The effects of light intensity on the growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a form of seafood poisoning that is widespread in tropical, subtropical and temperate regions worldwide. The distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3 have been reported for the waters surrounding the main island of Japan. To explore the bloom dynamics and the vertical distribution of these Japanese species and phylotypes of Gambierdiscus, the effects of light intensity on their growth were tested, using a photoirradiation-culture system. The relationship between the observed growth rates and light intensity conditions for the four species/phylotypes were formulated at R > 0.92 (p < 0.01) using regression analysis and photosynthesis-light intensity (P-L) model. Based on this equation, the optimum light intensity (L_max) and the semi-optimum light intensity range (L_s-opt) that resulted in the maximum growth rate (μ_max) and ≥80% μ _max values of the four species/phylotypes, respectively, were as follows: (1) the L_max and L_s-opt of G. australes were 208 μmol photons m⁻² s⁻¹ and 91–422 μmol photons m⁻² s⁻¹, respectively; (2) those of G. scabrosus were 252 and 120–421 μmol photons m⁻² s⁻¹, respectively; (3) those of Gambierdiscus sp. type 2 were 192 and 75–430 μmol photons m⁻² s⁻¹, respectively; and (4) those of Gambierdiscus sp. type 3 were ≥427 and 73–427 μmol photons m⁻² s⁻¹, respectively. All four Gambierdiscus species/phylotypes required approximately 10 μmol photons m⁻² s⁻¹ to maintain growth. The light intensities in coastal waters at a site in Tosa Bay were measured vertically at 1 m intervals once per season. The relationships between the observed light intensity and depth were formulated using Beer’s Law. Based on these equations, the range of the attenuation coefficients at Tosa Bay site was determined to be 0.058–0.119 m⁻¹. The values 1700 μmol photons m⁻² s⁻¹, 500 μmol photons m⁻² s⁻¹, and 200 μmol photons m⁻² s⁻¹ were substituted into the equations to estimate the vertical profiles of light intensity at sunny midday, cloudy midday and rainy midday, respectively. Based on the regression equations coupled with the empirically determined attenuation coefficients for each of the four seasons, the ranges of the projected depths of L_max and L_s-opt for the four Gambierdiscus species/phylotypes under sunny midday conditions, cloudy midday conditions, and rainy midday conditions were 12–38 m and 12–54 m, 1–16 m and 1–33 m, and 0 m and 0–16 m, respectively. These results suggest that light intensity plays an important role in the bloom dynamics and vertical distribution of Gambierdiscus species/phylotypes in Japanese coastal waters.     

Sex-specific light acclimation of Chara canescens (Charophyta)

Bisexual populations of the charophyte Chara canescens (Desv. et Loisel. in Loisel., 1810) containing male and female individuals are rarely found. Two experiments were carried out to study whether male and female algae from the same site exhibit different physiological capacities, especially with respect to light acclimation.Algae from two different shore levels and from laboratory cultures acclimated to six irradiance conditions (35–500 μmol photons m⁻² s⁻¹) were compared. Field measurements showed that both female and male algae of C. canescens are able to acclimate to daily changes in solar irradiance. The quantum yield of Photosystem II (PSII) decreased with increasing irradiance in the morning and increased with decreasing irradiance in the afternoon. Growth experiments showed increasing growth rates from 35 μmol photons m⁻² s⁻¹ (∼7 mg FW) up to 500 μmol photons m⁻² s⁻¹ (∼27 mg FW) in female and male C. canescens. The irradiance saturation point for photosynthesis (E_k) was about 140 μmol m⁻² s⁻¹ for both sexes within the whole range of acclimation irradiances. The maximum photosynthesis rate at saturating irradiances (P_max) of male algae was highest at E_k, whereas P_max of female algae was highest at 500 μmol photons m⁻² s⁻¹. The photosynthetic efficiency in the light-limited range (α) increased in female C. canescens and decreased in male C. canescens. The ratio of the non-photochemical quenching parameter (NPQ) to the relative electron transport rates rETR_(MT) increased in both sexes with irradiance, but showed a steeper increase in male than in female algae. Pigment analysis showed similar acclimation pattern for male and female C. canescens. Chl a/Chl b ratios of both sexes were constant over the whole range of E_g, whereas Chl a/carotenoid ratios in male and female C. canescens decreased from 70 μmol photons m⁻² s⁻¹ upwards. Pigment analysis pointed out that the carotenes α-, β- and γ-carotene were more prominent in male than in female algae.Our results indicate that female C. canescens are more efficient in light acclimation than male algae from the same site. Nevertheless, further investigations of bisexual C. canescens populations resolving CO₂-uptake mechanisms and/or genetic differences are needed.     

Ground level environmental protein concentrations in various ecuadorian environments: Potential uses of aerosolized protein for ecological research

Large quantities of free protein in the environment and other bioaerosols are ubiquitous throughout terrestrial ground level environments and may be integrative indicators of ecosystem status. Samples of ground level bioaerosols were collected from various ecosystems throughout Ecuador, including pristine humid tropical forest (pristine), highly altered secondary humid tropical forest (highly altered), secondary transitional very humid forest (regrowth transitional), and suburban dry montane deforested (suburban deforested). The results explored the sensitivity of localized aerosol protein concentrations to spatial and temporal variations within ecosystems, and their value for assessing environmental change. Ecosystem specific variations in environmental protein concentrations were observed: pristine 0.32 ± 0.09 μg/m³, highly altered 0.07 ± 0.05 μg/m³, regrowth transitional 0.17 ± 0.06 μg/m³, and suburban deforested 0.09 ± 0.04 μg/m³. Additionally, comparisons of intra-environmental differences in seasonal/daily weather (dry season 0.08 ± 0.03 μg/m³ and wet season 0.10 ± 0.04 μg/m³), environmental fragmentation (buffered 0.19 ± 0.06 μg/m³ and edge 0.15 ± 0.06 μg/m³), and sampling height (ground level 0.32 ± 0.09 μg/m³ and 10 m 0.24 ± 0.04 μg/m³) demonstrated the sensitivity of protein concentrations to environmental conditions. Local protein concentrations in altered environments correlated well with satellite-based spectral indices describing vegetation productivity: normalized difference vegetation index (NDVI) (r² = 0.801), net primary production (NPP) (r² = 0.827), leaf area index (LAI) (r² = 0.410). Moreover, protein concentrations distinguished the pristine site, which was not differentiated in spectral indices, potentially due to spectral saturation typical of highly vegetated environments. Bioaerosol concentrations represent an inexpensive method to increase understanding of environmental changes, especially in densely vegetated ecosystems with high canopies or in areas needing high spatial and temporal resolution. Further research to expand understanding of the applicability of bioaerosol concentrations for environmental monitoring is supported by this pilot study.     

Morphology and phylogeny of Henneguya jocu n. sp. (Myxosporea,Myxobolidae), infecting the gills of the marine fish Lutjanus jocu

Henneguya jocu n. sp. (Myxosporea, Myxobolidae) is described from the gill lamellae of the marine teleost fish Lutjanus jocu, with a focus on ultrastructural and molecular features. This myxosporean forms subspherical cysts up to ∼260 μm × 130 μm long, and develops asynchronously. Mature myxospores ellipsoidal with a bifurcated caudal process. Myxospore length 10.9 ± 0.4 μm (n = 50); width, 8.2 ± 0.3 μm (n = 50); and thickness, 2.9 ± 0.5 μm (n = 50). Two equal caudal processes, 34.1 ± 1.0 μm long (n = 50); and total myxospore length, 45.2 ± 1.0 μm (n = 50). Two symmetric valves surround two ellipsoidal polar capsules, 5.0 ± 0.3 × 1.4 ± 0.2 μm (n = 20), each containing an isofilar polar filament forming 4–5 coils along the inner wall of these structures, as well as a binucleated sporoplasm presenting a spherical vacuole and several globular sporoplasmosomes. Both the morphological data and molecular analysis of the SSU rDNA gene identify this parasite as a new species of the genus Henneguya. Maximum Likelihood and Maximum Parsimony analyses further indicate that the parasite clusters within others marine Myxobolidae species, forming a group alongside other Henneguya species described from marine hosts.     

Evaluation of the effects of light intensity on growth of the benthic dinoflagellate Ostreopsis sp. 1 using a newly developed photoirradiation-culture system and a novel regression analytical method

Benthic dinoflagellates of the genus Ostreopsis are found all over the world in temperate, subtropical, and tropical coastal regions. Our recent studies revealed that a putative “cryptic” species of Ostreopsis ovata is present widely along Japanese coasts. This organism, Ostreopsis sp. 1, possesses palytoxin analogs and thus its toxic blooms may be responsible for potential toxification of marine organisms. To evaluate the bloom dynamics of Ostreopsis sp. 1, the present study examined the growth responses of Ostreopsis sp. 1 strain s0716 to various light intensities (photon flux densities: μmol photons m⁻² s⁻¹) using a newly devised photoirradiation-culture system. This novel system has white light-emitting diodes (LEDs) capable of more closely simulating the wavelength spectrum of light entering the oceanic water column than do fluorescent tubes and halogen lamps. In this system, the light intensity of the white LEDs was reduced through two polarizing filters by varying the rotation angles of the filters. Thereby, the new system was capable of culturing microalgae under well-controlled light intensity conditions. Ostreopsis sp. 1 grew proportionally when light intensity was increased from 49.5 to 199 μmol photons m⁻² s⁻¹, but its growth appeared to be inhibited slightly at ≥263 μmol photons m⁻² s⁻¹. The relationship between observed growth rates and light intensity was calculated at R > 0.99 (P < 0.01) using a regression analysis with a modified equation of the photosynthesis-light intensity (P-L) model. The equation determined the critical light intensities for growth of Ostreopsis sp. 1 and the organism's growth potential as follows: (1) the threshold light intensity for growth: 29.8 μmol photons m⁻² s⁻¹; (2) the optimum light intensity (L_m) giving the maximum growth rate (μ_max = 0.659 divisions day⁻¹): 196 μmol photons m⁻² s⁻¹; (3) the optimum light intensity range (L_opt) giving ≥95% μ_max: 130–330 μmol photons m⁻² s⁻¹; (4) the semi-optimum range (L_sopt) giving ≥80% μ_max: 90 to over 460 μmol photons m⁻² s⁻¹. The L_sopt represents 4.5–23% ambient light intensity present in surface waters off of a temperate region of the Japanese coast, Tosa Bay; putatively, this semi-optimum range of light intensity appears at depth of 12.9–27.8 m. Considering these issues, our data indicate that Ostreopsis sp. 1 in coastal environments may form blooms at ca. ∼28 m depth in regions along Japanese coasts.     

Sources of variation contributing to production and quality attributes of Kyrgyz cashmere in Osh and Naryn provinces: Implications for industry development

We aimed to quantify the sources of variation contributing to the production and quality of cashmere produced in five districts in Osh and Naryn provinces of Kyrgyzstan. In early spring 2008 mid-side cashmere samples were taken from 719 cashmere adult females, and 41 cashmere adult males and castrates. Samples came from 53 villages and a total of 156 farmers’ flocks. For 91 goats from 33 farmers in 13 villages of two districts that had been sampled earlier, cashmere was combed from the goat at the time of a second visit (end of April 2008) when the cashmere would normally be harvested. Following standard cashmere objective measurement, data were examined using general linear modelling to quantify the effects of potential determinants. The mean fibre diameter (MFD) of cashmere differed between provinces (Osh 15.7 μm, Naryn 16.7 μm; P = 4.4 × 10⁻²⁰). About 42% of the cashmere was <16 μm, 48% was 16.0–18.0 μm and 9.5% was >18.0 μm. Most of the cashmere samples were coloured (81%), with 63% black and 19% white. The percentage of cashmere samples that were white declined as MFD increased (26% < 14 μm to 11% of >18 μm). The primary determinants of cashmere MFD of individual goats were age of goat (range 1.46 μm, P = 1.8 × 10⁻¹²) and farm (range 6.5 μm, P = 1.7 × 10⁻¹⁴). The lesser effects detected for sex (range 0.9 μm, P = 0.026) and colour of cashmere (range 1.8 μm, P = 0.023) were based on small sample sizes and are unreliable. Age of goat had important affects on fibre diameter variation (up to 1.7% in coefficient of variation, P = 5.8 × 10⁻⁶) and fibre curvature (2.5–5°/mm, P = 2.1 × 10⁻⁴). By far the greatest effect on fibre curvature was cashmere MFD (P = 3.0 × 10⁻¹⁰⁴) with a smaller effect of sex (about 5°/mm, P = 3.0 × 10⁻⁶). Village effects were detected on fibre diameter variability (range 4.5% in coefficient of variation, P = 0.027) and fibre curvature (range 15°/mm, P = 1.6 × 10⁻⁷). There was a strong negative association between increasing MFD and declining fibre curvature (−5.11 ± 0.181°/mm per 1 μm; P = 7.1 × 10⁻¹²¹; r² = 0.51). Average combed cashmere weight was 164 g, the clean cashmere content was 0.661 and median clean cashmere production was 110 g per goat (range 60–351 g). Combed cashmere production increased with altitude of the village, probably related to different moulting times as spring temperatures warmed up later in higher altitude villages up to 3200 masl. Measurements of combed cashmere MFD were coarser than the mid-side samples taken earlier in the year. There are farmers and cashmere goats in the sampled districts of Kyrgyzstan which produce the finest qualities of commercial cashmere as the vast majority of cashmere is fine, has low variation in fibre diameter and has fibre crimping (curvature) typical of Chinese and Mongolian cashmere. There is substantial scope to increase the production and commercial value of cashmere produced by Kyrgyz goats. In particular, some villages and farmers need to change their buck selection practices if they wish to produce acceptable cashmere. Farmers should separate their finer and white cashmere prior to sale.     

Antifeedant activity of numb and salty taste compounds against the larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

Helicoverpa armigera, an important polyphagous insect pest in agriculture, attacks more than 200 plant species of more than 30 families. Our previous study showed that the choice feeding percentages of H. armigera larvae to tobacco leaf discs treated with sweet, bitter, and hot taste substances were higher than the control leaf discs, while numb and salty substances could significantly inhibit their feeding. To quantitatively determine the synergistic effect of numb and salty substances, in this paper, the antifeeding activities of numb and salty substances and their mixtures blended in different doses or volume ratios were assayed on H. armigera larvae. The first bioassay was designed to elucidate the relative feeding preference of the larvae to the leaves from several common host species, each paired with tobacco leaf discs, and the result indicated that the most preferred host leaf by the larvae was tobacco leaf, followed by cotton and peanut leaves, suggesting that tobacco leaf was the most suitable matrix for the antifeeding bioassay, and the larval consumption of maize, pepper, or tomato leaves were significantly lower than that of tobacco leaf. The second bioassay was to test the choice feeding response of H. armigera larvae to tobacco leaf discs treated with Zanthoxylum bungeanum extracts obtained with different solvents, and the result showed that the antifeeding activity of the alcohol extracts was the strongest (93.38%), and the leaf consumption in the treatment and the control showed extremely significant difference (t = 4.23, t_0.01 = 3.25, P = 0.0022), followed by the dichloromethane extracts (47.64%), while the other three solvents (water, acetone, and n-hexane) could not extract the active antifeeding components from Z. bungeanum. The larval consumption of tobacco leaf discs treated with the alcohol extracts of Z. bungeanum and NaCl solution were significantly less than their corresponding controls. The mean larval consumption of the treated leaf discs decreased with ever-increasing dosage, and the consumption of tobacco leaf discs coated with different doses of alcohol extracts of Z. bungeanum or NaCl solution showed extremely significant difference (F_{alcohol extract of Z. bungeanum} = 3.88, F_0.01 = 3.58, P = 0.0064; F_{NaCl solution} = 54.29, F_0.01 = 3.58, P = 0.0000), with maximum antifeeding effects at a dosage of 30 μL per 1.5 cm ID leaf disc. We further tested the larval consumption of tobacco leaf discs treated with alcohol extracts of Z. bungeanum in saturated NaCl solution mixed in different volume ratios, and the result showed that the choice antifeeding percentages of the treatments with 15 μL or more Z. bungeanum alcohol extracts were higher than 90%, among which the mixture with 25:15 volume ratio of Z. bungeanum alcohol extracts and saturated NaCl solution exhibited the strongest antifeeding activity, and the mean consumed leaf area of tobacco leaf discs coated with this blend was only 0.10 mm². In the further test on feeding dose-response of the 25:15 mixture, the mean leaf consumption decreased linearly with ever-increasing dosage, with a regression equation y = ?3.9356x + 120.78(R² = 0.9998), and the 30 μL dose could completely inhibit H. armigera feeding.     

Synthesis,biological evaluation of novel 4,5-dihydro-2H-pyrazole 2-hydroxyphenyl derivatives as BRAF inhibitors

A series of novel 4,5-dihydropyrazole derivatives (3a–3t) containing hydroxyphenyl moiety as potential V600E mutant BRAF kinase (BRAF^V600E) inhibitors were designed and synthesized. Docking simulation was performed to insert compounds 3d (1-(5-(5-chloro-2-hydroxyphenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone) and 3m (1-(3-(4-chlorophenyl)-5-(3,5-dibromo-2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone) into the crystal structure of BRAF^V600E to determine the probable binding model, respectively. Based on the preliminary results, compound 3d and 3m with potent inhibitory activity in tumor growth may be a potential anticancer agent. Results of the bioassays against BRAF^V600E, MCF-7 human breast cancer cell line and WM266.4 human melanoma cell line all showed several compounds had potent activities IC₅₀ value in low micromolar range, among them, compound 3d and compound 3m showed strong potent anticancer activity, which were proved by that 3d: IC₅₀ = 1.31 μM for MCF-7 and IC₅₀ = 0.45 μM for WM266.5, IC₅₀ = 0.22 μM for BRAF^V600E, 3m: IC₅₀ = 0.97 μM for MCF-7 and IC₅₀ = 0.72 μM for WM266.5, IC₅₀ = 0.46 μM for BRAF^V600E, which were comparable with the positive control Erlotinib.     

Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass

A highly efficient laccase-producing fungus was isolated from soil and identified as Coltricia perennis SKU0322 by its morphology and by comparison of its internal transcribed spacer (ITS) rDNA gene sequence. Extracellular laccase (Cplac) from C. perennis was purified to homogeneity by anion-exchange and gel filtration chromatography. Cplac is a monomeric glycoprotein with 12% carbohydrate content and a molecular mass of 66 kDa determined by polyacrylamide-gel electrophoresis. Ultraviolet-visible absorption spectroscopy observed type 1 and type 3 copper signals from Cplac. The enzyme acted optimally at pH 3–4 and 75 °C. Its optimal activity was with 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS), it also oxidized various lignin-related phenols. The enzyme was characterized as a multi-copper blue laccase by its substrate specificity and internal amino acid sequence. It showed a higher catalytic efficiency towards ABTS (k_cat/K_m = 18.5 s^?1 μM^?1) and 2,6-dimethoxyphenol (k_cat/K_m = 13.9 s^?1 μM^?1) than any other reported laccase. Its high stability and catalytic efficiency suggest its suitability for industrial applications: it detoxified phenolic compounds in acid-pretreated rice straw and enhanced saccharification yield.     

Molecular cloning and functional analysis of the ortho-hydroxylases of p-coumaroyl coenzyme A/feruloyl coenzyme A involved in formation of umbelliferone and scopoletin in sweet potato,Ipomoea batatas (L.) Lam.

Ortho-hydroxylation of cinnamates is a key step in coumarin biosynthesis in plants. Ortho-hydroxylated cinnamates undergo trans/cis isomerization of the side-chain and then lactonization to form coumarins. Sweet potato [Ipomoea batatas (L.) Lam.] accumulates umbelliferone and scopoletin after biotic and abiotic stresses. To elucidate molecular aspects of ortho-hydroxylation involved in umbelliferone formation in sweet potato, isolation and characterization of cDNAs encoding 2-oxoglutarate-dependent dioxygenases (2OGD) was performed from sweet potato tubers treated with a chitosan elicitor. Five cDNAs (designated as Ib) encoding a protein of 358 amino acid residues were cloned, and these were categorized into two groups, Ib1 and Ib2, based on their amino acid sequences. Whether the recombinant Ib proteins had any enzymatic activity toward cinnamates was examined. Ib1 proteins exhibited ortho-hydroxylation activity toward feruloyl coenzyme A (CoA) to form scopoletin (K_m = ∼10 μM, k_cat = ∼2.7 s⁻¹). By contrast, Ib2 proteins catalyzed ortho-hydroxylation of feruloyl-CoA (K_m = 7.3–14.0 μM, k_cat = 0.28–0.55 s⁻¹) and also of p-coumaroyl-CoA (K_m = 6.1–15.2 μM, k_cat = 0.28–0.64 s⁻¹) to form scopoletin and umbelliferone, respectively. Fungal and chitosan treatments increased levels of umbelliferone and its glucoside (skimmin) in the tubers, and expression of the Ib2 gene was induced concomitantly.     

The harmful alga Aureococcus anophagefferens utilizes 19′-butanoyloxyfucoxanthin as well as xanthophyll cycle carotenoids in acclimating to higher light intensities

Aureococcus anophagefferens is a picoplanktonic microalga that is very well adapted to growth at low nutrient and low light levels, causing devastating blooms (“brown tides”) in estuarine waters. To study the factors involved in long-term acclimation to different light intensities, cells were acclimated for a number of generations to growth under low light (20 μmol photons m^? 2 s^? 1), medium light (60 or 90 μmol photons m^? 2 s^? 1) and high light (200 μmol photons m^? 2 s^? 1), and were analyzed for their contents of xanthophyll cycle carotenoids (the D pool), fucoxanthin and its derivatives (the F pool), Chls c₂ and c₃, and fucoxanthin Chl a/c polypeptides (FCPs). Higher growth light intensities resulted in increased steady state levels of both diadinoxanthin and diatoxanthin. However, it also resulted in the conversion of a significant fraction of fucoxanthin to 19′-butanoyloxyfucoxanthin without a change in the total F pool. The increase in 19′-butanoyloxyfucoxanthin was paralleled by a decrease in the effective antenna size, determined from the slope of the change in F₀ as a function of increasing light intensity. Transfer of acclimated cultures to a higher light intensity showed that the conversion of fucoxanthin to its derivative was a relatively slow process (time-frame of hours). We suggest the replacement of fucoxanthin with the bulkier 19′-butanoyloxyfucoxanthin results in a decrease in the light-harvesting efficiency of the FCP antenna and is part of the long-term acclimative response to growth at higher light intensities.     

Differences in the photoacclimation and photoprotection exhibited by two species of the ciguatera causing dinoflagellate genus,Gambierdiscus

In culture, Gambierdiscus spp. have been shown to prefer irradiances that are relatively low (≤250 μmol photons m⁻² s⁻¹) versus those to which they are frequently exposed to in their natural environment (>500 μmol photons m⁻² s⁻¹). Although several behavioral strategies for coping with such irradiances have been suggested, it is unclear as to how these dinoflagellates do so on a physiological level. More specifically, how do long term exposures (30 days) affect cell size and cellular chlorophyll content, and what is the photosynthetic response to short term, high irradiance exposures (up to 1464 μmol photons m⁻² s⁻¹)? The results of this study reveal that cell size and chlorophyll content exhibited by G. carolinianus increased with acclimation to increasing photon flux density. Additionally, both G. carolinianus and G. silvae exhibited reduced photosynthetic efficiency when acclimated to increased photon flux density. Photosynthetic yield exhibited by G. silvae was greater than that for G. carolinianus across all acclimation irradiances. Although such differences were evident, both G. carolinianus and G. silvae appear to have adequate biochemical mechanisms to withstand exposure to irradiances exceeding 250 μmol photons m⁻² s⁻¹ for at least short periods of time following acclimation to irradiances of up to 150 μmol photons m⁻² s⁻¹.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

Design and synthesis of a β-lactamase activated 5-fluorouracil prodrug

An efficient synthesis of a 5-fluorouracil-cephalosporin prodrug is described for use against colorectal and other cancers in antibody and gene-directed therapies. The compound shows stability in aqueous media until specifically activated by β-lactamase (βL). The kinetic parameters of the 5-fluorouracil-cephalosporin conjugate were determined in the presence of Enterobacter cloacae P99 βL (ECl βL) revealing a K_m = 95.4 μM and V_max = 3.21 μMol min^?1 mg^?1. The data compare favorably to related systems that have been reported and enable testing of this prodrug against cancer cell lines in vitro and in vivo.     

Improved retrieval of Secchi depth for optically-complex waters using remote sensing data

Water transparency is one of the ecological indicators for describing water quality and the underwater light field which determines its productivity. In the European Water Framework Directive (WFD) as well as in the European Marine Strategy Framework Directive (MSFD) water transparency is used for ecological status classification of inland, coastal and open sea waters and it is regarded as an indicator for eutrophication in Baltic Sea management (HELCOM, 2007). We developed and compared different empirical and semi-analytical algorithms for lakes and coastal Nordic waters to retrieve Secchi depth (Z_SD) from remote sensing data (MERIS, 300 m resolution). The algorithms were developed in water bodies with high coloured dissolved organic matter absorption (a_CDOM(442) ranging 1.7–4.0 m⁻¹), Chl a concentration (0.5–73 mg m⁻³) and total suspended matter (0.7–37.5 g m⁻³) and validated against an independent data set over inland and coastal waters (0.6 m < Z_SD < 14.8 m). The results indicate that for empirical algorithms, using longer wavelengths in the visible spectrum as a reference band decreases the RMSE and increases the coefficient of determination (R²). The accuracy increased (R² = 0.75, RMSE = 1.33 m, n = 134) when Z_SD was retrieved via an empirical relationship between Z_SD and K_d(490). The best agreement with in situ data was attained when Z_SD was calculated via both the diffuse and the beam attenuation coefficient (R² = 0.89, RMSE = 0.77 m, n = 89). The results demonstrate that transparency can be retrieved with high accuracy over various optical water types by the means of ocean color remote sensing, improving both the spatial and temporal coverage. The satellite derived Z_SD product could be therefore used as an additional source of information for WFD and MSFD reporting purposes.     

Liquid chromatography–tandem mass spectrometry assay for the quantification of free and total sialic acid in human cerebrospinal fluid

BackgroundAnalysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography–tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was validated.MethodsThe method utilized a simple sample-preparation procedure of protein precipitation for FSA and acid hydrolysis for TSA. Negative electrospray ionisation was used to monitor the transitions m/z 308.2 → 87.0 (SA) and m/z 311.2 → 90.0 (¹³C₃-SA). Conjugated sialic acid (CSA) was calculated by subtracting FSA from TSA. We established reference intervals for FSA, TSA and CSA in CSF in 217 control subjects. The method has been applied to patients’ samples with known differences in SA metabolites like meningitis (n = 6), brain tumour (n = 2), leukaemia (n = 5), and Salla disease (n = 1).ResultsLimit of detection (LOD) was 0.54 μM for FSA and 0.45 μM for TSA. Intra- and inter-assay variation for FSA (21.8 μM) were 4.8% (n = 10) and 10.4% (n = 40) respectively. Intra- and inter-assay variation for TSA (35.6 μM) were 9.7% (n = 10) and 12.8% (n = 40) respectively. Tested patients showed values of TSA above established reference value.ConclusionThe validated method allows sensitive and specific measurement of SA metabolites in CSF and can be applied for clinical diagnoses.     

Target Binding to S100B Reduces Dynamic Properties and Increases Ca2 +-Binding Affinity for Wild Type and EF-Hand Mutant Proteins

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca^2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With ^D63NS100B as an exception (^D63NK_D = 50 ± 9 μM), Ca^2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (^D61NK_D = 412 ± 67 μM, ^D65NK_D = 968 ± 171 μM, and ^E72AK_D = 471 ± 133 μM), when compared to wild-type protein (^WTK_D = 56 ± 9 μM). For the TRTK-12 complexes, the Ca^2 +-binding affinity to wild type (^WT + TRTKK_D = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (^{D61N + TRTK}K_D = 29 ± 1.2 μM, ^{D63N + TRTK}K_D = 10 ± 2.2 μM, ^{D65N + TRTK}K_D = 73 ± 4.4 μM, and ^{E72A + TRTK}K_D = 18 ± 3.7 μM). In addition, R_ex, as measured using relaxation dispersion for side‐chain ¹⁵N resonances of Asn63 (^D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, ^D61NS100B, ^D63NS100B, and ^D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca^2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) ^D63NS100B showed no change in Ca^2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca^2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca^2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.     

Effects of flooding on photosynthesis and root respiration in saltcedar (Tamarix ramosissima), an invasive riparian shrub

The introduced shrub Tamarix ramosissima invades riparian zones, but loses competitiveness under flooding. Metabolic effects of flooding could be important for T. ramosissima, but have not been previously investigated. Photosynthesis rates, stomatal conductance, internal (intercellular) CO₂, transpiration, and root alcohol dehydrogenase (ADH) activity were compared in T. ramosissima across soil types and under drained and flooded conditions in a greenhouse. Photosynthesis at 1500 μmol quanta m⁻² s⁻¹ (A₁₅₀₀) in flooded plants ranged from 2.3 to 6.2 μmol CO₂ m⁻² s⁻¹ during the first week, but A₁₅₀₀ increased to 6.4–12.7 μmol CO₂ m⁻² s⁻¹ by the third week of flooding. Stomatal conductance (g_s) at 1500 μmol quanta m⁻² s⁻¹ also decreased initially during flooding, where g_s was 0.018 to 0.099 mol H₂O m⁻² s⁻¹ during the first week, but g_s increased to 0.113–0.248 mol H₂O m⁻² s⁻¹ by the third week of flooding. However, photosynthesis in flooded plants was reduced by non-stomatal limitations, and subsequent increases indicate metabolic acclimation to flooding. Root ADH activities were higher in flooded plants compared to drained plants, indicating oxygen stress. Lower photosynthesis and greater oxygen stress could account for the susceptibility of T. ramosissima at the onset of flooding. Soil type had no effect on photosynthesis or on root ADH activity. In the field, stomatal conductance, leaf water potential, transpiration, and leaf δ¹³C were compared between T. ramosissima and other flooded species. T. ramosissima had lower stomatal conductance and water potential compared to Populus deltoides and Phragmites australis. Differences in physiological responses for T. ramosissima could become important for ecological concerns.     

Effects of pellet morphology on broth rheology in fermentations of Aspergillus terreus

Effects of pellet morphology on broth rheology are reported for pelleted submerged cultures of the lovastatin producing filamentous fungus Aspergillus terreus, growing in fluidized bed and stirred tank bioreactors. The pellet diameter and compactness were affected by the agitation intensity of the broth; however, the total biomass productivity was not affected. In fluidized beds and stirred tanks with agitation intensity of up to 300 rpm (impeller tip speed of 1.02 m s⁻¹), the fungal pellets were stable at diameters of up to about 2300 μm. In more intensely agitated stirred tanks (≥600 rpm; impeller tip speed of ≥2.03 m s⁻¹), the stable pellet size was only about ≤900 μm. The biomass concentration and the pellet diameter were the main factors that influenced the flow index and the consistency index of the power-law broths. Because the biomass productivity was the same in all experiments in a given type of reactor and the oxygen concentration was kept at ∼400% of air saturation, the pellet size and morphology were not influenced by oxygen mass transfer effects. Pellets were always dense in the core region and no necrosis of the biomass occurred.