the WT sensitive strain. METHODS Listeria monocytogenes INS7 resistant isolates R2 and R3 were characterized by 16S RNA gene sequencing and rep-PCR. Bacterial growth kinetic was studied in different culture media. Plasma membranes of sensitive and resistant bacteria were characterized by FTIR and Langmuir monolayer techniques. RESULTS The growth kinetic of the resistant isolates was slower as compared to the parental strain in TSB medium. Moreover, the resistant isolates barely grew in a glucose-based synthetic medium, suggesting that these cells had a major alteration in glucose transport. Resistant bacteria also had alterations in their cell wall and, most importantly, membrane lipids. In fact, even though enterocin CRL35 was able to bind to the membrane-water interface of both resistant and parental sensitive strains, this peptide was only able to get inserted into the latter membranes. CONCLUSIONS These results indicate that bacteriocin receptor is altered in combination with membrane structural modifications in enterocin CRL35-resistant L. monocytogenes strains. GENERAL SIGNIFICANCE Highly enterocin CRL35-resistant isolates derived from Listeria monocytogenes INS7 have not only an impaired glucose transport but also display structural changes in the hydrophobic core of their plasma membranes.