Transcriptomic and physiological analyses of the dinoflagellate Karenia mikimotoi reveal non‐alkaline phosphatase‐based molecular machinery of ATP utilisation

The ability to utilize dissolved organic phosphorus (DOP) is important for phytoplankton to survive the scarcity of dissolved inorganic phosphorus (DIP), and alkaline phosphatase (AP) has been the major research focus as a facilitating mechanism. Here, we employed a unique molecular ecological approach and conducted a broader search for underpinning molecular mechanisms of adenosine triphosphate (ATP) utilisation. Cultures of the dinoflagellate Karenia mikimotoi were set up in L1 medium (+P), DIP‐depleted L1 medium (–P) and ATP‐replacing‐DIP medium (ATP). Differential gene expression was profiled for ATP and +P cultures using suppression subtractive hybridisation (SSH) followed by 454 pyrosequencing, and RT‐qPCR methods. We found that ATP supported a similar growth rate and cell yield as L1 medium and observed DIP release from ATP into the medium, suggesting that K. mikimotoi cells were expressing extracellular hydrolases to hydrolyse ATP. However, our SSH, qPCR and enzymatic activity assays indicated that 5′‐nucleotidase (5NT), rather than AP, was responsible for ATP hydrolysis. Further gene expression analyses uncovered that intercellular purine metabolism was significantly changed following the utilisation of ATP. Our findings reveal a multi‐faceted machinery regulating ATP utilisation and P metabolism in K. mikimotoi, and underscore AP activity is not the exclusive indicator of DOP utilisation.     

Species-dependent effects of seawater acidification on alkaline phosphatase activity in dinoflagellates

Increases of atmospheric CO₂ cause ocean acidification (OA) and global warming, the latter of which can stratify the water column and impede nutrient supply from deep water. Phosphorus (P) is an essential nutrient for phytoplankton to grow. While dissolved inorganic phosphorus (DIP) is the preferred form of P, phytoplankton have evolved alkaline phosphatase (AP) to utilize dissolved organic phosphorus (DOP) when DIP is deficient. Although the function of AP is known to require pH > 7, how OA affects AP activity and hence the capacity of phytoplankton to utilize DOP is poorly understood. Here, we examined the effects of pH conditions (5.5–11) on AP activity from six species of dinoflagellates, an important group of marine phytoplankton. We observed a general pattern that AP activity declined sharply at pH 5.5, peaked between pH 7 and 8, and dropped at pH > 8. However, our data revealed remarkable interspecific variations in optimal pH and niche breadth of pH. Among the species examined, Fugacium kawagutii and Prorocentrum cordatum had an optimal pH at 8, and Alexandrium pacificum, Amphidinium carterae, Effrenium voratum, and Karenia mikimotoi showed an optimal pH of 7. However, whereas A. pacificum and K. mikimotoi had the broadest pH niche for AP (7–10) and F. kawagutii the second (8–10), Am. carterae, E. voratum, and P. cordatum exhibited a narrow pH range. The response of Am. carterae AP to pH changes was verified using purified AP heterologously expressed in Escherichia coli. These results in concert suggest OA will likely differentially impact the capacity of different phytoplankton species to utilize DOP in the projected more acidified and nutrient-limited future ocean.     

Detrimental impacts of the dinoflagellate Karenia mikimotoi in Fujian coastal waters on typical marine organisms

Blooms of the toxic dinoflagellate Karenia mikimotoi (K. mikimotoi) have occurred frequently in the East China Sea in recent decades and were responsible for massive mortalities of abalones in Fujian coastal areas in 2012, however, little is known about the effects of these blooms on other marine organisms. In this study, the toxic effects and the possible mechanisms of toxicity of K. mikimotoi from Fujian coastal waters on typical marine organisms at different trophic levels, including zooplankton (Brachionus plicatilis, Artemia salina, Calanus sinicus, and Neomysis awatschensis) and aquaculture species (Penaeus vannamei and Scophthalmus maximus) were investigated. At a bloom density of 3 × 10⁴ cells/mL, the Fujian strain of K. mikimotoi significantly affected the tested organisms, which had mortality rates at 96 h of 100, 23, 20, 97, 33, and 53%, respectively. Moreover, the intact cell suspension was toxic to all tested species, whereas cell-free culture and the ruptured cell suspension had no significant effects on the tested organisms. Possible mechanisms for this toxic effect, including reactive oxygen species (ROS) and hemolytic toxins, were evaluated. For K. mikimotoi, 0.014 ± 0.004 OD/(10⁴ cells) superoxide (O₂⁻) and 3.00 ± 0.00 nmol/(10⁴ cells) hydrogen peroxide (H₂O₂) were measured, but hydrogen peroxide did not affect rotifers at that concentration, and rotifers were not protected from the lethal effects of K. mikimotoi when the enzymes superoxide dismutase and catalase were added to counteract the ROS. The lipophilic extract of K. mikimotoi had a hemolytic effect on rabbit erythrocytes but exhibited no significant toxicity. These results suggest that this strain of K. mikimotoi can have detrimental effects on several typical marine organisms and that its toxicity may be associated with intact cells but is not related to ROS or hemolytic toxins.     

Allelopathic interactions between the macroalga Hizikia fusiformis (Harvey) and the harmful blooms-forming dinoflagellate Karenia mikimotoi

The effects of algal blooms on seaweeds have been rarely studied, although harmful algal blooms (HABs) are now normally regarded as worldwide incidents. In the present study, the effects of dense Karenia mikimotoi cells on the growth and photosynthesis of Hizikia fusiformis, a common and commercially cultivated macroalga in coastal waters of the East China Sea (ECS), were studied to understand the possible consequences when the mariculture encountered a dense harmful algal bloom. Furthermore, the counteraction of the latter on the growth and photosynthetic activities of K. mikimotoi was determined to evaluate the contribution of H. fusiformis commercial cultivation to environmental improvements. The results showed that the chlorophyll a (Chl a) contents, maximal photochemical efficiency (F_v/F_m) and relative electron transfer rate (rETR) of gas vesicles (specialized leaves), adult and young receptacles of H. fusiformis were all significantly (P < 0.05) inhibited compared with the mono-cultured ones. When compared with mono-cultured H. fusiformis (without K. mikimotoi), the Chl a contents in gas vesicles, adult and young receptacles decreased by 20.6%, 17.6% and 33.2% within 2 weeks. Correspondingly, the F_v/F_m decreased by 7.9%, 37.4% and 43.7%; the apparent photosynthetic efficiency (α) decreased by 9.4%, 47.1% and 48.3%; and rETR decreased by 19.5%, 52.6% and 68.2%, respectively. The Chl a concentration of the mono-cultured K. mikimotoi (without H. fusiformis) increased to 2247.97 μg l⁻¹ from 958.11 μg l⁻¹ within 14 d. Those of the co-cultivated ones (with H. fusiformis), however, increased to 1591.31 μg l⁻¹ on the 8th day and then decreased rapidly to 254.99 (±37.73) μg l⁻¹ after the next 6 days. Furthermore, compared with the mono-cultured K. mikimotoi cells, the F_v/F_m, α and rETR_max of co-cultivated ones decreased by 9.4%, 36.3% and 30.6%, respectively. The results indicated that the mature sporophytes of H. fusiformis were resistant to dense K. mikimotoi blooms and this resistance was organ-dependent as: gas vesicle > adult receptacles > young receptacles. On the other hand, commercial mariculture of H. fusiformis demonstrated the potential of preventing the occurrence of algal blooms.     

Potential impact of an exceptional bloom of Karenia mikimotoi on dissolved oxygen levels in waters off western Ireland

In the summer of 2005 an exceptional bloom of the dinoflagellate Karenia mikimotoi occurred along Ireland's Atlantic seaboard and was associated with the mass mortality of both benthic and pelagic marine life. Oxygen depletion, cellular toxicity and physical smothering, are considered to be the main factors involved in mortality. In this paper we use a theoretical approach based on stoichiometry (the Anderson ratio) and an average K. mikimotoi cellular carbon content of 329 pg C cell⁻¹ (n = 20) to calculate the carbonaceous and nitrogenous oxygen demand following bloom collapse. The method was validated against measurements of biochemical oxygen demand and K. mikimotoi cell concentration. The estimated potential oxygen utilisation (POU) was in good agreement with field observations across a range of cell concentrations. The magnitude of POU following bloom collapse, with the exception of three coastal areas, was considered insufficient to cause harm to most marine organisms. This indicates that the widespread occurrence of mortality was primarily due to other factors such as cellular toxicity and/or mucilage production, and not oxygen depletion or related phenomena. In Donegal Bay, Kilkieran Bay and inner Dingle Bay, where cell densities were in the order of 10⁶ cells L⁻¹, estimated POU was sufficient to cause hypoxia. Of the three areas, Donegal Bay is considered to be the most vulnerable due to its hydrographic characteristics (seasonally stratified, weak residual flow) and hypoxic conditions (2.2 mg L⁻¹ O₂) were directly observed in the Bay post bloom collapse. Here, depending on the time of bloom collapse, depressed DO levels could persist for weeks and continue to have a potentially chronic impact on the Bay.     

Efectos del tratamiento con ácido zoledrónico en pacientes adultos con osteogénesis imperfecta

Background and objectiveOsteogenesis imperfecta (OI) is a genetic disorder that results in bone fragility. Several studies have demonstrated the effectiveness of bisphosphonate therapy. The aim of this study was to evaluate the effects of intravenous zoledronic acid on bone mineral density (BMD) and biochemical markers of bone turnover in adults with OI.Material and methodsWe carried out a prospective non-randomized study in patients with osteoporosis or severe osteopenia (T score <?2) related to OI and intolerance or contraindication to oral bisphosphonates. The patients were treated with a zoledronic acid infusion every 6 months. Densitometry was carried out annually. Calcium (Ca), phosphate (P), intact parathormone (PTH), 25 hydroxyvitamin D and biochemical markers of bone turnover [bone alkaline phosphatase (BAP), beta-cross-laps (CTX) and urinary deoxypyridoxine (DOP)] were measured every year. Adverse events and new fractures were registered.ResultsTen patients (2 men and 8 women) were treated. Treatment increased BMD measured in the lumbar spine after 24 (0.738±0.141 vs 0.788±0.144 g/cm²; p=0.048) and 36 months (0.720±0.139 vs 0.820±0.128; p=0.01). Significant increases in BMD were also observed after 24 months in the femoral neck (0.677±0.121 vs 0.703±0.122 g/cm²; p<0.016). Serum Ca, P, BAP and CTX concentrations remained unchanged. PTH concentrations increased and vitamin D concentrations decreased after 36 months of treatment. DOP excretion decreased significantly after 24 months. Seven patients had mild influenza-like symptoms occurring within the first 24 h after the first infusion. No severe adverse events were observed. None of the patients had new fractures.ConclusionZoledronic acid seems to be a safe and effective treatment option in adults with osteoporosis related to OI.     

Molecular ecological responses of the dinoflagellate Karenia mikimotoi to phosphate stress

Karenia mikimotoi is a toxic, widespread dinoflagellate which could produce hemolytic toxins and ichthyotoxins affecting fisheries within the area of its bloom. Previous ecophysiological studies indicated that the enhance of environmental phosphate concentration could promote the growth of K. mikimotoi. Intrinsic mechanisms regarding the effects of external phosphate on its photosynthesis, cell cycle succession and differential proteins’ expressions are still unknown. K. mikimotoi was cultured in phosphate-deprived medium, while the culture in f/2 medium (Guillard, 1975) was introduced as phosphate-sufficient control experiment. Cell counts and phosphate concentration detection were performed every other day. Flowcytometry was applied to measure cell cycle succession and chlorophyll fluorescence intensity fluctuation. Differential proteomics expression was examined by SDS-PAGE tandem LTQ Orbitrap MS/MS spectrometry. Functions of each differential protein were searched within NCBInr protein database and Swissprot database. Our study demonstrated that phosphate stress inhibited growth and cell cycle succession of K. mikimotoi remarkably (p < 0.01). Algal chlorophyll fluorescence intensity was significantly affected by phosphate deprivation (p < 0.05). 11 species of differential proteins were detected only in phosphate-limited culture sample which related to stress signal transduction, vacuolar phosphate release, phospholipid degradation, organic acid synthesis and phagotrophy. 4 kinds of differential proteins were identified only in f/2 medium culture sample which referred to cell proliferation, glycolysis, SAM cycle and polyamine production. Based on analysis of differential proteomic functional annotation, we hypothesized proteomic response mechanism of K. mikimotoi to phosphate stress. Molecular biological responses of dinoflagellate K. mikimotoi to phosphate stress was explored.     

Synthesis and evaluation of benzo[b]thiophene derivatives as inhibitors of alkaline phosphatases

Presence of basic calcium phosphate in knee joints of osteoarthritis patients could be prevented by inhibiting tissue non-specific alkaline phosphatase (TNAP) activity. Levamisole or the L stereoisomer of tetramisole (a known TNAP inhibitor) has been used as a treatment for curing rheumatoid arthritis but its therapeutical use is limited due to side effects. We report the synthesis and the TNAP inhibition property of benzo[b]thiophene derivatives, among which benzothiopheno-tetramisole and benzothiopheno-2,3-dehydrotetramisole, which could be involved in a drug therapy for osteoarthritis. Two water soluble racemic benzothiopheno-tetramisole and -2,3-dehydrotetramisole with apparent inhibition constants K_i = 85 ± 6 μM and 135 ± 3 μM (n = 3) comparable to that of enantiomeric levamisole 93 ± 4 μM were found. Several novel derivatives showed more pronounced inhibition properties towards intestinal alkaline phosphatase than TNAP.     

Novel triclabendazole prodrug: A highly water soluble alternative for the treatment of fasciolosis

In this work we present the synthesis, aqueous solubility and stability, hydrolysis by alkaline phosphatase, and in vivo fasciolicidal activity in sheep of a highly water soluble phosphate salt prodrug of triclabendazole (MFR-5). The aqueous solubility of MFR-5 at pH 7 was 88,000-fold that of triclabendazole. MFR-5 showed excellent aqueous stability (>95% after 26 h) at pH 7, making it ideal for developing pharmaceutical compositions in the form of solutions that can easily be hydrolyzed by the enzyme alkaline phosphatase (t = 13.6 s) to liberate the precursor compound. An aqueous solution of MFR-5 administered intramuscularly to sheep at concentrations of 4, 6 and 8 mg/kg presented a fasciolicidal efficiency of 96.5%, 98.4% and 99.2%, respectively. In the in vivo experiments, MFR-5 reduced 100% the excretion of eggs in all of the above concentrations.     

Effect of tertiary sewage effluent additions on Prymnesium parvum cell toxicity and stable isotope ratios

We investigated the ability of the ichthyotoxic haptophyte Prymnesium parvum to use sewage-originated nutrients applying stable carbon (C) and nitrogen (N) isotope techniques. P. parvum was cultured under N and phosphorus (P) sufficient and deficient conditions in either sewage effluent-based medium or in a nitrate- and phosphate-based control. Cell densities and toxicities were monitored and stable carbon N isotopes signatures (δ¹³C and δ¹⁵N) of P. parvum and the sewage effluent analysed. Nitrogen and P sufficient cultures achieved the highest biomass followed by P and N deficient cultures, regardless of sewage effluent additions. The P deficient cultures with sewage effluent had higher toxicity, estimated as haemolytic activity (9.4 ± 0 × 10^?5 mg Saponin equiv. cell^?1) compared to the P deficient control and to all N deficient and NP sufficient cultures. Nutrient deficient conditions had no effect on the cell δ¹⁵N, but a decreasing effect on δ¹³C in the inorganic N deficient treatment. Growth in sewage-based media was followed by a substantial increase in the cell δ¹⁵N (10.4–16.1‰) compared to the control treatments (2.4–4.9‰), showing that P. parvum is capable of direct use of sewage-originated N, inorganic as well as organic. Uptake of terrestrial derived C in the sewage treatments was confirmed by a decrease in cell δ¹³C, implying that P. parvum is able to utilize organic nutrients in sewage effluent.     

Protective effect of Piper betle leaf extract against cadmium-induced oxidative stress and hepatic dysfunction in rats

The present study was undertaken to examine the attenuative effect of Piper betle leaf extract (PBE) against cadmium (Cd) induced oxidative hepatic dysfunction in the liver of rats. Pre-oral supplementation of PBE (200 mg/kg BW) treated rats showed the protective efficacy against Cd induced hepatic oxidative stress. Oral administration of Cd (5 mg/kg BW) for four weeks to rats significantly (P > 0.05) elevated the level of serum hepatic markers such as serum aspartate transaminase (AST), serum alanine transaminase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (GGT), bilirubin (TBRNs), oxidative stress markers viz., thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH), protein carbonyls (PC) and conjugated dienes (CD) and significantly (P > 0.05) reduced the enzymatic antioxidants viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) and non-enzymatic antioxidants Viz., reduced glutathione (GSH), total sulfhydryls (TSH), vitamin C and vitamin E in the liver. Pre-oral supplementation of PBE (200 mg/kg BW) in Cd intoxicated rats, the altered biochemical indices and pathological changes were recovered significantly (P > 0.05) which showed ameliorative effect of PBE against Cd induced hepatic oxidative stress. From the above findings, we suggested that the pre-administration of P. betle leaf extract exhibited remarkable protective effects against cadmium-induced oxidative hepatic injury in rats.     

Accumulation of phycocyanin in heterotrophic and mixotrophic cultures of the acidophilic red alga Galdieria sulphuraria

The relationship between light intensity, nitrogen availability and pigmentation was investigated in mixotrophic and heterotrophic cultures of the unicellular red alga Galdieria sulphuraria 074G, a potential host for production of the blue pigment, phycocyanin (PC). During the exponential growth phase of batch cultures, G. sulphuraria 074G contained 2–4 mg phycocyanin per g dry weight. In carbon-limited and nitrogen-sufficient batch cultures grown in darkness, this value increased to 8–12 mg g⁻¹ dry weight during the stationary phase, whereas the phycocyanin content in nitrogen-deficient cells decreased to values below 1 mg g⁻¹ dry weight during stationary phase. Light intensities between 0 and 100 μmol photons m⁻² s⁻¹ had no influence on phycocyanin accumulation in mixotrophic cultures grown on glucose or fructose, while light stimulated phycocyanin synthesis in cultures grown on glycerol, in which the phycocyanin content in stationary phase was increased from 10 mg g⁻¹ dry weight in darkness to 20 mg g⁻¹ dry weight at a light intensity of 80 μmol photons m⁻² s⁻¹. At higher light intensities, less phycocyanin accumulated than at lower intensities, irrespective of the carbon substrate used. In carbon-limited continuous flow cultures grown on glucose or glycerol at a dilution rate of 0.63 day⁻¹, corresponding to 50% of the maximum specific growth rate, the highest steady-state phycocyanin content of 15–28 mg g⁻¹ dry weight was found at 65 μmol photons m⁻² s⁻¹. In contrast to the apparent glucose repression of light-induced PC synthesis observed in batch cultures, no glucose repression of the light stimulation was observed in continuous flow cultures because the glucose concentration in the culture supernatant always remained at limiting levels. Despite the fact that G. sulphuraria 074G contains less phycocyanin than some other microalgae and cyanobacteria, the ability of G. sulphuraria 074G to grow and synthesize phycocyanin in heterotrophic or mixotrophic cultures makes it an interesting alternative to the cyanobacterium, Spirulina platensis presently used for synthesis of phycocyanin.     

16-Bromoepiandrosterone,an activator of the mammalian immune system,inhibits glucose 6-phosphate dehydrogenase from Trypanosoma cruzi and is toxic to these parasites grown in culture

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the pentose-phosphate pathway which supplies cells with ribose 5-phosphate (R5P) and NADPH. R5P is the precursor for the biosynthesis of nucleotides while NADPH is the cofactor of several dehydrogenases acting in a broad range of biosynthetic processes and in the maintenance of the cellular redox state. RNA interference-mediated reduction of G6PDH levels in bloodstream-form Trypanosoma brucei validated this enzyme as a drug target against Human African Trypanosomiasis. Dehydroepiandrosterone (DHEA), a human steroidal pro-hormone and its derivative 16α-bromoepiandrosterone (16BrEA) are uncompetitive inhibitors of mammalian G6PDH. Such steroids are also known to enhance the immune response in a broad range of animal infection models. It is noteworthy that the administration of DHEA to rats infected by Trypanosoma cruzi, the causative agent of Human American Trypanosomiasis (also known as Chagas’ disease), reduces blood parasite levels at both acute and chronic infection stages. In the present work, we investigated the in vitro effect of DHEA derivatives on the proliferation of T. cruzi epimastigotes and their inhibitory effect on a recombinant form of the parasite’s G6PDH (TcG6PDH). Our results show that DHEA and its derivative epiandrosterone (EA) are uncompetitive inhibitors of TcG6PDH, with K_i values of 21.5 ± 0.5 and 4.8 ± 0.3 μM, respectively. Results from quantitative inhibition assays indicate 16BrEA as a potent inhibitor of TcG6PDH with an IC₅₀ of 86 ± 8 nM and those from in vitro cell viability assays confirm its toxicity for T. cruzi epimastigotes, with a LD₅₀ of 12 ± 8 μM. In summary, we demonstrated that, in addition to host immune response enhancement, 16BrEA has a direct effect on parasite viability, most likely as a consequence of TcG6PDH inhibition.     

Stratification of alkaline phosphatase in sediments of two urban lakes and its effect on phosphorus cycle

Phosphorus loadings in sediments play an important role in lake eutrophication and the progress of its recovery. The phosphorus release is controlled by physical, chemical and biological mechanisms. Alkaline phosphatase catalyzes remineralization of organic phosphorus and then it may be an important factor accelerating phosphorus cycling in sediments. In this paper, distributions, properties and function of alkaline phosphatase with depths in sediments of two urban lakes were discussed. Alkaline phosphatase activity (APA) in the sediments of Lake Yuehu decreased with the sediment depth. APA in sediments of Lake Yuehu was, mostly, inhibited by Phe and L-Cys; and inhibiting ability of Phe could be stronger than L-Cys. APA in deeper layer (20–30 cm) of sediments was more sensitive to the inhibitors than other layers, but range of variation in APA was most wide in the subsurface layer (10–20 cm). All the facts implied that alkaline phosphatase occurred in various forms (isoenzymes). APA in the sediments with different depths of Lake Donghu responded Phe differently. Reacted with Phe and incubated for 1 day, the amounts of SRP released by these sediments varied correspondingly. SRP on the overlying water in deeper layers (5–10 cm and 15–20 cm) of Site T1 was higher than that in surface layer (0–5 cm) of the same site, 1 day after incubation. Hence, the SRP release resulted, at least partially, from the hydrolysis of some liable organic phosphorus mediated by APA. Alkaline phosphatase in lake sediments plays an important role in the release of internal phosphorus loadings and eutrophication. A possible explanation for the sensitivity at deeper layers could be another active region of hydrolysis by alkaline phosphatase from organic phosphorus, which added a new dimension in phosphorus cycling mediated by some biochemical mechanisms.     

Inhibitory effects of Chinese traditional herbs and herb-modified clays on the growth of harmful algae,Phaeocystis globosa and Prorocentrum donghaiense

In order to develop an improved method of mitigating harmful algal blooms (HABs), we assessed the inhibitory effects of five Chinese traditional herbs, Andrographis paniculata (Burm.f.) Nees, Galla chinensis, Punica granatum L., Cortex phellodendri chinensis and Radix scutellariae, as well as clay modified with herb extract on the growth of two harmful algae, Phaeocystis globosa and Prorocentrum donghaiense. The results showed that the five Chinese herbs had varying effects on the target microalgae and inhibitory rates ranged from −35% to 100% at concentration of 0.3 g dry wt./L during 96 h treatments. Among the five herbs, G. chinensis exhibited the strongest inhibitory effect, almost 100% on P. globosa and P. donghaiense. The growth of P. globosa and P. donghaiense was completely inhibited by G. chinensis extracts (0.3 g dry wt./L) during all growth phases, and lag phase cultures were more sensitive than exponential phase and stationary phase cultures. The highest inhibitory rate (100% inhibition) on P. globosa was observed at lag phase, followed by exponential phase (73.1% inhibition), and stationary phase (57% inhibition). The highest inhibitory rate of 100% on P. donghaiense was also found at lag phase, 40.3% at exponential phase, and 23.4% at stationary phase. Furthermore, modified clay with G. chinensis extract significantly enhanced the inhibitory impact. Modified clay (0.3 g L⁻¹) produced 95% of growth inhibition for both algae species at 24 h, and maintained the inhibition thereafter. Our study demonstrated that G. chinensis and clays modified with its extract significantly inhibited the growth of harmful species, therefore may provide ideas and another option for control of harmful algal blooms.     

Properties and mechanism of d-glucosaminate-6-phosphate ammonia-lyase: An aminotransferase family enzyme with d-amino acid specificity

Salmonella enterica serovar Typhimurium utilizes a wide range of growth substrates, some of which are relatively novel. One of these unusual substrates is d-glucosaminate, which is metabolized by the enzymes encoded in the dga operon. d-Glucosaminate is transported and converted to d-glucosaminate-6-phosphate (G6P) by a phosphotransferase system, composed of DgaABCD. The protein product of dgaE, d-glucosaminate-6-phosphate ammonia lyase (DGL), converts G6P to 2-keto-3-deoxygluconate-6-phosphate, which undergoes a retroaldol reaction catalyzed by the DgaF protein to give d-glyceraldehyde-3-phosphate and pyruvate. We have now developed an improved synthesis of G6P which gives a higher yield. The DGL reaction is of mechanistic interest because it is one of only a few enzymes in the pyridoxal-5′-phosphate (PLP) dependent aminotransferase superfamily known to catalyze reaction of a d-amino acid substrate. The pH dependence of DGL shows an optimum at 7.5–8.5, suggesting a requirement for a catalytic base. α-Glycerophosphate and inorganic phosphate are weak competitive inhibitors, with K_i values near 30 mM, and d-serine is neither a substrate nor an inhibitor. We have found in rapid-scanning stopped-flow experiments that DGL reacts rapidly with its substrate to form a quinonoid intermediate with λ_max = 480 nm, within the dead time (ca. 2 msec), which then rapidly decays (k = 279 s^− 1) to an intermediate with absorption between 330 and 350 nm, probably an aminoacrylate complex. We suggest a mechanism for DGL and propose that the unusual stereochemistry of the DGL reaction requires a catalytic base poised on the opposite face of the PLP-substrate complex from the other members of the aminotransferase superfamily.     

Culture filtrate of root endophytic fungus Piriformospora indica promotes the growth and lignan production of Linum album hairy root cultures

The effect of addition of autoclaved and filter-sterilized culture filtrate of Piriformospora indica (a root endophytic fungus) to the growing Linum album hairy root cultures on growth and lignan production was investigated. The addition resulted in a significant enhancement in lignan production and growth. The podophyllotoxin and 6-methoxypodophyllotoxin (the lignans) concentrations were maximally improved by 3.8 times (233.8 mg/L) and 4.4 times (131.9 mg/L) in comparison to control cultures, respectively, upon addition of 3.0% (v/v) filter-sterilized culture filtrate of P. indica to the hairy root cultures of L. album for exposure time of 48 h. This increase in the lignan content also coincided with the increase in phenylalanine ammonia lyase activity, which was 3.1-fold (371.4 μkat/kg protein) higher compared to control cultures under the same conditions. The maximal increase in hairy root biomass was, however, obtained under different conditions; it was enhanced by 1.4 times (21.8 g/L) in comparison to control cultures, when 2% (v/v) filter-sterilized culture filtrate was in contact with L. album cultures for 96 h.     

Alternative alert system for Ganga river eutrophication using alkaline phosphatase as a level determinant

Short-term variations in phosphorus (P) concentrations must be considered while assessing the long-term changes in trophic status and estimating the P load and export. Furthermore, given the challenges of conventional monitoring of river systems, a sediment-specific biomonitoring tool may be more successful inferring P related human controls. In this study, conducted along a 37 km river channel representing up-and downstream urban control, and through a trajectory from a major point source (Assi drain), we tested the patterns of concordance between alkaline phosphatase (AP) activity and soluble reactive-P (SRP) and between AP activity and trophic status in the Ganga River. To validate data comparison, we selected a reference site at Dev Prayag, situated ∼1130 km upstream to the main study stretch. Samples were collected for three consecutive year (March 2013 to February 2016) with respect to atmospheric deposition, surface runoff, point source loading, river water and sediment analysis. For trajectory analysis, samples were collected from 15 locations starting from the drain outlet (zero distance) upto 1.5 km downstream with sampling location 100 m away from the preceding one. We found marked spatial and temporal variations in P concentrations which could be traced by quantifying the AP activity. The AP activity, recorded highest at reference site, declined with increases in P; and at drain mouth it was close to zero reflecting strong influence of P level on alkaline phosphatase activity in the river. We used canonical correlation analysis (CCorA) to test the degrees of concordance and similarity in different variables. Most of the environmental variables and indicators of eutrophy appear largely clustered at one side of the coordinate separating AP activity and dissolved oxygen towards opposite side of the axis. The dynamic fit function relating AP activity with different variables showed significant positive correlation with DO (R² = 0.67; p < 0.001) and negative correlations with BOD (R² = 0.82; p < 0.001), Chl a biomass (R² = 0.52; p < 0.001) and trophic status index (R² = 0.54 (Chl a), 0.96 (DRP); p < 0.001). Furthermore, the enzyme activity did not show significant negative correlation with heavy metals in sediment. Because anthropogenic activities continue to enhance P loads; AP is inhibited directly by P availability; and eutrophy feedbacks sediment P release, our observations on P-AP activity relationship provide a valuable alternative means for detecting P related controls on water quality, trophic status and biogeochemical feedbacks in human impacted rivers.     

Pigment compositions and toxic effects of three harmful Karenia species,Karenia concordia,Karenia brevisulcata and Karenia mikimotoi (Gymnodiniales,Dinophyceae), on rotifers and brine shrimps

Karenia concordia, Karenia brevisulcata and Karenia mikimotoi are anomalously-pigmented gymnodinoids characterised by having fucoxanthin and two acyl-oxyfucoxanthin derivatives, instead of having peridinin as in the majority of photosynthetic dinoflagellates. HPLC pigment analyses of all three species, cultured in identical conditions, revealed two different patterns of pigmentation: (1) 19′-hexanoyl-oxyfucoxanthin-rich with very little fucoxanthin, as represented by K. concordia and K. brevisulcata, and (2) fucoxanthin-rich with much less acyl-oxyfucoxanthins, as represented by K. mikimotoi. Moreover, zeaxanthin was detected in both K. concordia and K. brevisulcata, but only trace amount in K. mikimotoi (generally not detected in this species). Both K. concordia and K. brevisulcata lacked carotene-β,ɛ and carotene-ɛ,ɛ. These differed from K. mikimotoi which generally produced all three carotenes (β,β; β,ɛ and ɛ,ɛ). At exponential growth phase, chlorophyll a content on a per cell basis of K. mikimotoi was more than double that of K. concordia and six times greater than that of K. brevisulcata. Toxicological tests conducted on rotifers elicited distinct responses – single-strength lipophilic cell extracts of both K. concordia and K. brevisulcata killed rotifers in tens of minutes (fast-acting), while that of double-strength K. mikimotoi, in tens of hours (slow-acting). Additionally, mature and nauplii forms of brine shrimps exposed to lipophilic cell extract of K. concordia appeared to be temporarily ‘anaesthetised’, but recovered in c. 45 min and 3 h respectively. No such ‘anaesthetic’ effect was observed on both forms of brine shrimps exposed to lipophilic extracts of either K. brevisulcata or K. mikimotoi. The former, however, killed both forms of brine shrimps in a matter of tens of hours, while those of the latter, did not cause any harm to either form tested.     

Examination of six commonly used laboratory fixatives in HAB monitoring programs for their use in quantitative PCR based on Taqman probe technology

Due to the need for more rapid and reliable detection, quantification and enumeration of harmful algal species the use of molecular methods are increasingly being used in monitoring and field studies. However, many studies often require sample fixation to allow for transportation before analyses are conducted. Here, we describe the effects of six fixatives (acidified Lugol's iodine with or without sodium thiosulphate, glutaraldehyde, paraformaldehyde (PFA), formalin and ethanol) on quantitative real-time polymerase chain reaction (qPCR) amplification with Taqman probes. We applied extracted total genomic DNA from four harmful algal species from Danish waters, representing three dinoflagellates (Alexandrium tamarense, Karenia mikimotoi, Karlodinium veneficum and a haptophyte (Prymnesium parvum). The C_q values generated on the qPCR amplification plot were compared to those of an unfixed sample that acted as a control. For all species positive amplifications were achieved from DNA templates from all preserved samples. However, amplification efficiencies between fixatives and species varied. Yet it was found that Lugol's iodine was the most ideal short-term fixative for enumeration of cells by qPCR as well as being the safest to handle. The effect of age on Lugol's iodine fixed samples was also addressed. Samples were fixed and stored at 5 °C in the dark and total genomic DNA extracted after 24 h, 72 h, 1 week, 2 weeks, 1 month and 2 months. Samples remained stable for 1 month for A. tamarense and K. veneficum and 2 months for K. mikimotoi and P. parvum.