Antibacterial and anti-biofilm activity, and mechanism of action of pleurocidin against drug resistant Staphylococcus aureus.

The abuse of antibiotics has resulted in the emergence of multi-drug-resistant bacteria. Staphylococcus aureus is a frequent cause of infections, and antibiotic-resistant S. aureus has become a serious problem. Antimicrobial peptides play an important role in innate immunity and are attracting increasing attention as alternative antibiotics. In a previous study, pleurocidin, derived from winter flounder, was identified as a 25-amino acid antimicrobial peptide with no cytotoxicity toward mammalian cells and low hemolytic activity. In the present study, pleurocidin was observed to exhibit antimicrobial activity against gram-positive and gram-negative bacteria, especially against drug resistant S. aureus. Pleurocidin retained its antibacterial activity against drug resistant S. aureus in the presence of a physiological salt concentration. Membrane depolarization assays and propidium iodide uptake indicated that pleurocidin kills bacteria by damaging the integrity of the bacterial membrane. DNA binding assays revealed that pleurocidin binds to DNA. Thus, pleurocidin targets not only the bacterial membrane, but also their DNA. S. aureus biofilms have become a serious problem because of increased resistance to antibiotics. Therefore, we investigated the effect of pleurocidin on biofilm inhibition and eradication using crystal violet staining and microscopic observation. Pleurocidin inhibited and eradicated biofilms at low concentrations. Taken together, the results suggested that pleurocidin is a promising candidate therapeutic agent to treat drug-resistant bacteria and biofilm-related infections.

Antibacterial and Antibiofilm Activity and Mode of Action of Magainin 2 against Drug-Resistant Acinetobacter baumannii.

Antimicrobial peptides (AMPs) are promising therapeutic agents for treating antibiotic-resistant bacterial infections. Previous studies showed that magainin 2 (isolated from African clawed fogs Xenopus laevis) has antimicrobial activity against gram-positive and gram-negative bacteria. The present study was conducted to investigate the antibacterial activity of magainin 2 against Acinetobacter baumannii. Magainin 2 showed excellent antibacterial activity against A. baumannii strains and high stability at physiological salt concentrations. This peptide was not cytotoxic towards HaCaT cells and showed no hemolytic activity. Biofilm inhibition and elimination were significantly induced in all A. baumannii strains exposed to magainin 2. We confirmed the mechanism of magainin 2 on the bacterial outer and inner membranes. Collectively, these results suggest that magainin 2 is an effective antimicrobial and antibiofilm agent against A. baumannii strains.

Effect of high free fatty acids on the anti-contractile response of perivascular adipose tissue in rat aorta.

To determine whether high free fatty acids (FFA) could affect the anti-contractile properties of perivascular adipose tissue (PVAT) in rat aortas. Wistar rats were divided into normal, obesity and fenofibrate groups and fed a normal, high-fat, and high-fat plus fenofibrate diet, respectively. Thoracic aortas with or without PVAT (PVAT+ and PVAT-) were prepared with either intact endothelium (E+) or with endothelium removed (E-). Aortas pre-treated with either 500µmol/L of palmitic acid (PA) or physiological salt solution (PSS), as a control, were used for in vitro study. Concentration-dependent responses of aortas to norepinephrine were measured. The anti-contractile effects of PVAT were attenuated in both obese rats with high FFA levels and in the PA group in the presence of endothelium, but not in the absence of endothelium. The attenuation of the anti-contractile effect was restored by reducing FFA levels in the fenofibrate group (P<0.05). Incubation of aortas (PVAT+ E+) with nitric oxide (NO) synthase inhibitor and tumor necrosis factor-alpha (TNF-α) in the normal group caused attenuation of the anti-contractile effect of PVAT (P<0.05). Incubation of aortas (PVAT+ E+) in the obese and PA groups with a NO donor, anti-TNF-α antibodies or free radical scavengers partially restored the anti-contractile effect of PVAT (P<0.05). Under both acute and chronic conditions, high FFA levels could attenuate the anti-contractile properties of PVAT by an endothelium-dependent rather than an endothelium-independent mechanism, in which inflammation and oxidative stress may play important roles.

Reduced angiotensin II levels cause generalized vascular dysfunction via oxidant stress in hamster cheek pouch arterioles.

OBJECTIVES: We investigated the effect of suppressing plasma angiotensin II (ANG II) levels on arteriolar relaxation in the hamster cheek pouch. METHODS: Arteriolar diameters were measured via television microscopy during short-term (3-6days) high salt (HS; 4% NaCl) diet and angiotensin converting enzyme (ACE) inhibition with captopril (100mg/kg/day). RESULTS: ACE inhibition and/or HS diet eliminated endothelium-dependent arteriolar dilation to acetylcholine, endothelium-independent dilation to the NO donor sodium nitroprusside, the prostacyclin analogs carbacyclin and iloprost, and the KATP channel opener cromakalim; and eliminated arteriolar constriction during KATP channel blockade with glibenclamide. Scavenging of superoxide radicals and low dose ANG II infusion (25ng/kg/min, subcutaneous) reduced oxidant stress and restored arteriolar dilation in arterioles of HS-fed hamsters. Vasoconstriction to topically-applied ANG II was unaffected by HS diet while arteriolar responses to elevation of superfusion solution PO2 were unaffected (5% O2, 10% O2) or reduced (21% O2) by HS diet. CONCLUSIONS: These findings indicate that sustained exposure to low levels of circulating ANG II leads to widespread dysfunction in endothelium-dependent and independent vascular relaxation mechanisms in cheek pouch arterioles by increasing vascular oxidant stress, but does not potentiate O2- or ANG II-induced constriction of arterioles in the distal microcirculation of normotensive hamsters.

Liposomal-delivery of phosphodiesterase 5 inhibitors augments UT-15C-stimulated ATP release from human erythrocytes.

The use of liposomes to affect targeted delivery of pharmaceutical agents to specific sites may result in the reduction of side effects and an increase in drug efficacy. Since liposomes are delivered intravascularly, erythrocytes, which constitute almost half of the volume of blood, are ideal targets for liposomal drug delivery. In vivo, erythrocytes serve not only in the role of oxygen transport but also as participants in the regulation of vascular diameter through the regulated release of the potent vasodilator, adenosine triphosphate (ATP). Unfortunately, erythrocytes of humans with pulmonary arterial hypertension (PAH) do not release ATP in response to the physiological stimulus of exposure to increases in mechanical deformation as would occur when these cells traverse the pulmonary circulation. This defect in erythrocyte physiology has been suggested to contribute to pulmonary hypertension in these individuals. In contrast to deformation, both healthy human and PAH erythrocytes do release ATP in response to incubation with prostacyclin analogs via a well-characterized signaling pathway. Importantly, inhibitors of phosphodiesterase 5 (PDE5) have been shown to significantly increase prostacyclin analog-induced ATP release from human erythrocytes. Here we investigate the hypothesis that targeted delivery of PDE5 inhibitors to human erythrocytes, using a liposomal delivery system, potentiates prostacyclin analog- induced ATP release. The findings are consistent with the hypothesis that directed delivery of this class of drugs to erythrocytes could be a new and important method to augment prostacyclin analog-induced ATP release from these cells. Such an approach could significantly limit side effects of both classes of drugs without compromising their therapeutic effectiveness in diseases such as PAH.

Erythropoietin has a restorative effect on the contractility of arteries following experimental hypoxia.

INTRODUCTION: The aim of this study was to investigate the effect of erythropoietin on vascular contractility using an in vitro model of hypoxia replicating the hypoxic environment of blood vessels and surrounding adipose tissue in obesity. METHODS AND RESULTS: Pharmacological in vitro studies were carried out on small mesenteric arterial segments from male Wistar rats with and without perivascular fat and endothelium. Contractile responses were investigated by wire myography under normoxia, experimental hypoxia ± erythropoietin and l-NNA. Perivascular fat exerted an anticontractile effect which was lost following the induction of experimental hypoxia. Erythropoietin prevented the loss of the anticontractile capacity when vessels were incubated for one hour before the induction of hypoxia or throughout the period of hypoxia; this was found to be independent of the function of perivascular fat, as fat denuded arteries had a similar reduction in contractility (artery no fat + hypoxia vs. artery no fat + hypoxia + erythropoietin). The mechanism by which erythropoietin was exerting its effect was found to be partially endothelium dependent and associated with an increase of nitric oxide bioavailability as nitric oxide synthase inhibition prevented the effect. CONCLUSIONS: Whilst erythropoietin is working downstream from perivascular fat, it is possible that it may be therapeutically useful in obesity when hypoxia and inflammation reduce the normal activity of perivascular fat.

Aging decreases the contribution of MaxiK channel in regulating vascular tone in mesenteric artery by unparallel downregulation of α- and ß1-subunit expression.

Vascular disease increases in incidence with age and is the commonest cause of morbidity and mortality among elderly people. Large-conductance Ca(2+)-activated K(+)(MaxiK) channel, with pore-forming α-subunit and modulatory ß1-subunit, is a key regulator of vascular tone. This study explored functional and molecular evidence of MaxiK alteration with aging in the mesenteric artery(MA). Young, Middle-aged, and Old male Wistar rats were used. Selective MaxiK channel blocker (Iberiotoxin) induced a significant increase of vascular tension in MA in all three age groups. However, these effects were greatly decreased in Old animals. The amplitude and frequency of spontaneous transient outward currents were significantly decreased with aging. Single channel recording revealed that aging induced a decrease of the open probability and the mean open time, but an increase of the mean closed time of MaxiK channel. The Ca(2+)/voltage sensitivity of MaxiK was also decreased. Western blotting showed that the protein expression of MaxiK ß1- and α-subunit was significantly reduced with aging, and the suppression of ß1 subunits was larger than that of α subunits. These data suggest that aging decreases capability of MaxiK channel in regulating vascular tone in the MA, which may be partially mediated by unparallel downregulation of α- and ß1-subunit expression.

Activation of D2-like dopamine receptors inhibits GABA and glycinergic neurotransmission to pre-motor cardiac vagal neurons in the nucleus ambiguus.

The parasympathetic control of heart rate arises from premotor cardiac vagal neurons (CVNs) located in the nucleus ambiguus (NA). Previous microinjection studies in NA show that dopamine evokes a decrease in heart rate, but the underlying mechanisms responsible for these responses were not identified. This study tested whether dopamine modulates inhibitory GABAergic and glycinergic and/or excitatory glutamatergic neurotransmission to CVNs. Retrogradely labeled CVNs were identified in an in vitro rat brainstem slice preparation and synaptic events were recorded using whole cell voltage clamp techniques. Bath application of dopamine (100 µM) had no effect on excitatory synaptic events, but reversibly inhibited the frequency (but not amplitude) of GABAergic inhibitory postsynaptic currents (IPSCs) in CVNs. Similarly, dopamine (10 µM and 100 µM) inhibited glycinergic IPSC frequency by ~50% and 70% respectively. The reduction in inhibitory neurotransmission to CVNs by dopamine was prevented by the sodium channel blocker TTX (1µM) indicating that the dopamine mediated effects were action potential dependent. Dopamine evoked responses were mimicked by the D2-like receptor agonist, Quinpirole but not D1-like receptor agonist, SKF 38393. In addition, the dopamine mediated depression of inhibitory synaptic responses were prevented by the D2-like receptor antagonist sulpiride, but not by D1-like or adrenergic or serotonergic receptor antagonists, suggesting that these responses were D2-like receptor mediated and not D1-like or adrenergic or 5-HT receptor mediated. These data suggest that dopamine acts via dis-inhibition, and diminishes inhibitory GABAergic and glycinergic neurotransmission to CVNs, which would be predicted to increase parasympathetic activity to the heart and evoke a bradycardia.