Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U     

The effect of compact formulations on the environmental profile of northern european granular laundry detergents

Regular (1988) and compact granular (1992, 1998) laundry detergents were compared on the basis of two distinct, complementary approaches: Environmental Risk Assessment and Life-Cycle Assessment. The results are presented in this paper and an accompanying paper in this volume (Part II: Life-Cycle Assessment). Exposure data from The Netherlands and Sweden were used for this retrospective analysis. The time period studied (1988–1998) spans many innovations in laundry detergents, one of which was the introduction of compact detergents. The aquatic risk assessment resulted in risk quotients below 1 for all detergent ingredients in both countries over the period studied. Furthermore, it showed that risk quotients decrease two to five-fold between 1988 and 1998 in each country due to the introduction of compact detergents. Slightly lower risk quotients were observed in Sweden, when compared to The Netherlands, attributable to the lower water hardness resulting in lower detergent usage per wash cycle in that country. If water hardnesses were equal, the outcome of the product risk assessments would also be the same in the two countries.     

Gene therapy legislation in The Netherlands

Several regulatory organisations are involved in the assessment of clinical gene therapy trials involving genetically modified organisms (GMOs) in The Netherlands. Medical, ethical and scientific aspects are, for instance, evaluated by the Central Committee on Research Involving Human Subjects (CCMO). The Ministry of Housing, Spatial Planning and the Environment (VROM) is the competent authority for the environmental risk assessment according to the deliberate release Directive 2001/18/EC. A Gene Therapy Office has been established in order to streamline the different national review processes and to enable the official procedures to be completed as quickly as possible. Although the Gene Therapy Office improved the application process at the national level, there is a difference of opinion between the EU member states with respect to the EU Directive according to which gene therapy trials are assessed, that urges for harmonisation. This review summarises the gene therapy legislation in The Netherlands and in particular The Netherlands rationale to follow Directive 2001/18/EC for the environmental risk assessment.     

The effect of compact formulations on the environmental profile of Northern European granular laundry detergents Part II: Life Cycle assessment

The environmental profile of laundry detergents at three time points (1988, 1992, and 1998) were compared on the basis of two distinct, complementary approaches: Environmental Risk Assessment (ERA) and Life-Cycle Assessment (LCA). The results are presented in this paper and its accompanying paper in this issue (Part I: Product Environmental Risk Assessment). Life-Cycle Inventory (LCI) data from The Netherlands and Sweden were used for this retrospective analysis. The chosen time period studied (1988 - 1998) spans significant, multiple formulation and process change in laundry detergents, including the introduction of compact, then super-compact, granular detergents. Cradle-to-Gate LCAs based on 1 kg of finished product (from raw material supply to packaged finished product leaving the suppliers site) revealed no significant differences between the products themselves, as manufactured between 1988, 1992 and 1998. Cradle-to-Grave LCAs based on 1000 wash cycles (from raw material supply to disposal of used product) indicated that the consumption of raw materials and energy, as well as environmental emissions (air, water and solid waste), decreased after the introduction of compact detergents in 1988. The LCAs revealed that a number of category indicator values decreased (for acidification, aquatic toxicity greenhouse effects, eutrophication, toxicity, ozone depletion and smog). Furthermore, the results of the LCAs support the conclusion that the differences between The Netherlands and Sweden are due to (1) differences in electrical generation between the countries, (2) differences in energy consumption during consumer use, (3) differences in detergent dosage per wash and (4) differences in the wastewater treatment infrastructure.     

Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.     

Optimal conditions for determination of cytochrome c oxidase activity in the rat heart

The determination of cytochrome c oxidase (COX) activity represents an important indicator for the evaluation of cell oxidative capacity. However, it has been shown repeatedly that different factors modify the rate of COX activity under various experimental conditions. The most important concern the ionic concentrations of the medium and the application of various detergents for the solubilization of mitochondrial membranes. We found the highest activity of COX in rat heart homogenates and mitochondria at 40-60 mM potassium phosphate. The rate of COX activity is dependent on the detergent/protein (P) ratio. Using n-dodecyl-beta-D-maltoside (lauryl maltoside, LM) as the detergent, we obtained the highest activity at LM/P ratios of (50:100):1. By kinetic measurements of low-affinity binding sites in heart mitochondria, we found Vlim values of 4.3 and 22.2 micromol cytochrome c per min per mg P in the presence or absence of lauryl maltoside, respectively. The Km values were 16.7 micromol in the presence or absence of lauryl maltoside. Our results thus indicate that 1) the exact assessment of COX activity in heart homogenates and mitochondria requires the determination of optimum phosphate concentrations in the medium used, and 2) even small modifications of the experimental procedure may induce significant differences in the maximum values of COX activity.     

Environmental capacity of receiving water as basis for regulating intensity of milkfish (Chanos chanos Forsskal) culture

This paper presents the results of the assessment of the environmental condition of a receiving water and demonstrates how the environmental capacity of the system can be estimated based on effluent discharge from milkfish ponds and water criteria from scientific literature and other studies. An estuary (average volume, 295 333 m³; average depth, 1.9 m) in Punta Pulao, Dumangas, Iloilo, Philippines served as discharge and irrigation system for commercial milkfish ponds and experimental/verification ponds owned by the Southeast Asian Fisheries Development Center. Total ammonia nitrogen, nitrite, nitrate, phosphate, and chlorophyll a (chl a) were determined monthly (during spring tide) for 4 months. Samples were taken at low and high tides that coincided with the draining and flooding of ponds, respectively. Monthly concentrations of these water quality variables generally increased from the first to fourth month of sampling (April–July 2001) during low tide. Very slight increases were observed during high tide. The magnitude of changes in the diurnal patterns of phosphate, chl a, and dissolved oxygen appeared to be higher at spring tide than at neap tide. This indicates that the inflowing river at low tide (during spring tide) brought effluents containing high amounts of nutrients from ponds located upstream. The water, salt, dissolved inorganic nitrogen (DIN), and phosphate (DIP) budgets of the entire estuary were determined following the one‐box model by LOICZ‐IGBP (2000, LOICZ biochemical budgeting procedure: a tutorial pamphlet. L. T. David, M. L. San Diego‐McGlone, C. J. Crossland and S. V. Smith (Eds). Publ. for LOICZ International Project Office, the Netherlands, 29 pp.). The budgets indicate that the system is net heterotrophic and nitrogen (N) fixing during the dry months, but that there could be no environmental impact during the rainy months because of short water exchange time. Results further suggest that the system is a source of N and phosphorus (P) during the dry and rainy months; the condition is largely influenced by the high amount of nutrient inputs in to the river. Linear regression analysis was performed to determine relationship between nutrient concentrations in the system and total DIN or DIP input to the system at low tide. Environmental capacity in terms of the maximum amount of DIN or DIP input to the system was predicted using regression analysis and following set criteria for nutrients, i.e. nitrite, nitrate, and phosphate. At present, the estuarine water quality has already reached its environmental capacity during the dry months. About 945 ha of commercial milkfish ponds are operating upstream, mostly as extensive systems. If these ponds are converted to semi‐intensive or intensive systems, it is recommended that the pond area be reduced to 122 ha if the DIP criterion is to be followed so as not to exceed the environmental capacity. Exceeding this environmental capacity may affect production through reduction of fish growth, occurrence of diseases, and fish mortalities.     

Environmental Factors Correlated with Size of Bacterial Populations in a Polluted Stream

Samples of water were taken from a polluted zone of the Gallinas River and analyzed as to numbers of total bacteria, coliforms, and fecal streptococci. Environmental factors measured were temperature, pH and concentrations of detergent, nitrate plus nitrite nitrogen, sulfate, chloride, bicarbonate, and phosphate. Thirty-two observations were made from 12 March through 22 July 1971. Stepwise multiple linear regression analyses of the data were carried out by computer to determine which of the environmental factors were significantly correlated with numbers of bacteria present. A multiple linear regression equation was constructed for each bacteriological parameter as a function of significant variables only. Log total bacteria was correlated positively with bicarbonate, phosphate, and detergent concentrations. Log coliforms was correlated positively with phosphate and sulfate concentrations and negatively with chloride concentration. Log fecal streptococci was correlated positively with bicarbonate and chloride concentrations.     

Equivalence analysis to support environmental safety assessment: Using nontarget organism count data from field trials with cisgenically modified potato

This paper considers the statistical analysis of entomological count data from field experiments with genetically modified (GM) plants. Such trials are carried out to assess environmental safety. Potential effects on nontarget organisms (NTOs), as indicators of biodiversity, are investigated. The European Food Safety Authority (EFSA) gives broad guidance on the environmental risk assessment (ERA) of GM plants. Field experiments must contain suitable comparator crops as a benchmark for the assessment of designated endpoints. In this paper, a detailed protocol is proposed to perform data analysis for the purpose of assessing environmental safety. The protocol includes the specification of a list of endpoints and their hierarchical relations, the specification of intended levels of data analysis, and the specification of provisional limits of concern to decide on the need for further investigation. The protocol emphasizes a graphical representation of estimates and confidence intervals for the ratio of mean abundances for the GM plant and its comparator crop. Interpretation relies mainly on equivalence testing in which confidence intervals are compared with the limits of concern. The proposed methodology is illustrated with entomological count data resulting from multiyear, multilocation field trials. A cisgenically modified potato line (with enhanced resistance to late blight disease) was compared to the original conventional potato variety in the Netherlands and Ireland in two successive years (2013, 2014). It is shown that the protocol encompasses alternative schemes for safety assessment resulting from different research questions and/or expert choices. Graphical displays of equivalence testing at several hierarchical levels and their interpretation are presented for one of these schemes. The proposed approaches should be of help in the ERA of GM or other novel plants.     

A dynamic phosphate budget model for a eutrophic lake

The relations between the external nutrient loading of lakes, recycling through sediments and the resulting productivity are complicated by feed-back mechanisms, seasonal variations and trends. Simulation is a useful tool for the identification of controlling factors and the assessment of the effects of management measures, supplementary to experimental research. The model variables in our dynamic phosphate budget model include inorganic and organic particulate phosphate and dissolved o-phosphate, in both sediments and overlying water. Sediments may be aerobic or anaerobic, depending on topography, temperature and composition. The major processes described are primary production, mineralisation, sedimentation, adsorption and diffusion. Several model parameters have been estimated directly for Lake Brielle (Netherlands). The sediment dilution rate, the extent of anaerobic conditions and the number and character of adsorption sites are important controlling factors.     

A Market‐Based Framework for Quantifying Displaced Production from Recycling or Reuse

The most significant environmental benefit of recycling or reusing a wide range of products and materials is typically the potential to displace primary material production; lack of displacement significantly reduces the environmental benefits of these activities. Because no consensus method to estimate displacement rate has emerged, environmental assessments have tended to assume that displacement occurs on a one‐to‐one basis. However, displaced production is a complex phenomenon governed primarily by market mechanisms, rather than physical relationships. This article advances the understanding of displacement by presenting a market‐based framework describing the displacement relationship and a methodology for quantifying displacement rate based on partial equilibrium modeling. Using this methodology, a general symbolic equation for displacement rate after an increase in recycling is derived. The model highlights the market mechanisms that govern displaced production and identifies five price response parameters that affect displacement rate. Results suggest that one‐to‐one displacement occurs only under specific parameter restrictions that are unlikely in competitive commodity markets, but zero displacement is possible if secondary materials are poor substitutes for primary materials; displacement is likely to be reduced if secondary materials have inferior technical properties. The presented methodology can be generally applied to any system in which recycled or reused materials are substitutes or complements for primary materials. Implications for improving recycling and reuse efficacy and environmental assessment methodology are discussed, and suggestions are presented for expanding the displacement methodology in future research.     

N1N8-bis(gamma-glutamyl)spermidine cross-linking in epidermal-cell envelopes. Comparison of cross-link levels in normal and psoriatic cell envelopes.

In order to explore the effect of electric charge on detergent solubilization of phospholipid bilayers, the interaction of nine electrically charged surfactants with neutral or electrically charged liposomes has been examined. The detergents belonged to the alkyl pyridinium, alkyl trimethylammonium or alkyl sulphate families. Large unilamellar liposomes formed by egg phosphatidylcholine plus or minus stearylamine or dicetyl phosphate were used. Solubilization was assessed as a decrease in light-scattering of the liposome suspensions. The results suggest that electrostatic forces do not play a significant role in the formation of mixed micelles and that hydrophobic interactions are by far the main forces involved in solubilization. In addition, from the study of thirty different liposome-surfactant systems, we have derived a series of empirical rules that may be useful in predicting the behaviour of untested surfactants: (i) the detergent concentration producing the onset of solubilization (Don) decreases as the alkyl chain length increases; the decrease follows a semi-logarithmic pattern in the case of alkyl pyridinium compounds; (ii) for surfactants with critical micellar concentrations (cmc) less than 6 x 10(-3) M, Don. is independent of the nature of the detergent and the bilayer composition; for detergents having cmc greater than 6 x 10(-3) M, Don. increases linearly with the cmc; and (iii) Don. varies linearly with the surfactant concentration that produces maximum solubilization.     

Structure of rhomboid protease in a lipid environment

Structures of the prokaryotic homologue of rhomboid proteases reveal a core of six transmembrane helices, with the active-site residues residing in a hydrophilic cavity. The native environment of rhomboid protease is a lipid bilayer, yet all the structures determined thus far are in a nonnative detergent environment. There remains a possibility of structural artefacts arising from the use of detergents. In an attempt to address the effect of detergents on the structure of rhomboid protease, crystals of GlpG, an Escherichia coli rhomboid protease in a lipid environment, were obtained using two alternative approaches. The structure of GlpG refined to 1. 7-Å resolution was obtained from crystals grown in the presence of lipid bicelles. This structure reveals well-ordered and partly ordered lipid molecules forming an annulus around the protein. Lipid molecules adapt to the surface features of protein and arrange such that they match the hydrophobic thickness of GlpG. Virtually identical two-dimensional crystals were also obtained after detergent removal by dialysis. A comparison of an equivalent structure determined in a completely delipidated detergent environment provides insights on how detergent substitutes for lipid. A detergent molecule is also observed close to the active site, helping to postulate a model for substrate binding and hydrolysis in rhomboids.     

LCA application to Russian Conditions

The environmental impact assessment existing in the Russian Federation at the present moment cannot provide potential scenarios of consequences for the environment from examined processes, since its goal is to calculate the money equivalent of emissions to the environment. Also, it cannot help the environmental specialist to choose the most environmentally sustainable scenario or process, proceeding from the whole life cycle of the object, because it is usually performed only for the use phase of an object. This study also aims to show possibilities for applying LCA methodology, as accepted in the ISO standards series 14040, and as applied to Russian conditions. The main purpose was to investigate a possibility of using the existing environmental impact assessment as the inventory stage in the LCA. As the minor goal, normalisation and weighting factor data for the Russian Federation were calculated on the basis of energy consumption extrapolation. In this paper, the environmental impacts are associated with a sewage wastewater facility. The inventory analysis is performed with data obtained from the MosvodokanalNIIproject (Moscow Research Institute for sewage wastewater treatment facilities) and supplemented with the SimaPro 5.0 database (the Netherlands). The environmental impact categories included and discussed in this study are eutrophication, global warming, landfill, acidification, ozone layer depletion and photochemical ozone creation. This study was performed for several design alternatives or scenarios of the wastewater facility. According to the LCA performed in this study, the most environmentally sustainable scenario is that which has the most effective and complicated treatment of sewage water and sludge.     

Bendiocarb and diflubenzuron as substitute insecticides for endosulfan in commercial mushroom growing

Diflubenzuron and bendiocarb treatments of compost and casing soil at a rate of 1 g a.i./m² each, resulted in comparable or even improved compost and casing soil colonisation over endosulfan treatments (at a rate of 1.5 g a.i./m²). Alternating diflubenzuron/bendiocarb treatments were preferable to the reverse treatments, which tended to diminish total yield of mushrooms significantly by 1.4–3.9 kg/m². This effect was due to the casing soil treatment with diflubenzuron and not due to the compost treatment with bendiocarb after spawning. The effectiveness of these substitutes for endosulfan was tested on five commercial mushroom farms, where a similar or improved control of Megaselia halterata (Diptera: Phoridae) was obtained over control (endosulfan) treatments. If present, Lycoriella auripila (Diptera: Lycoriidae) was very effectively controlled by the substitute insecticides compared with endosulfan. Relatively high numbers of M. halterata were occasionally observed in endosulfan-treated houses, indicating that a certain level of resistance towards this insecticide may already have developed. This assessment of tolerance in M. halterata emphasised the need for substitute insecticides with different modes of action, in addition to environmental reasons. The chemicals should be alternately applied within individual crops to avoid resistance development.     

Evidence for a phospholipid requirement in the specific binding of glucocorticoids to receptors of fibroblasts and thymic lymphocytes.

The specific steroid binding capacity of soluble preparations from mouse fibroblasts and rat thymic lymphocytes is inactivated by incubation with phospholipases. Receptor binding is drastically reduced by very low concentrations of boiled phospholipase A preparations from bee venom and snake venoms. The enzyme effect is calcium-dependent and is blocked by both phospholipid and a substrate analog that is a competitive inhibitor of phospholipase A. The specific binding capacity is also sensitive to digestion by phospholipase C. Two possible mechanisms are considered for the phospholipase A effect: (a) the receptor protein may be associated with a phospholipid component which is required for specific hormone binding; (b) phospholipase A may be producing detergent products that are indirectly inactivating the receptor. Examination of the effects of lysophosphatide on the receptor and assay of lipid phosphate in the receptor preparation do not support a mechanism based solely on detergent effects. Because phospholipase C, which does not produce detergent products, also inactivates the binding, we propose that the phospholipases may be digesting the phospholipid which is a requisite component of the glucocorticoid receptor.     

Banning Fisheries Discards Abruptly Has a Negative Impact on the Population Dynamics of Charismatic Marine Megafauna

Food subsidies have the potential to modify ecosystems and affect the provision of goods and services. Predictable Anthropogenic Food Subsidies (PAFS) modify ecosystems by altering ecological processes and food webs. The global concern over the effects of PAFS in ecosystems has led to development of environmental policies aimed at curbing the production or ultimately banning of PAFS. However, the effects of reducing or banning PAFS are not known. We explore the consequences of PAFS removal in a marine ecosystem under two scenarios: 1) gradual reduction, or 2) an abrupt ban, using a mass balance model to test these hypotheses–The reduction or loss of PAFS will: i) modify trophic levels and food webs through effects on foraging by opportunistic species, ii) increase the resilience of opportunistic species to food shortages, and iii) modify predator–prey interactions through shifts in prey consumption. We found that PAFS lower the trophic levels of opportunistic scavengers and increase their food pathways. Scavengers are able to switch prey when PAFS are reduced gradually but they decline when PAFS are abruptly banned. PAFS reduction to a certain minimal level causes a drop in the ecosystem’s stability. We recommend gradual reduction of PAFS to a minimal level that would maintain the ecosystem’s stability and allow species exploiting PAFS to habituate to the food subsidy reduction.     

Kinetics of the reconstituted dicarboxylate carrier from rat liver mitochondria

The dicarboxylate carrier from rat liver mitochondria was purified by the Amberlite/hydroxyapatite procedure and reconstituted in egg yolk phospholipid vesicles by removing the detergent with Amberlite. The efficiency of reconstitution was optimized with respect to the ratio of detergent/phospholipid, the concentration of phospholipid and the number of Amberlite column passages. In the reconstituted system the incorporated dicarboxylate carrier catalyzed a first-order reaction of malate/phosphate exchange. V of the reconstituted malate/phosphate exchange was determined to be 6000 mumol/min per g protein at 25 degrees C. This value was independent of the type of substrate present at the external or internal space of the liposomes (malate, phosphate or malonate). The half-saturation constant was 0.49 mM for malate, 0.54 mM for malonate and 1.41 mM for phosphate. The activation energy of the exchange reaction was determined to be 95.8 kJ/mol. The transport was independent of the external pH in the range between pH 6 and 8.     

Anionic carbohydrate-containing cell wall polymers of Streptomyces melanosporofaciens and related species

The structures of cell wall anionic carbohydrate-containing polymers in Streptomyces melanosporofaciens VKM Ac-1864T and phylogenetically close organisms-S. hygroscopicus subsp. hygroscopicus VKM Ac-831T, S. violaceusniger VKM Ac-583T, S. endus VKM Ac-1331, S. endus VKM Ac-129, and S. rutgersensis subsp. castelarensis VKM Ac-832T--have been comparatively studied by chemical and NMR spectroscopic methods. The natural polymer of a new, previously unknown structure, Kdn (3-deoxy-D-glycero-Dgalacto-non-2-ulopyranosonic acid) with beta-galactose residues at C-9, has been found in the cell walls of all the strains under study. The cell walls of all the studied organisms contain three teichoic acids (TA): a predominant TA (1,3-poly(glycerol phosphate) with N-acetylated alpha-glucosaminyl substitutes by C-2 of glycerol, and minor TAs, 1,3- and 2,3-poly(glycerol phosphate) polymers without substitution. Their chains have O-acetyl and O-lysyl groups. Microorganisms of the above-mentioned species differ in the number of alpha-glucosaminyl substitutes and in the degree of their acetylation in the predominant teichoic acid.