Ectoines as novel anti-inflammatory and tissue protective lead compounds with special focus on inflammatory bowel disease and lung inflammation.

The compatible solute ectoine is one of the most abundant and powerful cytoprotectant in the microbial world. Due to its unique ability to stabilize biological membranes and macromolecules it has been successfully commercialized as ingredient of various over-the-counter drugs, achieving primarily epithelial protection. While trying to elucidate the mechanism of its cell protective properties in in-vitro studies, a significant anti-inflammatory effect was documented for the small molecule. The tissue protective potential of ectoine considerably improved organ quality during preservation. In addition, ectoine and derivatives have been demonstrated to significantly decrease inflammatory cytokine production, thereby alleviating the inflammatory response following organ transplantation, and launching new therapeutic options for pathologies such as Inflammatory Bowel Disease (IBD) and Chronic Obstructive Pulmonary Disease (COPD). In this review, we aim to summarize the knowledge of this fairly nascent field of the anti-inflammatory potential of diverse ectoines. We also point out that this promising field faces challenges in its biochemical and molecular substantiations, including defining the molecular mechanisms of the observed effects and their regulation. However, based on their potent cytoprotective, anti-inflammatory, and non-toxic properties we believe that ectoines represent promising candidates for risk free interventions in inflammatory pathologies with steeply increasing demands for new therapeutics.

How the Neurotoxin ß-N-Methylamino-l-Alanine Accumulates in Bivalves: Distribution of the Different Accumulation Fractions among Organs.

The environmental neurotoxin ß-methylamino-l-alanine (BMAA) may represent a risk for human health. BMAA accumulates in freshwater and marine organisms consumed by humans. However, few data are available about the kinetics of BMAA accumulation and detoxification in exposed organisms, as well as the organ distribution and the fractions in which BMAA is present in tissues (free, soluble bound or precipitated bound cellular fractions). Here, we exposed the bivalve mussel Dreissena polymorpha to 7.5 µg of dissolved BMAA/mussel/3 days for 21 days, followed by 21 days of depuration in clear water. At 1, 3, 8, 14 and 21 days of exposure and depuration, the hemolymph and organs (digestive gland, the gills, the mantle, the gonad and muscles/foot) were sampled. Total BMAA as well as free BMAA, soluble bound and precipitated bound BMAA were quantified by tandem mass spectrometry. Free and soluble bound BMAA spread throughout all tissues from the first day of exposure to the last day of depuration, without a specific target organ. However, precipitated bound BMAA was detected only in muscles and foot from the last day of exposure to day 8 of depuration, at a lower concentration compared to free and soluble bound BMAA. In soft tissues (digestive gland, gonad, gills, mantle and muscles/foot), BMAA mostly accumulated as a free molecule and in the soluble bound fraction, with variations occurring between the two fractions among tissues and over time. The results suggest that the assessment of bivalve contamination by BMAA may require the quantification of total BMAA in whole individuals when possible.

ß-Methylamino-L-alanine substitution of serine in SOD1 suggests a direct role in ALS etiology.

Exposure to the environmental toxin ß-methylamino-L-alanine (BMAA) is linked to amyotrophic lateral sclerosis (ALS), but its disease-promoting mechanism remains unknown. We propose that incorporation of BMAA into the ALS-linked protein Cu,Zn superoxide dismutase (SOD1) upon translation promotes protein misfolding and aggregation, which has been linked to ALS onset and progression. Using molecular simulation and predictive energetic computation, we demonstrate that substituting any serine with BMAA in SOD1 results in structural destabilization and aberrant dynamics, promoting neurotoxic SOD1 aggregation. We propose that translational incorporation of BMAA into SOD1 is directly responsible for its toxicity in neurodegeneration, and BMAA modification of SOD1 may serve as a biomarker of ALS.

Transfer of a cyanobacterial neurotoxin, ß-methylamino-l-alanine from soil to crop and its bioaccumulation in Chinese cabbage.

Most cyanobacteria can synthesize the notorious neurotoxin ß-methylamino-l-alanine (BMAA) that is transferred and bioaccumulated through natural food webs of aquatic ecosystems and ultimately arises the potential human health risks by the consumption of BMAA-contaminated aquatic products. Fertilization of cyanobacterial composts in farmlands may also lead to BMAA contamination in soil and its possible transfer and bioaccumulation within major crops, thereby threatening human health. In this study, we first detected a high level of BMAA (1.8-16.3 µg g-1) in cyanobacterial composts. In order to assess the health risks from cyanobacterial composts, we planted Chinese cabbage, a favourite vegetable in China, in BMAA-contaminated soil (4.0 µg BMAA/g soil) and detected the levels of free and protein-associated BMAA in soil and crop organs during the whole growth cycle by HPLC-MS/MS, respectively. Our results demonstrated that BMAA indeed transferred from soil to root, stem and leaf of Chinese cabbage during the growth cycle. The BMAA level finally accumulated in the edible portions was much higher than the initial level in soil, including 13.82 µg g-1 in leaf and 4.71 µg g-1 in stem. The discovery of the neurotoxin BMAA in this vegetable, an ending in human consumption, not only provides a BMAA transfer pathway in farmland ecosystems, but also is alarming and requires attention due to the potential risk of cyanobacterial composts to human health.

Environmental neurotoxin interaction with proteins: Dose-dependent increase of free and protein-associated BMAA (ß-N-methylamino-L-alanine) in neonatal rat brain.

ß-Methylamino-L-alanine (BMAA) is implicated in the aetiology of neurodegenerative disorders. Neonatal exposure to BMAA induces cognitive impairments and progressive neurodegenerative changes including intracellular fibril formation in the hippocampus of adult rats. It is unclear why the neonatal hippocampus is especially vulnerable and the critical cellular perturbations preceding BMAA-induced toxicity remains to be elucidated. The aim of this study was to compare the level of free and protein-associated BMAA in neonatal rat brain and peripheral tissues after different exposures to BMAA. Ultra-high performance liquid chromatography-tandem mass spectrometry analysis revealed that BMAA passed the neonatal blood-brain barrier and was distributed to all studied brain areas. BMAA was also associated to proteins in the brain, especially in the hippocampus. The level in the brain was, however, considerably lower compared to the liver that is not a target organ for BMAA. In contrast to the liver there was a significantly increased level of protein-association of BMAA in the hippocampus and other brain areas following repeated administration suggesting that the degradation of BMAA-associated proteins may be lower in neonatal brain than in the liver. Additional evidence is needed in support of a role for protein misincorporation in the neonatal hippocampus for long-term effects of BMAA.

Assessment of the non-protein amino acid BMAA in Mediterranean mussel Mytilus galloprovincialis after feeding with estuarine cyanobacteria.

To determine whether 2-amino-3-methylaminopropanoic acid (BMAA) could be taken up by marine organisms from seawater or their diet mussels Mytilus galloprovincialis, collected from the North Atlantic Portuguese shore, were exposed to seawater doped with BMAA standard (for up to 48 h) or fed with cyanobacteria (for up to 15 days). Mussels were able to uptake BMAA when exposed to seawater. Mussels fed with cyanobacteria Synechocystis salina showed a rise in BMAA concentration during feeding and a decline in concentration during the subsequent depuration period. Cells from the gills and hepatopancreas of mussels fed with S. salina showed lessened metabolic activity in mussels fed for longer periods of time. A hot acidic digestion (considered to account for total BMAA) was compared with a proteolytic digestion, using pepsin, trypsin and chymotrypsin. The latter was able to extract from mussels approximately 30% of total BMAA. Implications for BMAA trophic transfers in marine ecosystems are discussed.

Pharmacokinetics of the natural antibiotic negamycin.

1. Negamycin exerts its antimicrobial activity by inhibiting bacterial protein synthesis and is efficacious in animal models of infection. In order to optimize negamycin exposure for therapeutic purposes, its pharmacokinetics in pre-clinical species were determined. 2. Negamycin has a dipeptide-like structure with logD7.4 < -1, causing low permeation into Caco-2 cells, low-oral bioavailability in rats of 6% and low-plasma protein binding of 10% in mouse, rat, dog and human plasma. Negamycin degradation rates in microsomes and hepatocytes predicted low-hepatic intrinsic clearance in pre-clinical species, which was confirmed in vivo where clearance varied between 3.4 and 11.5 mL/min/kg and virtually all negamycin was cleared unchanged renally. The similar behavior in multiple animal species allowed for the prediction of systemic clearance and volume of distribution in humans using multiple-scaling methods and physiological-based pharmacokinetic modeling and simulation. 3. Only 0.05-0.25% (mol/mol) of administered negamycin was recovered as 2-(1-methylhydrazinyl)acetic acid, a potential reactive metabolite, from rat and dog urine, respectively. 4. In summary, negamycin is a very polar molecule with low-plasma protein binding and low-oral bioavailability that is slowly and exclusively cleared into the urine. Its physicochemical properties make intravenous or intramuscular administration, or a derivative thereof, for therapeutic purposes most likely.

Detection of cyanotoxins, ß-N-methylamino-L-alanine and microcystins, from a lake surrounded by cases of amyotrophic lateral sclerosis.

A cluster of amyotrophic lateral sclerosis (ALS) has been previously described to border Lake Mascoma in Enfield, NH, with an incidence of ALS approximating 25 times expected. We hypothesize a possible association with cyanobacterial blooms that can produce ß-N-methylamino-L-alanine (BMAA), a neurotoxic amino acid implicated as a possible cause of ALS/PDC in Guam. Muscle, liver, and brain tissue samples from a Lake Mascoma carp, as well as filtered aerosol samples, were analyzed for microcystins (MC), free and protein-bound BMAA, and the BMAA isomers 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl)glycine (AEG). In carp brain, BMAA and DAB concentrations were 0.043 µg/g ± 0.02 SD and 0.01 µg/g ± 0.002 SD respectively. In carp liver and muscle, the BMAA concentrations were 1.28 µg/g and 1.27 µg/g respectively, and DAB was not detected. BMAA was detected in the air filters, as were the isomers DAB and AEG. These results demonstrate that a putative cause for ALS, BMAA, exists in an environment that has a documented cluster of ALS. Although cause and effect have not been demonstrated, our observations and measurements strengthen the association.

[Therapeutic readthrough strategy for suppression of nonsense mutations in duchenne muscular dystrophy].

Effective treatment for Duchenne muscular dystrophy (DMD) is currently unavailable. Readthrough of disease-causing premature termination codons might alleviate the symptoms of genetic diseases caused by nonsense mutations. Several ribosome-binding compounds, including selective antibiotics and synthetic novel small molecules, induce translational readthrough, restoring full-length functional proteins. Here in this innovative therapeutic strategy has been summarized with a focus on DMD. We have previously reported that negamycin restored dystrophin expression with less toxicity than gentamicin in mdx mice. To explore more potent readthrough inducers, we established the transgenic mouse called READ (readthrough evaluation and assessment by dural receptor) for readthrough-specific detection. Using READ mice, we discovered drug candidates, including sterically negamycin-like small molecules and aminoglycoside derivatives. The newly developed small molecules induced dose-dependent readthrough with greater potency than ataluren in vitro and promoted the expression of dystrophin and reduction in serum creatine kinase activity in mdx mice. Moreover, the aminoglycoside derivative restored both dystrophin protein and contractile function of mdx skeletal muscles with appreciably higher readthrough activity and lower toxicity than that of gentamicin. Furthermore, we confirmed the efficacy of a thioglycolate-based depilatory agent to enhance the topical delivery of skin-impermeable drugs, including aminoglycosides. These promising new chemotherapeutic agents with beneficial effects on readthrough action, lower toxicity, and transdermal delivery may have significant value in treating or preventing genetic diseases caused by nonsense mutations.

Transfer of a cyanobacterial neurotoxin within a temperate aquatic ecosystem suggests pathways for human exposure.

beta-methylamino-L-alanine (BMAA), a neurotoxic nonprotein amino acid produced by most cyanobacteria, has been proposed to be the causative agent of devastating neurodegenerative diseases on the island of Guam in the Pacific Ocean. Because cyanobacteria are widespread globally, we hypothesized that BMAA might occur and bioaccumulate in other ecosystems. Here we demonstrate, based on a recently developed extraction and HPLC-MS/MS method and long-term monitoring of BMAA in cyanobacterial populations of a temperate aquatic ecosystem (Baltic Sea, 2007-2008), that BMAA is biosynthesized by cyanobacterial genera dominating the massive surface blooms of this water body. BMAA also was found at higher concentrations in organisms of higher trophic levels that directly or indirectly feed on cyanobacteria, such as zooplankton and various vertebrates (fish) and invertebrates (mussels, oysters). Pelagic and benthic fish species used for human consumption were included. The highest BMAA levels were detected in the muscle and brain of bottom-dwelling fishes. The discovery of regular biosynthesis of the neurotoxin BMAA in a large temperate aquatic ecosystem combined with its possible transfer and bioaccumulation within major food webs, some ending in human consumption, is alarming and requires attention.

Cycloheximide treatment induces the uptake of neutral and dibasic amino acids via the activation of system b(0,+) in human intestinal Caco-2 cells.

Amino acids in enterocytes are thought to be absorbed in the intestinal epithelium via various types of amino acid transport, although the regulation of these amino acid transport systems has not been elucidated. We examined in the present study the effect of several inhibitors involved in mRNA and protein synthesis, and of protein translocation on the L-leucine (Leu) uptake in human intestinal epithelial-like Caco-2 cells. Culturing Caco-2 cells with cycloheximide (CHX) enabled the L-Leu uptake to be significantly increased in a dose- and time-dependent manner. The uptake of L-lysine (Lys) was also increased by the CHX treatment, whereas the uptake of L-glutamate, taurine, and Gly-Gln was not changed. Among the two transport systems, b(0,+) and y+, which are known to be involved in L-Lys uptake by Caco-2, the system b(0,+) component was greatly increased by the CHX treatment, suggesting that system b(0,+) was mainly responsible for the increase in L-Leu and L-Lys uptake. The mRNA levels of rBAT and b(0,+) AT, whose molecules comprise system b(0,+), were both significantly increased by the CHX treatment in a time-dependent manner. These results strongly suggest that the CHX treatment increased the Leu and Lys uptake by activating system b(0,+) and inducing rBAT and b(0,+) AT mRNA expression in human intestinal epithelial Caco-2 cells.

Selective brain uptake and behavioral effects of the cyanobacterial toxin BMAA (beta-N-methylamino-L-alanine) following neonatal administration to rodents.

Cyanobacteria are extensively distributed in terrestrial and aquatic environments all over the world. Most cyanobacteria can produce the neurotoxin beta-N-methylamino-L-alanine (BMAA), which has been detected in several water systems and could accumulate in food chains. The aim of the study was to investigate the transfer of BMAA to fetal and neonatal brains and the effects of BMAA on the development of behavioral characteristics during the brain growth spurt (BGS) in rodents. Pregnant and neonatal mice were given an injection of (3)H-BMAA on gestational day 14 and postnatal day (PND) 10, respectively, and processed for tape-section autoradiography. The study revealed transplacental transfer of (3)H-BMAA and a significant uptake in fetal mouse brain. The radioactivity was specifically located in the hippocampus, striatum, brainstem, spinal cord and cerebellum of 10-day-old mice. The effect of repeated BMAA treatment (200 or 600 mg/kg s.c.) during BGS on rat behavior was also studied. BMAA treatment on PND 9-10 induced acute alterations, such as impaired locomotor ability and hyperactivity, in the behavior of neonatal rats. Furthermore, rats given the high dose of BMAA failed to habituate to the test environment when tested at juvenile age. In conclusion, the results demonstrated that BMAA was transferred to the neonatal brain and induced significant changes in the behavior of neonatal rats following administration during BGS. The observed behavioral changes suggest possible cognitive impairment. Increased information on the long-term effects of BMAA on cognitive function following fetal and neonatal exposure is required for assessment of the risk to children's health.

The multifunctional role of ectoine as a natural cell protectant.

The protective properties of ectoine, formerly described for only extremophilic microorganisms, can be transferred to human skin. Our present data show that the compatible solute ectoine protects the cellular membrane from damage caused by surfactants. Transepidermal water loss measurements in vivo suggest that the barrier function of the skin is strengthened after the topical application of an oil in water emulsion containing ectoine. Ectoine functions as a superior moisturizer with long-term efficacy. These findings indicating that ectoine is a strong water structure-forming solute are explained in silico by means of molecular dynamic simulations. Spherical clusters containing (1) water, (2) water with ectoine, and (3) water with glycerol are created as model systems. The stronger the water-binding activity of the solute, the greater the quantity of water molecules remaining in the cluster at high temperatures. Water clusters around ectoine molecules remain stable for a long period of time, whereas mixtures of water and glycerol break down and water molecules diffuse out of the spheres. On the basis of these findings, we suggest that the hydrogen bond properties of solutes are not solely responsible for maintaining the water structure form. Moreover, the particular electrostatic potential of ectoine as an amphoteric molecule with zwitterionic character is the major cause for its strong affinity to water. Because of its outstanding water-binding activity, ectoine might be especially useful in preventing water loss in dry atopic skin and in recovering skin viability and preventing skin aging.

Enantioselective determination of dencichine in rabbit plasma by high-performance liquid chromatography-electrospray mass spectrometry.

An analytical method was developed for the determination of enantiomers of dencichine in plasma. Sample extraction from plasma was achieved by a solid-phase extraction (SPE) procedure using a C(18) cartridge, with carbocisteine as the internal standard. Plasma was deproteinized using inorganic acid and derivatizated before the SPE. Chiral separation of dencichine enantiomers was achieved by pre-column derivatization using o-phthaldialdehyde (OPA) and the chiral thiol N-isobutanoyl-L-cysteine (NIBC) to form diastereoisomeric isoindole derivatives that were separable by ODS column using a gradient solvent programme. The column eluent was monitored using mass spectrometry (MS). The conditions of MS detection were optimized, and selected ion monitoring was used to selectively detect D-dencichine and its arrangement isomer. High sensitivity and selectivity were obtained using this method. The limit of detection was determined to be 10 ng/ml for D-dencichine and 8 ng/ml for L-dencichine in plasma. The linearity was demonstrated over a wide range of concentrations, from 0.5 to 50 microg/ml for both enatiomers. The intra- and inter-day precision (C.V.), studied at four concentrations, was less than 7.0%. No interferences from endogenous amino acids and isomers of dencichine were found. The method was suitable for pharmacokinetic studies of dencichine enantiomers.

Ectoines as compatible solutes and carbon and energy sources for the halophilic bacterium Chromohalobacter salexigens.

AIMS: To investigate the catabolism of ectoine and hydroxyectoine, which are the major compatible solutes synthesized by Chromohalobacter salexigens. METHODS AND RESULTS: Growth curves performed in M63 minimal medium with low (0.75 mol l(-1) NaCl), optimal (1.5 mol l(-1) NaCl) or high (2.5 mol l(-1) NaCl) salinity revealed that betaine and ectoines were used as substrate for growth at optimal and high salt. Ectoine transport was maximal at optimal salinity, and showed 3- and 1.5-fold lower values at low and high salinity respectively. The salt-sensitive ectA mutant CHR62 showed an ectoine transport rate 6.8-fold higher than that of the wild type. Incubation of C. salexigens in a mixture of glucose and ectoine resulted in a biphasic growth pattern. However, CO(2) production due to ectoine catabolism was lower, but not completely abolished, in the presence of glucose. When used as the sole carbon source, glycine betaine effectively inhibited ectoine and hydroxyectoine synthesis at any salinity. CONCLUSIONS: The catabolic pathways for ectoine and hydroxyectoine in C. salexigens operate at optimal and high (although less efficiently) salinity. Endogenous ectoine(s) may repress its own transport. Ectoine utilization was only partially repressed by glucose. Betaine, when used as carbon source, suppresses synthesis of ectoines even under high osmolarity conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is a previous step to the subsequent isolation and manipulation of the catabolic genes, so as to generate strains with enhanced production of ectoine and hydroxyectoine.

Metabolism of dietary ODAP in humans may be responsible for the low incidence of neurolathyrism.

OBJECTIVES: The reasons for the very low incidence of the disease neurolathyrism in humans even after excessive consumption of the pulse, Lathyrus sativus, under severe drought and famine conditions, and its continued consumption by large populations during normal periods without any deleterious effects have been examined in the context of a possible metabolism or detoxification of beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (ODAP), the major neurotoxic amino acid of L. sativus. DESIGN AND METHODS: ODAP in urine samples from 54 subjects habitually consuming the pulse and in three volunteers on an L. sativus diet was determined by the OPT method following clean up of the samples on an alumina column. Urinary oxalate was also determined in these individuals. RESULTS: Twenty-five subjects showed no excretion of ODAP and it was only less than 0.7% of the dietary intake in the remaining 29 subjects. Urinary excretion of ODAP in three volunteers was also less than 1% with a peak excretion in the 4-h sample. The 4-h blood sample from one volunteer had a maximum ODAP concentration of 177 microM. The urinary oxalate content in the volunteers was nearly 3-fold higher compared to controls. CONCLUSIONS: The low excretion of dietary ingested ODAP in humans is in sharp contrast to that seen in animals and indicates a metabolism or detoxification of ODAP which may be unique to humans and may explain the low incidence of neurolathyrism.

Structure-based design of diaminopyranosides as a novel inhibitor core unit of HIV proteases.

Novel HIV PR inhibitors, which contain a diaminopyranoside moiety as an inhibitor core unit, were designed based on the 3D structures of complexes of HIV-1 PR with transition-state mimics. These compounds were examined for their ability to inhibit the hydrolytic activity of a recombinant HIV-1 PR.

Receptor interactions of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, the Lathyrus sativus putative excitotoxin, with synaptic membranes.

Direct evidence for the excitotoxicity of 3-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin has been studied by examining the binding of chemically synthesized [2,3 3H]ODAP ([3H]ODAP) to synaptic membranes. [3H]ODAP binding to membranes was mostly nonspecific, with only a very low specific binding (15-20% of the total binding) and was also not saturable. The low specific binding of [3H]ODAP remained unaltered under a variety of assay conditions. A low Bmax of 3.2 +/- 0.4 pmol/mg and Kd 0.2 +/- 0.08 microM could be discerned for the high affinity interactions under conditions wherein more than 80-90% of the binding was nonspecific. While ODAP could inhibit the binding of [3H]glutamate to chick synaptic membranes with a Ki of 10 +/- 0.9 microM, even L-DAP, a non neurotoxic amino acid was also equally effective in inhibiting the binding of [3H]glutamate. The very low specific binding of [3H]ODAP to synaptic membranes thus does not warrant considering its interactions at glutamate receptors as a significant event. The results thus suggest that the reported in vitro excitotoxic potential of ODAP may not reflect its true mechanism of neurotoxicity.

In vivo metabolism of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid: the Lathyrus sativus neurotoxin in experimental animals.

A comparative study of the metabolism of 1,2,3 (14)C-ODAP and 4,5 (14)C-ODAP in mice, rats and chicks has been carried out. Following oral administration of 1,2,3 (14)C-ODAP to either black or white mice, nearly 16% of the radioactivity appeared in the expired CO2 within 8 h, while in the rat only 3% of it appeared and in chicks it was less than 2%. No 14CO2 appeared in the expired air in mice given 4,5 (14)C-ODAP. Electrophoregrams of the spot urine samples from the animals given 1,2,3 (14)C-ODAP showed the presence of one radioactive metabolite (metabolite-1) in addition to ODAP. While the urine from rats and mice given 4,5 (14)C-ODAP indicated the presence of metabolite-1 as well as 14C-oxalate, in chicks, however, no 14C-oxalate was present and only metabolite-1 could be detected. The results indicate that ODAP can to some extent undergo oxidation in vivo in mice (and to a lesser extent in rats) leading to the formation of CO2 and oxalate and a similar pathway might be more prominent in humans leading to a near complete oxidation of ODAP.