Dermaseptin-S1 decreases Candida albicans growth, biofilm formation and the expression of hyphal wall protein 1 and aspartic protease genes.

AIMS: This study aimed to investigate the effect of synthetic antimicrobial peptide dermaseptin-S1 (DS1) (ALWKTMLKKLGTMALHAGKAALGAADTISQGTQ) on the growth of Candida albicans, its transition from blastospore to hyphae, and its biofilm formation. We also analysed the expression of different genes (HWP1 and SAPs) involved in C. albicans virulence. METHODS AND RESULTS: Using cell count we showed that in addition to decreasing C. albicans growth, peptide DS1 inhibited its transition from blastospore to hyphal form. These effects are comparable to those obtained with amphotericin B (AmB). Electron microscopy analyses showed that C. albicans cells treated with either DS1 or AmB displayed a distorted cell wall surface, suggesting that the effect of DS1 was similar to that of AmB on C. albicans cell membrane structure. These observations were confirmed by our results with biofilms showing that both DS1 peptide and AmB significantly inhibited biofilm formation after 2 and 4 days. The effect of DS1 on C. albicans growth, transition and biofilm formation may occur through gene modulation, as the expression of HWP1, SAP1, SAP2, SAP3, SAP9 and SAP10 genes involved in C. albicans pathogenesis were all downregulated when C. albicans was treated with DS1. CONCLUSIONS: DS1 inhibits the growth and hyphal transition of C. albicans. DS1 was also able to decrease the expression of and gene expression of hyphal wall protein 1 and aspartic proteases genes by C. albicans. SIGNIFICANCE AND IMPACT OF THE STUDY: These data provide new insight into the efficacy of DS1 against C. albicans and its potential for use as an antifungal therapy.

Human immunodeficiency virus type 1 gp120 and ethanol coexposure in rat organotypic brain slice cultures: Curtailment of gp120-induced neurotoxicity and neurotoxic mediators by moderate but not high ethanol concentrations.

Human immunodeficiency virus type 1 (HIV-1) envelope protein gp120, implicated with other retroviral proteins in acquired immunodeficiency syndrome (AIDS)-related dementia, causes neuronal degeneration by inciting cascades of neurotoxic mediators from glia. It also may facilitate neuronal glutamate (N-methyl-D-aspartate, NMDA) receptor-mediated excitotoxicity by interacting at the glycine coagonist site. The authors reported that preconditioning rat organotypic hippocampal-cortical slice cultures subchronically with ethanol at concentrations occurring during moderate drinking (20 to 30 mM) prevented gp120's induction of neurotoxic mediators and intracellular calcium, as well as neuronal death. The authors now find that the acute copresence of ethanol in moderate as opposed to high concentrations similarly blocks the retroviral protein's neurotoxic effects in brain slice cultures, assessed with lactate dehydrogenase (LDH) release and propidium iodide (PI) labeling. As with ethanol preconditioning, neuroprotection against gp120 by moderate ethanol coexposure appears secondary to abrogation of the retroviral protein's early induction of arachidonic acid (AA), glutamate, and superoxide (but not nitric oxide) elevations/release. Additionally, experiments indicate that 30 mM ethanol is sufficient to inhibit the NMDA receptor, particularly in the presence of added glycine, thus hindering potential direct neuronal stimulation by gp120. However, in contrast to moderate ethanol, 100 mM ethanol, a concentration tolerated only in chronic alcoholics, potentiates gp120-dependent neurotoxicity (PI labeling) in the hippocampal CA1 region, augments LDH release, and fails to curtail gp120's actions on AA, glutamate, and superoxide-but does suppress nitric oxide induction. The results indicate dominant roles for AA, superoxide, and glutamate-mediated oxidative stress in gp120's neurotoxic mechanism, but perhaps a less important role for NMDA receptor stimulation, which would be constrained at both ethanol concentrations employed. We suggest that ethanol's concentration-dependent, two-edged sword behavior could alter the development of dementia in HIV-1-infected individuals during social consumption or abuse. Further studies are needed to elucidate the differing apparently glial effects of the two concentrations of ethanol.

Ethanol pre-exposure suppresses HIV-1 glycoprotein 120-induced neuronal degeneration by abrogating endogenous glutamate/Ca2+-mediated neurotoxicity.

The neurotoxic mechanism of HIV-1 envelope glycoprotein 120 (gp120) involves glutamatergic (NMDA) receptor/Ca2+-dependent excitotoxicity, mediated in part via glia. Pro-inflammatory cytokines also may have roles. We have reported that pre-exposure of brain cultures to 'physiological' ethanol concentrations (20-30 mM) protects against neuronal damage from HIV-1 gp120, but not from the direct receptor agonist, NMDA. Using lactate dehydrogenase assays and propidium iodide staining of rat organotypic hippocampal-entorhinal cortical slice cultures we determined that ethanol's suppression of gp120 neurotoxicity required at least 4 days of pretreatment. The gp120-induced neurotoxicity was accompanied by interleukin-6 elevations that were not affected by the pretreatment. However, gp120 induced substantial, early increases in extracellular glutamate levels that were blocked by ethanol pretreatment, conceivably abrogating excitotoxicity. Consistent with abrogation of excitotoxic pathways, fura-2 imaging showed selective deficits in gp120-dependent intracellular Ca2+ responses in ethanol-pretreated slices. Gp120 is believed to increase glutamate levels by both stimulating release and inhibiting (re)uptake. Results with a labeled glutamate analog, D-[3H]aspartate, revealed that gp120's inhibition of glutamate uptake, rather than its stimulation of release, was abolished after ethanol. Further studies indicated that two converging effects of ethanol pretreatment may underlie the abolishment of gp120-mediated glutamate uptake inhibition: (a) blockade of gp120-induced release (ostensibly from glia) of arachidonic acid, an inhibitor of astroglial glutamate reuptake, and (b) modest proliferation and activation of astroglia upon gp120 stimulation--which are likely to augment glutamate transporters. Thus, as with gp120 itself, glia and glutamate/arachidonic acid regulation appear to be important targets for ethanol. Since moderate ethanol consumption is as common among HIV-infected individuals as in the general population, this newly recognized neuroprotective (and apparently anti-excitotoxic) effect of ethanol withdrawal in vitro could be important, but it requires further study before its significance, if any, is understood.

Selective toxicity of ochratoxin A in primary cultures from different brain regions.

Ochratoxin A (OTA) is a mycotoxin produced by moulds from the Aspergillus and Penicillium genera. It is a natural contaminant of a wide variety of both human and animal foodstuffs. Via dietary intake, OTA passes into the blood of both humans and animals and accumulates in several organs, such as the kidney and the brain with selective toxicity in the ventral mesencephalon and in the cerebellum. In order to confirm the regional selectivity to OTA cytotoxicity in rat brain, investigations were designed to study the mechanism of the cytotoxicity of OTA in primary cultures of the above-mentioned structures (ventral mesencephalon and cerebellum), and to compare their sensitivity to the toxin. Protein and DNA synthases, lactate dehydrogenase (LDH) release and production of malondialdehyde (MDA) were assayed in astrocytes and neurones of the selected structures in the presence of OTA. After 48 h incubation, OTA (10-150 microM) induced an inhibition of protein and DNA syntheses in a concentration-dependent manner with a selective higher toxicity in the cells of the ventral mesencephalon [50% inhibitory concentrations (IC50) of protein and DNA syntheses were 14 +/- 2 microM for neurones and 40 +/- 5 microM for astrocytes] compared to the cerebellum values (24 +/- 7 microM for neurones and 69 +/- 9 microM for astrocytes). In parallel, a significant increase in levels of MDA and LDH release were noted. Altogether these results indicate that OTA is also a neurotoxic substance in addition to its well-documented nephrotoxicity and that the effects are likely to be restricted within particular structures of the brain.

Regional selectivity to ochratoxin A, distribution and cytotoxicity in rat brain.

Ochratoxin A (OTA) a chlorodihydro-isocoumarin linked through an amide bond to phenylalanine, is a mycotoxin found as a contaminant in foodstuffs and shown to be nephrotoxic, teratogenic, immunosuppressive, genotoxic, mutagenic and carcinogenic in rodents. Ochratoxin A is known to induce teratogenic effects in neonates (rats and mice) exposed in utero, characterised by microcephaly and modification of the brain levels of free amino acids. Since OTA has been found to accumulate in the brain according to the duration of exposure to doses in the range of natural contamination of feedstuffs, experiments were designed to determine more precisely the structural target of OTA in the brain. After intracerebral injection, OTA (403 ng/10 microl) was not found in the following parts of the brain: the frontal cortex (FC), striatum (ST), ventral mesencephalon (VM) and the cerebellum (CB) in contrast to the rest of the brain, probably due to the detection limit of 0.1 ng/g of tissue. However lactate dehydrogenase (LDH) was increased in extracellular space in the VM to a greater extent than in the rest of the brain, indicating that this structure could be one of the targets of OTA in the brain. Contents of free amino acids were morever similarly modified in the VM and in the rest of the brain. Male rats were given OTA (289 microg/kg per 24 h) by gastric intubation for 8 days and the main brain structures analysed for OTA content and cytotoxicity. OTA was found in the following structures in decreasing order: rest of the brain (50.3%), cerebellum (34.4%), VM (5.1%), striatum (3.3%) and hippocampus (2.9%) of the total OTA amount found in the brain, which represents 0.022% to 0.028% of the given dose. Interestingly cytotoxicity as measured by lactate dehydrogenase (LDH) release in the extracellular space was much more pronounced in the VM, hippocampus, and striatum than in the cerebellum, whereas no cytotoxicity was observed in the rest of the brain. Similarly deoxyribonuclease (DNase) activity in relation to possible necrotic cells was increased in the VM and cerebellum. Altogether these results designated the ventral mesencephalon, hippocampus, striatum and cerebellum as the main OTA-targets in the brain of adult rats and excluded the rest of the brain.

Subchronic effects of ochratoxin A on young adult rat brain and partial prevention by aspartame, a sweetener.

1. Ochratoxin A (OTA) is a mycotoxin produced by several fungi, especially Aspergillus and Penicillium species. Many food and foodstuffs can be contaminated by ochratoxin A, which is consequently found in blood of animals and humans. 2. The distribution into the brain of young adult rats fed OTA for 1 to 6 weeks and some consequences have been investigated in the present study. 3. Our results on rats given OTA (289 microg/kg/48 h) indicated that OTA accumulated in the whole brain as function of time according to a regression curve, Y=-8.723 a+16.72 with a correlation coefficient of r=0.989, where Y-axis is the OTA concentration in ng/g of brain and X-axis is the duration of the treatment in weeks. The brain OTA contents was 11.95 +/- 2.2, 23.89 +/- 4.4, 39.9 +/- 4.5, 50.3 +/- 7.3, 78.8 +/- 6.3, 94 +/- 16 ng/g of brain in the mycotoxin-treated animals for respectively 1, 2, 3, 4, 5 and 6-weeks treatment. OTA induced modifications of free amino-acid concentrations in the brain, mainly, Tyrosine (Tyr) and phenylalanine (Phe). Tyr decreased significantly as compared to control (p < 0.05). Phe increased significantly as compared to control (p < 0.05). 4. Aspartame, (25 mg/kg/48 h) a structural analogue of OTA largely modified the distribution and prevented the accumulation of OTA in the brain since the respective brain OTA contents decreased respectively to 9.6 +/- 7.9, 19.2 +/- 3.0, 26.8 +/- 4.2, 19.7 +/- 1.9, 13.7 /- 5.6 and 11.0 +/- 6.0 ng/g of tissue, for the same duration of treatment. It also prevented the modifications of Tyr and Phe levels. 5. The histological investigations showed several necrotic cells with pyknotic nucleus, detected in OTA treated animals with higher frequency as compared to the controls and Aspartame treated ones. Aspartame appeared to significantly prevent this nuclear effect as well, the meaning of which is discussed.

Potassium channel modulation by the pseudopeptide ochratoxin A in rat nerve fibers.

Ochratoxin A (OTA), a naturally occurring mycotoxin produced by Aspergillus and Penicillium genera, blocks anion conductance in Madin-Darby canine kidney (MDCK) cells and reduces the potassium concentration gradient in the same cells. So far, a direct effect of OTA on cation channels has not been reported. Experiments were then designed to explore the OTA action on ion channels of rat nerve fibres. Voltage clamp technique has shown that OTA (0.1-1 microM) does not significantly affect sodium current in the intact myelinated axon. After paranodal demyelination (using 0.2% pronase) large K+ outward and K+ tail currents are elicited upon depolarization and repolarization. OTA application on pronase treated fibres produces the following effects: 1) reduction in the amplitude of the tail current with a negligible effect on the time constant of its fast decaying component (modified by pronase application); 2) reduction in potassium conductance to 30% of the control value (i.e., the value in pronase-treated fibres); and 3) reduction in the leakage conductance. Our findings point at the paranodal region of myelinated nerve fibres as a target for the neurotoxic pseudopeptide OTA found as contaminant in food. The uncleaved OTA is effectively acting since pronase fails to cleave it into phenylalanine and OT alpha.

High performance liquid chromatography with ultraviolet detection used for laboratory routine determination of fluvoxamine in human plasma.

Fluvoxamine is an antidepressant drug introduced into the clinic in 1986. It acts by selectively inhibiting neuronal serotonin recapture. It can be quantified by several methods, including high performance liquid chromatography. The HPLC method used so far needs special equipment and has poor sensitivity. The technique is difficult and time consuming. An easier, quicker and more sensitive HPLC assay for the routine determination of fluvoxamine in human plasma has therefore been developed. After alkalinisation and direct extraction by a mixture of n-hexane-isoamylic alcohol 985: 15 (v/v) of plasma samples, the organic phases were further extracted by HCl 0.1 N. Thirty microL of the final extract (with loxapine as internal standard) were injected directly into a C-8 column with a mobile phase consisting of 370 mL acetonitrile, 0.4 mL diethylamine, 630 mL of distilled water, 25 mL pic B5. UV detection at 254 nm was used. The whole process was completed in 40 min. The detection limit was 10 ng mL-1. No interference was found either with several benzodiazepines or with antidepressant drugs commonly associated during treatments.