Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y.

Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10pM. Acrylamide significantly reduced the number of neurons starting at 1µM and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide.

Fungicide prochloraz induces oxidative stress and DNA damage in vitro.

Prochloraz is widely used in horticulture and agriculture, e.g. as a post-harvest anti-mold treatment. Prochloraz is a known endocrine disruptor causing developmental toxicity with multiple mechanisms of action. However, data are scarce concerning other toxic effects. Since oxidative stress response, with formation of reactive oxygen species (ROS), is a common mechanism for different toxic endpoints, e.g. genotoxicity, carcinogenicity and teratogenicity, the aim of this study was to investigate if prochloraz can induce oxidative stress and/or DNA damage in human cells. A cell culture based in vitro model was used to study oxidative stress response by prochloraz, as measured by the activity of the nuclear factor erythroid 2-related factor 2 (Nrf2), a key molecule in oxidative defense mechanisms. It was observed that prochloraz induced oxidative stress in cultured human adrenocortical H295R and hepatoma HepG2 cells at non-toxic concentrations. Further, we used Comet assay to investigate the DNA damaging potential of prochloraz, and found that non-toxic concentrations of prochloraz induced DNA damage in HepG2 cells. These are novel findings, contradicting previous studies in the field of prochloraz and genotoxicity. This study reports a new mechanism by which prochloraz may exert toxicity. Our findings suggest that prochloraz might have genotoxic properties.

In vitro screening of inhibition of PPAR-γ activity as a first step in identification of potential breast carcinogens.

Alcohol consumption and increased estrogen levels are major risk factors for breast cancer, and peroxisome proliferator-activated receptor γ (PPAR-γ) plays an important role in alcohol-induced breast cancer. PPAR-γ activity is inhibited by ethanol, leading to increased aromatase activity and estrogen biosynthesis ultimately leading to breast cancer. If other organic solvents inhibit PPAR-γ activity, they should also lead to increased oestrogen biosynthesis and thus be potential breast carcinogens. Ten commonly used hydrophilic organic solvents were first tested in a cell-based screening assay for inhibitory effects on PPAR-γ transactivation. The chemicals shown to inhibit PPAR-γ were tested with vectors encoding PPAR-γ with deleted AB domains and only the ligand-binding domain to rule out unspecific toxicity. Next, the effects on biosynthesis of estradiol, testosterone and oestrone sulphate were measured in the H295R steroidogenesis assay after incubation with the chemicals. Ethylene glycol, ethyl acetate, and dimethyl sulphoxide inhibited PPAR-γ transactivation in a dose-dependent manner. The inhibitory effect on PPAR-γ was specific for PPAR-γ since the AB domain of PPAR-γ was required for the inhibitory effect. In the second step, ethylene glycol significantly increased production of oestradiol by 19% (p < 0.05) and ethyl acetate inhibited production of testosterone (p < 0.05). We here show that screening of 10 commonly used organic solvents for the ability to inhibit PPAR-γ transactivation followed by a well-established steroidogenesis assay for production of sex hormones in exposed H295 R cells may provide a screening tool for potential breast carcinogens. This initial screening thus identified ethylene glycol and possibly ethyl acetate as potential breast carcinogens.

Anionic linear aliphatic surfactants activate TRPV1: a possible endpoint for estimation of detergent induced eye nociception?

The transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway. Activation of the receptor causes a Ca(2+) influx in sensory C-fibres with secondary effects leading to neurogenic inflammation in the surrounding tissue. We have earlier reported specific activation of TRPV1 by surfactant-containing hygiene products. We have continued this project by investigating activation of the TRPV1 by shampoo and soap ingredients in low concentrations measured as intracellular Ca(2+) influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. As a TRPV1 specific control, the TRPV1 antagonist capsazepine was used. The response was quantified as the product induced Ca(2+) influx during 2 min in relation to the maximum response induced by the TRPV1 agonist capsaicin. The results show that anionic alkyl linear surfactant ingredients such as sodium lauryl sulphate, sodium laureth sulphate, ammonium lauryl sulphate, sodium C12-15 pareth sulphate and N-lauroylsarcosine concentration-dependently induced Ca(2+) influx that could be addressed to TRPV1. The cationic surfactants benzalkonium chloride and cetylpyridinium chloride induced a Ca(2+) influx that was not TRPV1 mediated as well as the zwitterionic surfactant cocamidopropyl betaine, the non-linear anionic surfactant sodium deoxycholate and the non-ionic surfactant Triton-X. These results reveal a new mechanistic pathway for surfactant-induced nociception.

The burden of rheumatoid arthritis and access to treatment: determinants of access.

As part of the study "The Burden of Rheumatoid Arthritis (RA) and Patient Access to Treatment", this paper reviews the impact on access to RA drugs of the approval processes, pricing and funding decisions and times to market (access) in different countries. In addition, an overview of health technology assessments (HTA) and the economic literature related to RA treatments is provided. The time from approval to market access ranged from immediate to over 500 days in the countries included in the study. A total of 55 HTA reports were identified, 40 of them in the period between 2002 and 2006; 29 were performed by European HTA agencies, 14 in Canada and 7 in the United States. A total of 239 economic evaluations related to RA were identified in a specialized health economic database (HEED).

Workshop 7 (synthesis): role and governance implications of virtual water trade.

Virtual water trade (VWT) is a powerful concept which stimulates fresh thinking about water scarcity and management. It is a potential solution for water-short countries to achieve food security. VWT may also have the potential to play a key role in international negotiations over management of transboundary water resources.

Workshop 3 (synthesis): water, poverty alleviation and social programs.

Poverty is a complex issue, which must be understood in a holistic manner. Low and variable income is certainly a key element, but it is far from enough to portray poverty. The various characteristics of poverty and their relative strength are determined through contextually specific circumstances, in terms of history, environmental preconditions, socio-cultural traits, etc. Much of this context is made up of local and national circumstances. The consequences of globalisation must, however, increasingly to be taken into account. At a larger scale, it is also relevant to mention that climate change will have a negative, although largely unpredictable, impact for the people in some parts of the world. For those who are already living on marginal lands or who are exposed to water problems, climate change is likely to create considerable adverse effects.

Workshop 1 (synthesis): steps towards increased effectiveness in water use--possibilities and replicability.

Inevitable increases in human needs and wants leading to mounting water stress necessitate that each unit of water is used as efficiently as possible. Excessive use of water cannot be accepted. Similarly, the least possible impact on water quality is a must. Yet, regional development and urgent social and environmental objectives may motivate an allocation of scarce water resources where the output is lower as compared to what could have been accomplished with an alternative allocation. In essence, the effective use of water presumes a broad and long-term perspective on societal development and environmental sustainability. Presentations illuminated that technical and institutional arrangements must be applied to reach an effective use. In practice, although not ncessarily in policy statements, technical arrangements continue to have a pivotal position in this regard.

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy.

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.

Hydrolytic properties of a beta-mannosidase purified from Aspergillus niger.

A beta-mannosidase was purified to homogeneity from the culture filtrate of Aspergillus niger. A specific activity of 500 nkat mg-1 and a 53-fold purification was achieved using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The isolated enzyme has an isoelectric point of 5.0 and appears to be a dimer composed of two 135-kDa subunits. It is a glycoprotein and contains 17% N-linked carbohydrate by weight. Maximal activity was observed at pH 2.4 5.0 and at 70 degrees C. The beta-mannosidase hydrolyzed beta-1,4-linked manno-oligosaccharides of degree of polymerization (DP) 2-6 and also released mannose from polymeric ivory nut mannan and galactomannan. The Km and Vmax values for p-nitrophenyl-beta-D-mannopyranoside were 0.30 mM and 500 nkat mg-1, respectively. Hydrolysis of D-galactose substituted manno-oligosaccharides showed that the beta-mannosidase was able to cleave up to, but not beyond, a side group. An internal peptide sequence of 15 amino acids was highly similar to that of an Aspergillus aculeatus beta-mannosidase belonging to family 2 of glycosyl hydrolases.

Separation and characterization of amines from individual atrial gland vesicles of Aplysia californica.

Several amine-containing components of individual vesicles from the atrial gland of Aplysia californica were identified with capillary electrophoresis (CE). On-line derivatization with naphthalene-2,3-dicarboxaldehyde was performed, and the derivatized amine-containing components were detected with laser-induced fluorescence (LIF). Amino acids, including taurine, that had not been determined previously in atrial gland vesicles were observed by using CE-LIF, and their identities were confirmed with CE, HPLC, NMR, and electrospray ionization mass spectrometry. The finding that taurine is packaged and stored into secretory vesicles supports the hypothesis that taurine may exhibit neuromodulatory activity. The bioactive peptides, well-known to be in atrial gland vesicles, were detected in lysed vesicle samples fractionated with HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These peptides were also observed in single-vesicle runs with CE-LIF. The atrial gland vesicles (ranging from 0.5 to 2 microns diameter and 65 aL to 4 fL volume, respectively) studied in this work represent the smallest biological entities to be analyzed chemically on an individual basis.

Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes.

We describe an efficient technique for the selective chemical and biological manipulation of the contents of individual cells. This technique is based on the electric-field-induced permeabilization (electroporation) in biological membranes using a low-voltage pulse generator and microelectrodes. A spatially highly focused electric field allows introduction of polar cell-impermeant solutes such as fluorescent dyes, fluorogenic reagents, and DNA into single cells. The high spatial resolution of the technique allows for design of, for example, cellular network constructions in which cells in close contact with each other can be made to possess different biochemical, biophysical, and morphological properties. Fluorescein, and fluo-3 (a calcium-sensitive fluorophore), are electroporated into the soma of cultured single progenitor cells derived from adult rat hippocampus. Fluo-3 also is introduced into individual submicrometer diameter processes of thapsigargin-treated progenitor cells, and a plasmid vector cDNA construct (pRAY 1), expressing the green fluorescent protein, is electroporated into cultured single COS 7 cells. At high electric field strengths, observations of dye-transfer into organelles are proposed.

Probing single secretory vesicles with capillary electrophoresis.

Secretory vesicles obtained from the atrial gland of the gastropod mollusk Aplysia californica were chemically analyzed individually with a combination of optical trapping, capillary electrophoresis separation, and a laser-induced fluorescence detection. With the use of optical trapping, a single vesicle that had attoliters (10(-18) liters) of volume was introduced into the tapered inlet of a separation capillary. Once the vesicle was injected, it was lysed, and its components were fluorescently labeled with naphthalene-2, 3-dicarboxaldehyde before separation. The resultant electropherograms indicated distinct variations in the contents of single vesicles.

Macro-scale water scarcity requires micro-scale approaches. Aspects of vulnerability in semi-arid development.

PIP: 4 types of water scarcity exist. Aridity and intermittent droughts consist of the natural types while land desiccation and water stress are man-made types. Climatic aridity, intermittent droughts, land degradation, and population growth link to create growing critical water scarcity conditions. Specifically, in arid lands where only a limited growing season exists anyhow, increased and nonsustaining activities spurred on by population growth degrade soils resulting in interference with water recharge of the root zone. This combination precipitates intermittent droughts upsetting the water supply for plants and people. This occurs now in Africa to the degree that by 2025, 66% of people will experience severe water shortages. Policymakers in developing countries and bilateral and multilateral development agencies providing technical assistance need to understand these relationships. They must develop a new strategy which includes water resource assessments followed by upgraded water plans for optimal use of available water resources and by the creation of best land use criteria. Their challenge is to balance the acute needs of people with conserving the productivity of the resource base. Experience shows that maximizing agricultural production per unit of water instead of per unit of land can increase income and employment. For example, in India, a semiarid area produced, with a given amount of water, as much as 30 times the amount of crops if the crops had a low water demand (e.g., grapes and potatoes) rather than those with a high water demand (e.g., sugarcane). This microscale approach and other such approaches could help semiarid Africa. Yet decision makers must seriously consider the transferability of these approaches to the African cultural and geographical environment. More essential than that, however, is the very high levels of water stress caused by the rapid population growth in famine-prone African countries.^ieng