Immobilization of enzymes based on hydrophobic interaction. III. Adsorbent substituent density and its impact on the immobilization of β-amylase

Hexyl-groups have been introduced into crosslinked Sepharose 6B, yielding gels with degrees of substitution which range from 0.02 to 0.70 mol hexyl-side chain per mole galactose residue. The gels were exposed to β-amylase in solution, and the resulting adsorbates indicated a monotonic increase in adsorption capacity with an increasing hexyl-content. Adsorbate activity, by contrast, displayed a maximum for a carrier gel with a hexyl–galactose ratio of 0.51. Adsorbates based on gels with different hexyl-content were used in column reactors for continuous maltose production from a soluble starch substrate.     

Mechanisms of Basin-Scale Nitrogen Load Reductions under Intensified Irrigated Agriculture

Irrigated agriculture can modify the cycling and transport of nitrogen (N), due to associated water diversions, water losses, and changes in transport flow-paths. We investigate dominant processes behind observed long-term changes in dissolved inorganic nitrogen (DIN) concentrations and loads of the extensive (465,000 km2) semi-arid Amu Darya River basin (ADRB) in Central Asia. We specifically considered a 40-year period (1960–2000) of large irrigation expansion, reduced river water flows, increased fertilizer application and net increase of N input into the soil-water system. Results showed that observed decreases in riverine DIN concentration near the Aral Sea outlet of ADRB primarily were due to increased recirculation of irrigation water, which extends the flow-path lengths and enhances N attenuation. The observed DIN concentrations matched a developed analytical relation between concentration attenuation and recirculation ratio, showing that a fourfold increase in basin-scale recirculation can increase DIN attenuation from 85 to 99%. Such effects have previously only been observed at small scales, in laboratory experiments and at individual agricultural plots. These results imply that increased recirculation can have contributed to observed increases in N attenuation in agriculturally dominated drainage basins in different parts of the world. Additionally, it can be important for basin scale attenuation of other pollutants, including phosphorous, metals and organic matter. A six-fold lower DIN export from ADRB during the period 1981–2000, compared to the period 1960–1980, was due to the combined result of drastic river flow reduction of almost 70%, and decreased DIN concentrations at the basin outlet. Several arid and semi-arid regions around the world are projected to undergo similar reductions in discharge as the ADRB due to climate change and agricultural intensification, and may therefore undergo comparable shifts in DIN export as shown here for the ADRB. For example, projected future increases of irrigation water withdrawals between 2005 and 2050 may decrease the DIN export from arid world regions by 40%.     

FCS cell surface measurements--photophysical limitations and consequences on molecular ensembles with heterogenic mobilities.

BACKGROUND: Fluorescence Correlation Spectroscopy is a powerful method to analyze densities and diffusive behavior of molecules in membranes, but effects of photodegradation can easily be overlooked. METHOD: Based on experimental photophysical parameters, calculations were performed to analyze the consequences of photobleaching in fluorescence correlation spectroscopy (FCS) cell surface experiments, covering a range of standard measurement conditions. RESULTS: Cumulative effects of photobleaching can be prominent, although an absolute majority of the fluorescent molecules would pass the laser excitation beam without being photo-bleached. Given a distribution of molecules on a cell surface with different diffusive properties, the fraction of molecules that is actually analyzed depends strongly on the excitation intensities and measurement times, as well as on the size of the reservoir of freely diffusing molecules. Both the slower and the faster diffusing molecules can be disfavored. CONCLUSIONS: Apart from quantifying photobleaching effects, the calculations suggest that the effects can be used to extract additional information, for instance about the size of the reservoirs of free diffusion. By certain choices of measurement conditions, it may be possible to more specifically analyze certain species within a population, based on their different diffusive properties, different areas of free diffusion, or different kinetics of possible transient binding.     

Environmental levels of the antiviral oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC₅₀ for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.     

Antipredator phenotype in crucian carp altered by a psychoactive drug

Predator‐inducible defenses constitute a widespread form of adaptive phenotypic plasticity, and such defenses have recently been suggested linked with the neuroendocrine system. The neuroendocrine system is a target of endocrine disruptors, such as psychoactive pharmaceuticals, which are common aquatic contaminants. We hypothesized that exposure to an antidepressant pollutant, fluoxetine, influences the physiological stress response in our model species, crucian carp, affecting its behavioral and morphological responses to predation threat. We examined short‐ and long‐term effects of fluoxetine and predator exposure on behavior and morphology in crucian carp. Seventeen days of exposure to a high dose of fluoxetine (100 µg/L) resulted in a shyer phenotype, regardless of the presence/absence of a pike predator, but this effect disappeared after long‐term exposure. Fluoxetine effects on morphological plasticity were context‐dependent as a low dose (1 µg/L) only influenced crucian carp body shape in pike presence. A high dose of fluoxetine strongly influenced body shape regardless of predator treatment. Our results highlight that environmental pollution by pharmaceuticals could disrupt physiological regulation of ecologically important inducible defenses.     

Disrupted oogenesis in the frog Xenopus tropicalis after exposure to environmental progestin concentrations

Levonorgestrel is a synthetic progesterone commonly used in pharmaceuticals (e.g., in contraceptives). It is found in sewage treatment plant effluents at concentrations up to 30 ng/L and was recently shown to pose a threat to egg laying in fish. Information on the susceptibility of adult amphibians to progestin toxicity is lacking. The present study aimed to 1) characterize progestogenic effects on the full cycle of oogenesis (egg development) in frogs and 2) determine female amphibians' susceptibility to reproductive impacts from progestogenic compounds in the environment. Sexually mature female Xenopus tropicalis were exposed to levonorgestrel via the surrounding water for 7 days (0, 51, or 307 ng/L) or 28 days (0, 1.3, 18, 160, or 1240 ng/L). Their ovaries were analyzed histologically with respect to frequencies of immature (in early meiotic prophase I), previtellogenic, vitellogenic, mature, and atretic oocytes. The 28-day exposure caused reduced proportions of oocytes at immature, vitellogenic, and mature stages, and increased proportions of previtellogenic oocytes compared with the control. The lowest tested concentration, 1.3 ng/L, increased the proportions of previtellogenic oocytes and reduced the proportions of vitellogenic oocytes, indicating inhibited vitellogenesis. The present study shows that progestin concentrations found in the aquatic environment impaired oogenesis in adult frogs. Our results indicate that progestogenic effects on oocyte development include interrupted germ cell progression into meiosis and inhibited vitellogenesis. Considering the crucial role of oogenesis in female fertility, our results indicate that progestogenic pollutants may pose a threat to reproduction in wild amphibian populations.     

Salt-independent adsorption of human serum proteins on cyanocarbon gels

Electron donor acceptor gels based on cyanocarbons have been tested for human serum protein adsorption in the absence of salt-promotion by water-structuring salt. This phenomenon was compared with a normal adsorption process in the presence of salt. The tricyanoaminopropene–divinyl sulfone–agarose displayed unusual protein adsorption properties as binding could occur both independently or dependently of the salt-promotion. The absence of hydrophobic or ionic character of the salt-independent interaction suggests an electron donor acceptor adsorption mechanism which is shown, for the first time, to occur independently of salt-promotion in aqueous solution. Study of the protein adsorption specificity showed similar protein selectivity for the fractions adsorbed in both conditions.     

Detection of the Antiviral Drug Oseltamivir in Aquatic Environments

Oseltamivir (Tamiflu®) is the most important antiviral drug available and a cornerstone in the defence against a future influenza pandemic. Recent publications have shown that the active metabolite, oseltamivir carboxylate (OC), is not degraded in sewage treatment plants and is also persistent in aquatic environments. This implies that OC will be present in aquatic environments in areas where oseltamivir is prescribed to patients for therapeutic use. The country where oseltamivir is used most is Japan, where it is used to treat seasonal flu. We measured the levels of OC in water samples from the Yodo River system in the Kyoto and Osaka prefectures, Japan, taken before and during the flu-season 2007/8. No OC was detected before the flu-season but 2–58 ng L⁻¹ was detected in the samples taken during the flu season. This study shows, for the first time, that low levels of oseltamivir can be found in the aquatic environment. Therefore the natural reservoir of influenza virus, dabbling ducks, is exposed to oseltamivir, which could promote the evolution of viral resistance.     

Risks of hormonally active pharmaceuticals to amphibians: a growing concern regarding progestagens

Most amphibians breed in water, including the terrestrial species, and may therefore be exposed to water-borne pharmaceuticals during critical phases of the reproductive cycle, i.e. sex differentiation and gamete maturation. The objectives of this paper were to (i) review available literature regarding adverse effects of hormonally active pharmaceuticals on amphibians, with special reference to environmentally relevant exposure levels and (ii) expand the knowledge on toxicity of progestagens in amphibians by determining effects of norethindrone (NET) and progesterone (P) exposure to 0, 1, 10 or 100 ng l⁻¹ (nominal) on oogenesis in the test species Xenopus tropicalis. Very little information was found on toxicity of environmentally relevant concentrations of pharmaceuticals on amphibians. Research has shown that environmental concentrations (1.8 ng l⁻¹) of the pharmaceutical oestrogen ethinylestradiol (EE₂) cause developmental reproductive toxicity involving impaired spermatogenesis in frogs. Recently, it was found that the progestagen levonorgestrel (LNG) inhibited oogenesis in frogs by interrupting the formation of vitellogenic oocytes at an environmentally relevant concentration (1.3 ng l⁻¹). Results from the present study revealed that 1 ng NET l⁻¹ and 10 ng P l⁻¹ caused reduced proportions of vitellogenic oocytes and increased proportions of previtellogenic oocytes compared with the controls, thereby indicating inhibited vitellogenesis. Hence, the available literature shows that the oestrogen EE₂ and the progestagens LNG, NET and P impair reproductive functions in amphibians at environmentally relevant exposure concentrations. The progestagens are of particular concern given their prevalence, the range of compounds and that several of them (LNG, NET and P) share the same target (oogenesis) at environmental exposure concentrations, indicating a risk for adverse effects on fertility in exposed wild amphibians.