Plasma levels of intermedin (adrenomedullin-2) in healthy human volunteers and patients with heart failure

Intermedin/adrenomedullin-2 (IMD) is a member of the adrenomedullin/CGRP peptide family. Less is known about the distribution of IMD than for other family members within the mammalian cardiovascular system, particularly in humans. The aim was to evaluate plasma IMD levels in healthy subjects and patients with chronic heart failure. IMD and its precursor fragments, preproIMD_25–56 and preproIMD_57–92, were measured by radioimmunoassay in 75 healthy subjects and levels of IMD were also compared to those of adrenomedullin (AM) and mid-region proadrenomedullin_45–92 (MRproAM_45–92) in 19 patients with systolic heart failure (LVEF < 45%). In healthy subjects, plasma levels (mean + SE) of IMD (6.3 + 0.6 pg ml⁻¹) were lower than, but correlated with those of AM (25.8 + 1.8 pg ml⁻¹; r = 0.49, p < 0.001). Plasma preproIMD_25–56 (39.6 + 3.1 pg ml⁻¹), preproIMD_57–92 (25.9 + 3.8 pg ml⁻¹) and MRproAM_45–92 (200.2 + 6.7 pg ml⁻¹) were greater than their respective bioactive peptides. IMD levels correlated positively with BMI but not age, and were elevated in heart failure (9.8 + 1.3 pg ml⁻¹, p < 0.05), similarly to MRproAM_45–92 (329.5 + 41.9 pg ml⁻¹, p < 0.001) and AM (56.8 + 10.9 pg ml⁻¹, p < 0.01). IMD levels were greater in heart failure patients with concomitant renal impairment (11.3 + 1.8 pg ml⁻¹) than those without (6.5 + 1.0 pg ml⁻¹; p < 0.05). IMD and AM were greater in patients receiving submaximal compared with maximal heart failure drug therapy and were decreased after 6 months of cardiac resynchronization therapy. In conclusion, IMD is present in the plasma of healthy subjects less abundantly than AM, but is similarly correlated weakly with BMI. IMD levels are elevated in heart failure, especially with concomitant renal impairment, and tend to be reduced by high intensity drug or pacing therapy.     

Impact of microcarrier coverage on using permittivity for on-line monitoring high adherent Vero cell densities in perfusion bioreactors

The paper presents the interest of on-line permittivity monitoring to estimate the density of Vero cells growing on microcarriers (MCs), even when high cell densities were reached in perfusion bioreactors (4.5 × 10⁶ cells ml⁻¹). Cultures were performed with various MCs concentrations in a reactor equipped with a settling tube. A linear correlation between on-line permittivity and off-line volumetric cell concentration was observed provided that MCs are not fully covered by cells. High permittivities such as 250 pF cm⁻¹ could be measured without signal saturation of the Fogale Biomass system^®. The correlation was no longer linear when cell density per carrier exceeded 100% cell confluency corresponding to 150 cells MC⁻¹ (0.15 × 10⁶ cells cm⁻²). This behaviour was attributed to the decrease of cell volume when cells saturated MCs surface. It mainly happened when low MCs concentration and continuous medium renewing were used. Therefore, permittivity sensor can be considered as a reliable tool to monitor on-line adherent cell densities not exceeding total cell confluency. Moreover, it could be useful to detect when cell confluency occurs.     

Metabolic engineering of Corynebacterium glutamicum for the production of itaconate

The capability of Corynebacterium glutamicum for glucose-based synthesis of itaconate was explored, which can serve as building block for production of polymers, chemicals, and fuels. C. glutamicum was highly tolerant to itaconate and did not metabolize it. Expression of the Aspergillus terreus CAD1 gene encoding cis-aconitate decarboxylase (CAD) in strain ATCC13032 led to the production of 1.4 mM itaconate in the stationary growth phase. Fusion of CAD with the Escherichia coli maltose-binding protein increased its activity and the itaconate titer more than two-fold. Nitrogen-limited growth conditions boosted CAD activity and itaconate titer about 10-fold to values of 1440 mU mg⁻¹ and 30 mM. Reduction of isocitrate dehydrogenase activity via exchange of the ATG start codon to GTG or TTG resulted in maximal itaconate titers of 60 mM (7.8 g l⁻¹), a molar yield of 0.4 mol mol⁻¹, and a volumetric productivity of 2.1 mmol l⁻¹ h⁻¹.     

Amperometric xanthine biosensors based on electrodeposition of platinum on polyvinylferrocenium coated Pt electrode

Novel xanthine biosensors were successfully fabricated by immobilizing xanthine oxidase on polyvinylferrocenium perchlorate matrix (PVF⁺ClO₄⁻) and platinum electrodeposited polyvinylferrocenium perchlorate matrix. PVF⁺ClO₄⁻ film was coated on Pt electrode at +0.7 V vs. Ag/AgCl by electrooxidation of polyvinylferrocene (PVF). Platinum nanoparticles were deposited on PVF⁺ClO₄⁻ electrode by electrochemical deposition in 2.0 mM H₂PtCl₆ solution at −0.2 V. Xanthine oxidase was incorporated into the polymer matrix via ion exchange process by immersing modified Pt electrodes in the enzyme solution. The amperometric responses of the biosensors were measured via monitoring oxidation current of hydrogen peroxide at +0.5 V. Under the optimal conditions, the linear ranges of xanthine detection were determined as 1.73 × 10⁻³–1.74 mM for PVF⁺XO⁻ and 0.43 × 10⁻³–2.84 mM for PVF⁺XO⁻/Pt. The detection limits of xanthine were 5.20 × 10⁻⁴ mM for PVF⁺XO⁻ and 1.30 × 10⁻⁴ mM for PVF⁺XO⁻/Pt. Moreover, the effects of applied potential, electrodeposition potential, H₂PtCl₆ concentration, amount of electrodeposited Pt nanoparticles, thickness of polymeric film, temperature, immobilization time, xanthine and xanthine oxidase concentrations on the response currents of the biosensors were investigated in detail. The effects of interferents, the operational and storage stabilities of biosensors and the applicabilities to drug samples of the biosensors analysis were also evaluated.     

Effects of Yucca schidigera extract on fermentation and degradation of steroidal saponins in the rumen simulation technique (RUSITEC)

A rumen simulation technique (RUSITEC) apparatus with eight 940 ml fermentation vessels was used to study the effects of the steroidal saponins in Yucca schidigera extract (YE) on ruminal microbial activity and saponin degradation. The YE contained approximately 4.4% (w/w) saponin, as smilagenin equivalents, and was included at 0 (control) or 0.5 mg ml⁻¹ (n=4) in the McDougall's buffer infused continuously into the vessels (dilution rate=0.75 day⁻¹). Each vessel received 5 g chopped alfalfa hay and 5 g concentrate (as-fed basis) daily for 22 days. Ammonia concentrations were lower (P<0.05) in effluent from vessels receiving YE than from controls for the first half of the study, but did not differ thereafter. Total amounts of VFA in effluent were not affected (P>0.05) by YE, but molar proportions of iso-butyric and iso-valeric acids were lower (P<0.05) in the YE vessels than in the controls in the first half of the experiment. Yucca extract at 0.5 mg ml⁻¹ did not affect (P>0.05) dry matter disappearance (DMD) from hay or from concentrate, nor did it affect total gas or methane production, or bacterial numbers (total or cellulolytic populations) in homogenates prepared from fermenter vessel liquid and feed particles. Protozoal numbers in the homogenates were substantially reduced (P<0.01) by YE (at 0.5 mg ml⁻¹), protease activity was increased (P<0.05), deaminase activity and activity against Ala₂ were unaffected (P>0.05) and activity against Ala₅ was reduced by 25% (P>0.05). When the homogenates from control and YE-supplemented (0.5 mg ml⁻¹) vessels were used to inoculate roll tubes containing 0 or 5 mg ml⁻¹ of YE, fewer colonies developed (P<0.01) in roll tubes containing YE than in those without YE, irrespective of the source of inoculum. Homogenates were also assayed for saponin degradation and for protease, peptidase and deaminase activities. Inoculum from the vessels receiving YE degraded saponin slightly during a 2 h incubation. Yucca extract at 0.5 mg ml⁻¹ altered proteolytic activity and reduced protozoal numbers, but did not affect DMD or bacterial activity, and did not induce resistance to YE at a concentration of 5 mg ml⁻¹.     

THE EFFECT OF OXYGEN DEPRIVATION ON INORGANIC NITROGEN UPTAKE IN AN ANTARCTIC MACROLICHEN

Snow meltwater containing 36 ng ml⁻¹NO₃⁻-N (raised here to between 95–101 ng ml⁻¹NO₃⁻-N) and 112 ng ml⁻¹NH₄⁺-N was sprayed onto illuminatedUsnea sphacelataat 2°C in a 2-1 capacity transparent perspex chamber force-ventilated with either air or O₂- (and CO₂-) free N₂. The NO₃-concentration in meltwater recirculated through a layer ofU. sphacelatafell toc. 8 ng ml⁻¹after 1·25 h. Although the pattern of decline was broadly comparable in both air and N₂, the initial rate of decline was lower in N₂. When undepleted meltwater was continuously sprayed onto the lichen and the effluent collected for analysis, the lichen was found to retain 55% of the wet deposited NO₃⁻in air but only 27% under N₂. Up to 90% of NH₄⁺supplied in a continuous spray of meltwater was retained by the lichen but this was affected little by O₂and CO₂deprivation.     

Structural characterization of thermostable,solvent tolerant,cytosafe tannase from Bacillus subtilis PAB2

Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K_m = 0.445 mM, V_max = 125.8 mM/mg/min and K_cat = 2.88 min^–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t_1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q₁₀, ΔG_d and ΔS_d values of 2.08, 99.37 KJ/mol and 252.38 J mol⁻¹ K⁻¹, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study.     

In situ quantification of microcarrier animal cell cultures using near-infrared spectroscopy

In-line monitoring tools are still required to understand and control animal cell processes, particularly in the case of vaccine production. Here, in situ near-infrared spectroscopy (NIRS) quantification of components in culture media was performed using microcarrier-based cultivations of adherent Vero cells. Because microcarriers were found to interfere with NIRS spectra acquisition, a suitable and innovative in situ calibration was developed for bioreactor cultures. A reliable and accurate NIRS technique for the quantification of glucose and lactate was established, with a calibration standard error of 0.30 and 0.21 g l⁻¹, respectively. The robustness of this method was evaluated by performing NIRS calibration with operating conditions similar to those of industrial processes, including parameters such as microcarrier concentrations, cell seeding states and changes in analyte concentration due to feed and harvest strategies. Based on this calibration procedure, the predicted analyte concentrations in unknown samples was measured by NIRS analyses with an accuracy of 0.36 g l⁻¹ for glucose and 0.29 g l⁻¹ for lactate.     

In situ quantification of microcarrier animal cell cultures using near-infrared spectroscopy

In-line monitoring tools are still required to understand and control animal cell processes, particularly in the case of vaccine production. Here, in situ near-infrared spectroscopy (NIRS) quantification of components in culture media was performed using microcarrier-based cultivations of adherent Vero cells. Because microcarriers were found to interfere with NIRS spectra acquisition, a suitable and innovative in situ calibration was developed for bioreactor cultures. A reliable and accurate NIRS technique for the quantification of glucose and lactate was established, with a calibration standard error of 0.30 and 0.21 g l⁻¹, respectively. The robustness of this method was evaluated by performing NIRS calibration with operating conditions similar to those of industrial processes, including parameters such as microcarrier concentrations, cell seeding states and changes in analyte concentration due to feed and harvest strategies. Based on this calibration procedure, the predicted analyte concentrations in unknown samples was measured by NIRS analyses with an accuracy of 0.36 g l⁻¹ for glucose and 0.29 g l⁻¹ for lactate.     

Glucocorticoid receptors on and in a unicellular organism,Cryptobia salmositica

This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50 ng ml⁻¹ of RU486 was more effective in inhibiting parasite replication in cultures with 7,000 parasites ml⁻¹ than in cultures with 14,000 parasites ml⁻¹. Also, 100 ng ml⁻¹ of RU486/ml was more effective than 50 ng ml⁻¹ in inhibiting parasite multiplication in the 14,000 parasites ml^-1 cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand–receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host–parasite interaction.     

A comparative study of the use of inorganic carbon resources by Chara aspera and Potamogeton pectinatus

We studied the potential role of dissolved inorganic carbon (DIC) in determining vegetation dominance of Potamogeton pectinatus L. and Chara aspera Deth. ex Willd. by monitoring the seasonal dynamics of DIC in a shallow lake and comparing the use of DIC of the two species. The HCO₃⁻-concentration in summer dropped from 2.5 to <0.5 mM with seasonally increasing Chara biomass, whereas outside the vegetation concentrations remained at 2.5 mM. Inside Potamogeton spp. vegetation DIC decreased from 2.5 to ca. 0.75 mM HCO₃⁻. A growth experiment showed ash-free biomass for P. pectinatus was nearly two times as high as for C. aspera at 3 mM HCO₃⁻, but almost two times lower at 0.5 mM than at 3.0. In a separate experiment, P. pectinatus precultured at a relatively low HCO₃⁻-level had a lower net photosynthetic rate (P_max, 0.1 mmol O₂ g⁻¹ DW h⁻¹) than C. aspera (P_max, 0.1 mmol O₂ g⁻¹ DW h⁻¹) over the range of HCO₃⁻-concentrations tested (P_max, 0.14 mmol O₂ g⁻¹ DW h⁻¹). In response to CO₂ no significant differences between the compensation points (P. pectinatus, 28 mM; C. aspera 66 mM), were observed, but the photosynthetic rate increased faster than for C. aspera than for P. pectinatus. Under field conditions, the use of CO₂ is not important since inside vegetation CO₂-concentrations were below 10 μM, and thus, not available for photosynthesis of either species during the main part of the growth season. It is suggested that C. aspera may be a better competitor for HCO₃⁻ than P. pectinatus in conditions with a low HCO₃⁻ supply. As HCO₃⁻ is a strong limiting factor for growth inside the vegetation and probably the only carbon source available, the superior ability of C. aspera to use HCO₃⁻ may be an important factor explaining its present dominance in Veluwemeer.     

Batch and continuous fermentative production of hydrogen with anaerobic sludge entrapped in a composite polymeric matrix

Cell immobilization techniques were adopted to biohydrogen production using immobilized anaerobic sludge as the seed culture. Sucrose-based synthetic wastewater was converted to H₂ using batch and continuous cultures. A novel composite polymeric material comprising polymethyl methacrylate (PMMA), collagen, and activated carbon was used to entrap biomass for H₂ production. Using the PMMA immobilized cells, the favorable conditions for batch H₂ fermentation were 35 °C, pH 6.0, and an 20 g COD l⁻¹ of sucrose, giving a H₂ production rate of 238 ml h⁻¹ l⁻¹ and a H₂ yield of 2.25 mol H₂ mol sucrose⁻¹. Under these optimal conditions, continuous H₂ fermentation was conducted at a hydraulic retention time (HRT) of 4–8 h, giving the best H₂-producing rate of 1.8 l h⁻¹ l⁻¹ (over seven-fold of the best batch result) at a HRT of 6 h and a H₂ yield of 2.0 mol H₂ mol sucrose⁻¹. The sucrose conversion was essentially over 90% in all runs. The biogas consisted of only H₂ and CO₂. The major soluble metabolites were butyric acid, acetic acid, and 2,3-butandiol, while a small amount of ethanol also detected. The PMMA-immobilized-cell system developed in this work seems to be a promising H₂-producing process due to the high stability in continuous operations and the capability of achieving a competitively high H₂ production rate under a relatively low organic loading rate.     

Characterization and overproduction of a thermo-alkaline pectate lyase from alkaliphilic Bacillus licheniformis with potential in ramie degumming

A thermo-alkaline pectate lyase (BliPelA) gene from an alkaliphilic Bacillus licheniformis strain was cloned and overexpressed in Escherichia coli. Mature BliPelA exhibited maximum activity at pH 11 and 70 °C, and demonstrated cleavage capability on a broad range of substrates such as polygalacturonic acid, pectins, and methylated pectins. The highest specific activity, of 320 U mg⁻¹, was towards polygalacturonic acid. Significant ramie (Boehmeria nivea) fiber weight loss (21.5%) was obtained following enzyme treatment and combined enzyme-chemical treatment (29.3%), indicating a high ramie degumming efficiency of BliPelA. The total activity of recombinant BliPelA reached 1450.1 U ml⁻¹ with a productivity of 48.3 U ml⁻¹ h⁻¹ under high-cell-density cultivation with a glycerol exponential feeding strategy for 30 h in 1-l fed-batch fermenter, and 1380.1 U ml⁻¹ with a productivity of 57.5 U ml⁻¹ h⁻¹ after 24 h under constant glucose feeding in a 20-l fermenter using E. coli as the host. The enzyme yields reached 4.5 and 4.3 g l⁻¹ in 1-l and 20-l fed-batch fermenters, respectively, which are higher than those of most reported alkaline Pels. Based on these promising properties and high-level production, BliPelA shows great potential for application in ramie degumming in textile industry.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

Characterization of constitutive and acid-induced outwardly rectifying chloride currents in immortalized mouse distal tubular cells

Thiazides block Na⁺ reabsorption while enhancing Ca²⁺ reabsorption in the kidney. As previously demonstrated in immortalized mouse distal convoluted tubule (MDCT) cells, chlorothiazide application induced a robust plasma membrane hyperpolarization, which increased Ca²⁺ uptake. This essential thiazide-induced hyperpolarization was prevented by the Cl⁻ channel inhibitor 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), implicating NPPB-sensitive Cl⁻ channels, however the nature of these Cl⁻ channels has been rarely described in the literature. Here we show that MDCT cells express a dominant, outwardly rectifying Cl⁻ current at extracellular pH 7.4. This constitutive Cl⁻ current was more permeable to larger anions (Eisenman sequence I; I⁻ > Br⁻ ≥ Cl⁻) and was substantially inhibited by > 100 mM [Ca²⁺]_o, which distinguished it from ClC-K2/barttin. Moreover, the constitutive Cl⁻ current was blocked by NPPB, along with other Cl⁻ channel inhibitors (4,4′-diisothiocyanatostilbene-2,2′-disulfonate, DIDS; flufenamic acid, FFA). Subjecting the MDCT cells to an acidic extracellular solution (pH < 5.5) induced a substantially larger outwardly rectifying NPPB-sensitive Cl⁻ current. This acid-induced Cl⁻ current was also anion permeable (I⁻ > Br⁻ > Cl⁻), but was distinguished from the constitutive Cl⁻ current by its rectification characteristics, ion sensitivities, and response to FFA. In addition, we have identified similar outwardly rectifying and acid-sensitive currents in immortalized cells from the inner medullary collecting duct (mIMCD-3 cells). Expression of an acid-induced Cl⁻ current would be particularly relevant in the acidic IMCD (pH < 5.5). To our knowledge, the properties of these Cl⁻ currents are unique and provide the mechanisms to account for the Cl⁻ efflux previously speculated to be present in MDCT cells.     

Expression and purification of recombinant feline interferon in the baculovirus-insect larvae system

Feline interferons (FeIFNs) are cytokines with antiviral, antitumor and immunomodulatory functions used as therapeutic agents in a variety of veterinary diseases. In this work, FeIFN-α7 and FeIFN-α7xArg containing eight residues of arginine were expressed in Sf9 cells and insect larvae. At 4 days post-infection (dpi), the concentrations of FeIFN-α7 and FeIFN-α7xArg in suspension culture were (1.28 ± 0.15) × 10⁶ U ml⁻¹ and (1.3 ± 0.2) × 10⁶ U ml⁻¹ respectively. The maximum expression levels of FeIFN-α7 and FeIFN-α7xArg were (3.7 ± 0.2) × 10⁶ U ml⁻¹ and (3.5 ± 0.4) × 10⁶ U ml⁻¹ at 2 dpi in Rachiplusia nu larvae and (1.1 ± 0.2) × 10⁶ U ml⁻¹ and (1.0 ± 0.15) × 10⁶ U ml⁻¹ at 5 dpi in Spodoptera frugiperda larvae respectively. R. nu was a better host for FeIFN-α7 and FeIFN-α7xArg expression. The 8xArg tag did not affect the biological activity of FeIFN-α7 and was useful to promote the FeIFN-α7xArg adsorption on ion exchange chromatography (IEC), allowing its purification in a single step from supernatant culture and R. nu larvae. FeIFN-α7xArg was purified from the larval extract with a yield of 70% and a purification factor of 25 free of viruses. We conclude that R. nu larvae are new low-cost hosts for the expression of recombinant FeIFN-α7.     

Cyanide hydratase from Aspergillus niger K10: Overproduction in Escherichia coli,purification, characterization and use in continuous cyanide degradation

A cyanide hydratase from Aspergillus niger K10 was expressed in Escherichia coli and purified. Apart from HCN, it transformed some nitriles, preferentially 2-cyanopyridine and fumaronitrile. V_max and K_m for HCN were ca. 6.8 mmol min⁻¹ mg⁻¹ protein and 109 mM, respectively. V_max for fumaronitrile and 2-cyanopyridine was two to three orders of magnitude lower than for HCN (ca. 18.8 and 10.3 μmol min⁻¹ mg⁻¹, respectively) but K_m was also lower (ca. 14.7 and 3.7 mM, respectively). Both cyanide hydratase and nitrilase activities were abolished in truncated enzyme variants missing 18–34 C-terminal aa residues. The enzyme exhibited the highest activity at 45 °C and pH 8–9; it was unstable at over 35 °C and at below pH 5.5. The operational stability of the whole-cell catalyst was examined in continuous stirred membrane reactors with 70-mL working volume. The catalyst exhibited a half-life of 5.6 h at 28 °C. A reactor loaded with an excess of the catalyst was used to degrade 25 mM KCN. A conversion rate of over 80% was maintained for 3 days.     

Simultaneous determination of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam in human serum using gas chromatography–mass spectrometry

A method for the quantitation of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam from human serum capable of monitoring concentrations achieved under therapeutic conditions is presented. The substances were extracted under basic conditions with toluene and the hydroxy metabolites transformed to their tert-butyldimethylsilyl derivatives with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. The samples were measured by gas chromatography–mass spectrometry. The limits of detection are 0.2 ng ml⁻¹ for midazolam and 0.1 ng ml⁻¹ for 1-hydroxy- and 4-hydroxymidazolam. The coefficients of variation are 3.9% at 5 ng ml⁻¹ for midazolam, 6.7% at 2 ng ml⁻¹ for 1-hydroxymidazolam and 8.8% (22.2%) at 0.5 (0.2) ng ml⁻¹ for 4-hydroxymidazolam.     

Killing potential protist predators as a survival strategy of the newly described dinoflagellate Alexandrium pohangense

Blooms caused by some species belonging to the dinoflagellate genus Alexandrium are known to cause large-scale mortality of fish. Thus, the dynamics of these species is important and of concern to scientists, officials, and people in the aquaculture industry. To understand the dynamics of such species, their growth and mortality due to predation need to be assessed. The newly described dinoflagellate Alexandrium pohangense is known to grow slowly, with a maximum autotrophic growth rate of 0.1 d⁻¹. Thus, it may not form bloom patches if its mortality due to predation is high. Therefore, to explore the mortality of A. pohangense due to predation, feeding on this species by the common heterotrophic dinoflagellates Gyrodinium dominans, Gyrodinium moestrupii, Luciella masanensis, Noctiluca scintillans, Oxyrrhis marina, Oblea rotunda, Polykrikos kofoidii, and Pfiesteria piscicida, as well as by the ciliate Tiarina fusus, was examined. None of these potential predators was able to feed on A. pohangense. In contrast, these potential predators were killed and their bodies were dissolved when incubated with A. pohangense cells or cell-free culture filtrates. The survival of G. moestrupii, O. marina, P. kofoidii, and T. fusus on incubation with 10 cells ml⁻¹ of A. pohangense was 20–60%, while that at the equivalent culture filtrates was 20–70%. With increasing A. pohangense cell-concentration (up to 1000 cells ml⁻¹ or equivalent culture filtrates), the survival rate of G. moestrupii, O. marina, P. kofoidii, and T. fusus rapidly decreased. The lethal concentration (LC₅₀) for G. moestrupii, O. marina, P. kofoidii, and T. fusus at the elapsed time of 24 h with A. pohangense cells (cultures of 11.4, 13.3, 1.6, and 3.3 cells ml⁻¹, respectively) was lower than that with A. pohangense filtrates (culture filtrates of 35.5, 30.6, 5.5, and 5.0 cells ml⁻¹, respectively). Furthermore, most of the ciliates and heterotrophic dinoflagellates in the water collected from the coast of Tongyoung, Korea, were killed when incubated with cultures of 1000 A. pohangense cells ml⁻¹ and equivalent culture filtrates. The relatively slow growing A. pohangense may form blooms by reducing mortality due to predation through killing potential protist predators.     

Removal of two pathogenic scuticociliates Miamiensis avidus and Miamiensis sp. using cells or culture filtrates of the dinoflagellate Alexandrium andersonii

Scuticociliatosis, which is caused by parasitic protistan pathogens known as scuticociliates, is one of the most serious diseases in marine aquaculture worldwide. Thus, elimination of these ciliates is a primary concern for scientists and managers in the aquaculture industry. To date, formalin and other toxic chemicals have been used as anti-scuticociliate agents, but issues regarding their secondary effects often arise. Consequently, development of safer methods is necessary. To find out a safe method of controlling scuticociliate populations in aqua-tanks or small-scale natural environments, cultures of 14 phototrophic dinoflagellates were tested to determine whether they were able to control populations of the common scuticociliates Miamiensis avidus and Miamiensis sp. isolated from Korean waters. Among the dinoflagellates tested, both cells and culture filtrates of Alexandrium andersonii effectively killed M. avidus and Miamiensis sp. The minimal concentration of cells and equivalent culture filtrates of A. andersonii to kill all M. avidus cells within 48 h of incubation was ca. 2500 and 4500 cells ml⁻¹, respectively; whereas those needed to kill all Miamiensis sp. cells were ca. 1000 and 4500 cells ml⁻¹, respectively. It was estimated that 1 m³ of the stock culture containing 20,000 A. andersonii cells ml⁻¹ could eliminate all M. avidus cells in 7 m³ of waters within the aqua-tanks on land and all Miamiensis sp. cells in 19 m³ of waters within 48 h. None of the brine shrimp Artemia salina nauplii incubated with concentrations of 50–4500 A. andersonii cells ml⁻¹ for 24 h was dead. Furthermore, none of the flounder Paralichthys olivaceus juveniles incubated with a mean concentration of ca. 2280 A. andersonii cells ml⁻¹ for 96 h was dead. Therefore, A. andersonii cultures may be used as a safe biological method for controlling populations of scuticociliates and can replace toxic formalin. The results of this study provided the basis for developing the method to control scuticociliate populations and understanding interactions between scuticociliates and phototrophic dinoflagellates in marine ecosystems.