IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

Adaptation of the nematode Caenorhabditis elegans to medium high temperature

Action of high temperature (36°C) on the nematode Caenorhabditis elegans organism was manifested in errors of the motor program of swimming induced by a mechanical stimulus (37 ± 2 min), the complete, but reversible cessation of locomotion (57 ± 3 min), while damage—in thermal death (215 ± 5 min). Addition into medium of atropine (10^?8–10^?9 M) and chemical stimuli (10^?8–10^?6 cAMP or lysine) causes considerable changes of thermal stability of the worm locomotion. Analysis of these data has shown that the cause of the reversible thermal disturbance of the C. elegans locomotion is disintegration of neurons in the nervous centers regulating behavior. The obtained data indicate the presence in the simple organism of C. elegans of adaptations increasing stability of processes of integration of neurons to a high temperature, which were found earlier in arthropods and vertebrates.     

Methadone but not Morphine Inhibits Lubiprostone-Stimulated Cl− Currents in T84 Intestinal Cells and Recombinant Human ClC-2, but not CFTR Cl− Currents

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl^? channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl^? currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl^? currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl^? currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl^? currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl₂-inhibited Cl^? currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl^? currents with half-maximal inhibition at 100 and 200–230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl^? currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl^? currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl^? currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl^? currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.     

Toxicity of Chemically Spiked Sediment to the Carpet Shell Clam Ruditapes decussatus Embryos and Larvae

The objective of this research was to assess the toxicity of sediment contaminated with cadmium, DDT, chlorpyrifos, and fluoranthene to embryos and larvae of the European clam Ruditapes decussatus, exposed to two sediment fractions, the whole sediment and elutriate. The percentages of abnormal D-shaped larvae and larval mortality have been investigated. The median effective concentration (EC50) values, reducing 50% of the percentage of D-shaped larvae, in whole sediments and elutriates were, respectively, 1.17 mg/kg and 417.1 μgl^?1 (3.71 μM) for cadmium, 1.66 mg/kg and 97.8 μgl^?1 (0.48 μM) for fluoranthene, 1.71 mg/kg and 384.8 μgl^?1 (1.08 μM) for DDT, and 0.96 mg/kg and 339.5 μgl^?1 (0.96 μM) for chlorpyrifos. The 96h-median lethal concentrations (LC50) reducing larval survival by 50% were 4.04 mg/kg 654.3 μgl^?1 (5.82 μM) for cadmium, 17.41 mg/kg 8666.6 μgl^?1 (42.84 μM) for fluoranthene, 3.93 mg/kg and 457.4 μgl^?1 (1.29 μM) for DDT, and 2.53 mg/kg and 308.06 μgl^?1 (0.87 μM) for chlorpyrifos. Based on EC₅₀ and LC₅₀ comparisons to toxicity data for other marine species, these findings suggest that the R. decussatus embryotoxicity and larvae mortality bioassay were among the most sensitive tools for sediment quality assessment.     

Effects of Arsenic on Growth, Photosynthetic Activity, and Accumulation in Two New Hyperaccumulating Populations of Isatis cappadocica Desv.

The effect of arsenic on leaf photosynthetic rate, growth responses, and accumulation capability of Isatis cappadocica Desv., a Brassica collected from Iranian arsenic-contaminated mine spoils and control populations, was investigated. Both populations of I. cappadocica were considerably more tolerant than the reference Brassica species (Descurainia sophia). The 1,000 μM arsenate exposure inhibited root growth completely in D. sophia, but only by 50 and 40 % in the nonmine and mine populations of I. cappadocica, respectively. Furthermore, the chlorophyll contents of both populations of I. cappadocica were not statistically different, especially when plants were exposed to 5–800 μM As. The chlorophyll a fluorescence kinetics (F _v/F _m) and electron transfer rate values of treated I. cappadocica populations remained unaffected, indicating normal photosynthetic efficiency and strength of plants in the presence of arsenic. After 28 days of exposure to 1,300 μM As, shoot arsenic concentrations of mine and nonmine populations reached 310 and 345 mg kg^?1, respectively, with the arsenic transfer factor and bioaccumulation greater than 1.0. According to these results, it was shown that I. cappadocica had strong tolerance to and the capability to hyperaccumulate arsenic; therefore, it is a potential As hyperaccumulator.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

Feeding elicitors and precursors enhance colchicine accumulation in morphogenic cultures of <Emphasis Type="Italic">Gloriosa superba</Emphasis> L.

Morphogenic cultures of Gloriosa superba were initiated on Murashige and Skoog’s medium fortified with 2 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L^?1 naphthaleneacetic acid (NAA), 4% sucrose and 0.1% activated charcoal. To enhance the content of the alkaloid colchicine, morphogenic cultures were treated with different concentrations of abiotic elicitors like signalling compounds, metals, biotic elicitors, precursors and a combination of elicitors. Signalling molecules like acetyl salicylic acid (ASA) and sodium nitroprusside improved the production of colchicine. Abiotic elicitors have markedly (p?≤?0.05 or ≤?0.01) enhanced the colchicine content either at lower or higher concentrations. Among the metals, the highest amount of 11.67 mg of colchicine g^?1 dry wt was noticed at 60 mM rubidium chloride, followed by 60 mM NaCl (11.18 mg g^?1). Contrarily, in the presence of biotic elicitors such as Fusarium oxysporum, Alternaria solani, and Saccharomyces cerevisiae, colchicine content ranged only between 2 and 5.32 mg g^?1, but Bacillus subtilis repressed it. Among the aromatic amino acids, phenylalanine at 500 mg L^?1 influenced the highest accumulation of 19.48 mg g^?1 dry tissue, followed by tryptophan (12.47 mg g^?1), and tyrosine (9.87 mg g^?1), a direct precursor of colchicine biosynthesis, while intact tubers and leaves contained 4.65 and 4.16 mg of colchicine g^?1 dry tissue respectively. A combination of 10 µM AlCl₃ and 50 µM salicylic acid (SA) registered 17.34 mg g^?1 followed by 16.24 mg g^?1 tissue in presence of 1 µM HgCl₂ and 50 µM SA. The results suggest that the elicitor-stimulated colchicine accumulation was a stress response and can be exploited further for commercial production.     

Screening and characterization of Isochrysis strains and optimization of culture conditions for docosahexaenoic acid production

Isochrysis is a genus of marine unicellular microalgae that produces docosahexaenoic acid (DHA, C22:6), a very long chain polyunsaturated fatty acid (PUFA) of significant health and nutritional value. Mass cultivation of Isochrysis for DHA production for human consumption has not been established due to disappointing low DHA productivity obtained from commonly used Isochrysis strains. In this study, 19 natural Isochrysis strains were screened for DHA yields and the results showed that the cellular DHA content ranged from 6.8 to 17.0 % of total fatty acids with the highest DHA content occurring in the exponential growth phase. Isochrysis galbana #153180 exhibited the greatest DHA production potential and was selected for further investigation. The effects of different light intensities, forms, and concentrations of nitrogen, phosphorus, and salinity on growth and DHA production of I. galbana #153180 were studied in a bubble column photobioreactor (PBR). Under favorable culture conditions, I. galbana #153180 contained DHA up to 17.5 % of total fatty acids or 1.7 % of cell dry weight. I. galbana #153180 was further tested in outdoor flat-plate PBRs varying in light path length, starting cell density (SCD), and culture mode (batch versus semicontinuous). When optimized, record high biomass and DHA productivity of I. galbana #153180 of 0.72 g L^?1?day^?1 and 13.6 mg?L^?1?day^?1, or 26.4 g?m^?2?day^?1 and 547.7 mg?m^?2?day^?1, respectively, were obtained, suggesting that I. galbana #153180 may be a desirable strain for commercial production of DHA.     

Factors affecting biohydrogen production by unicellular halotolerant cyanobacterium Aphanothece halophytica

The effects of several physiological parameters on H₂ production rate in the unicellular halotolerant cyanobacterium Aphanothece halophytica were investigated. Under nitrogen deprivation, the growth of cells was inhibited, but H₂ production rate was enhanced approximately fourfold. Interestingly, cells grown under sulfur deprivation exhibited a decrease in cell growth, H₂ production rate, and bidirectional hydrogenase activity. Glucose was the preferred sugar source for H₂ production by A. halophytica, but H₂ production decreased at high glucose concentrations. H₂ production rate was optimum when cells were grown in the presence of 0.75 M?NaCl, or 0.4 μM?Fe³⁺, or 1 μM?Ni²⁺. The optimum light intensity and temperature for H₂ production were 30 μmol photons m^?2?s^?1 and 35 °C, respectively. A two-stage culture of A. halophytica was performed in order to overcome the reduction of cell growth in N-free medium. In the first stage, cells were grown in normal medium to accumulate biomass, and in the second stage, H₂ production by the obtained biomass was induced by growing cells in N-free medium supplemented with various chemicals for 24 h. A. halophytica grown in N-free medium containing various MgSO₄ concentrations had a high H₂ production rate between 11.432 and 12.767 μmol H₂ mg?chlorophyll a (chl a)^?1?h^?1, a 30-fold increase compared to cells grown in normal medium. The highest rate of 13.804 μmol H₂ mg?chl a ^?1?h^?1 was obtained when the N-free growth medium contained 0.4 μM Fe³⁺. These results suggested the possibility of using A. halophytica and some other halotolerant cyanobacteria thriving under extreme environmental conditions in the sea as potential sources for H₂ production in the future.     

Evaluation of 5-HT7 Receptor Trafficking on In Vivo and In Vitro Model of Lipopolysaccharide (LPS)-Induced Inflammatory Cell Injury in Rats and LPS-Treated A549 Cells

This study aimed to investigate the effects of the 5-HT7 receptor agonist (LP44) and antagonist (SB269970) on LPS-induced in vivo tissue damage and cell culture by molecular methods. This study was conducted in two steps. For in vivo studies, 24 female rats were divided into four groups. Group I: healthy; II (2nd h): LPS 5 mg/kg administered intraperitoneally (i.p.); III (4th h): LPS 5 mg/kg administered i.p.; IV (8th h): LPS 5 mg/kg administered i.p. For in vitro studies, we used the A549 cell line. Groups: I control (healthy) (2–4 h); II LPS: 1 µg/ml E. Coli O55:B5 strain (2–4 h); III agonist (LP44) 10^?9 M (2–4 h); IV antagonist (SB269970) 10^?9 M (2–4 h); V LPS+agonist 10^?9 M (LP44 1 µg/ml) (2–4 h); VI LPS+antagonist 10^?9 M (2–4 h). In molecular analyses, we determined increased TNF-α, IL-1β, NF-κB, and 5-HT7 mRNA expressions in rat lung tissues and increased TNF-α, iNOS, and 5-HT7 mRNA expressions in the A549 cell line. In in vitro parameters, LP44 agonist administration-related decrease was observed. Our study showed that lung 5-HT7 receptor expression is increased in LPS-induced endotoxemia. All this data suggest that 5-HT7 receptor overexpression is an important protective mechanism during LPS-induced sepsis-related cell damage.     

Bioethanol production from the hydrolysate of Palmaria palmata using sulfuric acid and fermentation with brewer’s yeast

Seaweeds, particularly species of red macroalgae, are promising resources for bioethanol production because of their exceptionally high carbohydrate content. Of 20 seaweeds evaluated, Palmaria palmata (Rhodymenia palmata) contained the highest carbohydrate content (469.8 mg g^?1 seaweed) with a carrageenan content of 354 mg g^?1 seaweed. Such a high carrageenan content makes the high-volume production of bioethanol feasible. Acid hydrolysis of P. palmata in 0.4 M H₂SO₄ at 125 °C for 25 min released 27 mg of glucose, 218.4 mg of reducing sugars, and 127.6 mg of galactose per gram of seaweed. Ethanol fermentation of these hydrolysis products using an inoculum concentration of 1.5 mg mL^?1 at 30 °C and 72 h in a shaking incubator at 130 rpm yielded 17.3 mg of ethanol per gram of seaweed.     

Improvement of 5-aminolevulinic acid production by Rubrivivax benzoatilyticus PS-5 with self-flocculation by co-fermentation of precursors and volatile fatty acids under pH-controlled conditions

Photosynthetic bacteria are known to utilize volatile fatty acids as a carbon source for growth and product formation. In this study, a new isolate, Rubrivivax benzoatilyticus PS-5, possessing self-flocculation properties, was cultivated in modified glutamate-malate (GM) medium containing glutamate and malate as carbon sources. The effect of acetic acid, propionic acid and butyric acid (at 1–4 g L^?1) as co-substrates and 7.5 mM glycine, 10 mM succinic acid as precursors for 5-aminolevulinic acid (ALA) production from R. benzoatilyticus PS-5 was investigated. Among the volatile fatty acids tested, acetic acid was preferred to butyric acid and propionic acid, with the optimum concentrations of 3 g L^?1, 1 g L^?1 and 3 g L^?1, respectively. The highest ALA production was 169.71 μM, 162.16 μM and 46.18 μM, respectively, while the highest productivity was 2.57 μM h^?1, 2.25 μM h^?1 and 0.96 μM h^?1, respectively. The precursor was consumed completely (100 %) while the assimilation of the acetic acid and butyric acid was 62.50 % and 48.65 %, respectively. Supplementation of propionic acid (at 1–4 g l^?1) had a negative effect on growth and ALA production. To increase production efficiency, the pH-control strategy (at pH 6.0–8.0) during fermentation was tested. The optimum pH was 7.0, giving the maximum ALA production of 286.18 μM and a productivity of 3.97 μM h^?1. These values were 1.68-fold and 1.54-fold higher, respectively, than those under uncontrolled pH conditions.     

Dietary Supplementation of Zinc Nanoparticles and Its Influence on Biology,Physiology and Immune Responses of the Freshwater Prawn,Macrobrachium rosenbergii

The present study was conducted to assess the influence of dietary zinc nanoparticles (size 50 nm) on the growth, biochemical constituents, enzymatic antioxidant levels and the nonspecific immune response of the freshwater prawn, Macrobrachium rosenbergii post larvae (PL). The concentrations of dietary supplement zinc nanoparticles (ZnNPs) were 0, 10, 20, 40, 60 and 80 mg kg^?1 with the basal diet, and the level of Zn in ZnNP-supplemented diets were 0.71, 10.61, 20.73, 40.73, 60.61 and 80.60 mg kg^?1, respectively. ZnNP-incorporated diets were fed to M. rosenbergii PL (initial body weight, 0.18?±?0.02 g) in a triplicate experimental setup for a period of 90 days. ZnNP supplemented feed fed PL up to 60 mg kg^?1 showed significantly (P?<?0.05) improved performance in survival, growth and activities of digestive enzymes (protease, amylase and lipase). The concentrations of biochemical constituents (total protein, total amino acid, total carbohydrate and total lipid), total haemocyte count and differential haemocyte count were elevated in 10–60 mg kg^?1 ZnNP supplemented feed fed PL. However, the PL fed with 80 mg ZnNPs kg^?1 showed negative results. Activities of enzymatic antioxidants [superoxide dismutase (SOD) and catalase (CAT)], metabolic enzymes [glutamate–oxaloacetate transaminase (GOT) and glutamate–pyruvate transaminase (GPT)] and the process of lipid peroxidation (LPO) in the hepatopancreas and muscle showed no significant alterations in 10–60 mg kg^?1 ZnNP supplemented feed fed PL. Whereas, 80 mg ZnNPs kg^?1 supplemented feed fed PL showed significant elevations in SOD, CAT, LPO, GOT and GPT. Therefore, 80 mg ZnNPs kg^?1 was found to be toxic to M. rosenbergii PL. Thus, the study suggests that up to 60 mg ZnNPs kg^?1 can be supplemented for regulating survival, growth and immunity of M. rosenbergii.     

Genome-wide association study identified the human leukocyte antigen region as a novel locus for plasma beta-2 microglobulin

Beta-2 microglobulin (B2M) is a component of the major histocompatibility complex (MHC) class I molecule and has been studied as a biomarker of kidney function, cardiovascular diseases and mortality. Little is known about the genes influencing its levels directly or through glomerular filtration rate (GFR). We conducted a genome-wide association study of plasma B2M levels in 6738 European Americans from the Atherosclerosis Risk in Communities study to identify novel loci for B2M and assessed its association with known estimated GFR (eGFR) loci. We identified 2 genome-wide significant loci. One was in the human leukocyte antigen (HLA) region on chromosome 6 (lowest p value = 1.8 × 10^?23 for rs9264638). At this locus, 6 index SNPs accounted for 3.2 % of log(B2M) variance, and their association with B2M could largely be explained by imputed classical alleles of the MHC class I genes: HLA-A, HLA-B, or HLA-C. The index SNPs at this locus were not associated with eGFR based on serum creatinine (eGFRcr). The other locus of B2M was on chromosome 12 (rs3184504 at SH2B3, beta = 0.02, p value = 3.1 × 10^?8), which was previously implicated as an eGFR locus. In conclusion, although B2M is known to be a component of MHC class I molecule, the association between HLA class I alleles and plasma B2M levels in a community-based population is novel. The identification of the two novel loci for B2M extends our understanding of its metabolism and informs its use as a kidney filtration biomarker.     

Sensitivity of Amoeba proteus to Morphine and N-Allylnormorphine. A Pressure Study*

SYNOPSIS. Amoebae exposed to 2 × 10^?3 M morphine appeared to have normal form and activity; however, a 1–2 fold increase in concentration resulted in retraction of pseudopodia and surface blebbing, then cytolysis. Amoebae cultured in 2 × 10^?3 morphine for 5–7 days, then transferred to toxic concentrations of morphine (5 × 10^?2 M) had their survival time increased 4–5 fold over the controls. As indicated by resistance to the solational action of high pressure, morphine distinctly increased pseudopodial stability, depending upon morphine concentration and, to a lesser degree, duration of exposure. Altho N-allylnormorphine did not appreciably affect pseudopodial stability, it did antagonize the action of morphine on pseudopodial stability, N-Allylnormorphine apparently competes for the same site as morphine. It is suggested that morphine affects the sol-gel equilibrium within the cell and thus may affect the integrity of proteins associated with pseudopodial stability.     

Effect of salinity on filtration rates of mussels Mytilus edulis with special emphasis on dwarfed mussels from the low-saline Central Baltic Sea

The effect of salinity on the filtration rate of blue mussels, Mytilus edulis, from the brackish Great Belt (Denmark) and the low-saline Central Baltic Sea, respectively, was studied. First, we measured the effect of long-term (weeks) constant ambient salinities between 5 and 30 psu on the filtration rate of M. edulis collected in the Great Belt where the mean salinity is 17 psu. At salinities between 10 and 30 psu, the filtration rates did not vary much, but at 5 psu the filtration rates were significantly lower. Next, we studied dwarfed M. edulis (<25 mm shell length) from Central Baltic Sea (Askö, Sweden) where the mean salinity is 6.5 psu. The maximum filtration rate (F, ml min^?1 ind.^?1) as a function of shell length (L, mm) and dry weight of soft parts (W, mg) were found to be: F = 0.003L ^2.71 and F = 0.478W ^0.92, respectively, and these results indicate that the filtration rates of dwarfed Baltic Sea mussels are comparable to filtration rates of Great Belt mussels of similar size exposed to salinities >10 psu. When Baltic Sea mussels acclimatized to 20 psu in the laboratory were exposed to 6.5 psu this caused a drastic reduction in the filtration rate, but after about 2 days the previous high filtration rate was regained at 6.5 psu, and further, a similar pattern was observed when the 6.5 psu exposed mussels were finally re-exposed to 20 psu. The observed lack of Great Belt mussels to completely adjust to 5 psu, in contrast to the ease of Baltic Sea mussels to adjust back and forth between 6.5 and 20 psu, is remarkable and may perhaps be explained by different genotypes of Great Belt and Baltic Sea mussels.     

Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn.

In vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. were successfully cryopreserved by vitrification. Shoot tips (0.5–1 mm) excised from 6-week-old shoots were precultured in hormone-free Woody Plant Medium (WPM) supplemented with 0.2 M sucrose, for 2 days at 4 °C in the dark, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 °C. Osmoprotected shoot tips were first dehydrated with 50 % vitrification solution (PVS2), for 30 min at 0 °C, and then placed in 100 % PVS2, for 30 min at 0 °C. The solution was replaced with fresh 100 % PVS2, and the shoot tips were plunged directly into liquid nitrogen. The shoot tips were rewarmed in a water bath at 40 °C for 2 min, and then washed twice, for 10 min at 25 °C, with 1.2 M sucrose solution, before being transferred onto WPM supplemented with 0.5 mg l^?1 N ⁶-benzyladenine, 0.5 mg l^?1 indole-3-acetic acid, 0.2 mg l^?1 zeatin, 20 g l^?1 glucose and 6 g l^?1 Difco Bacto agar. The shoot tips were kept in darkness for 1 week and under dim lighting for another week, before being exposed to standard culture conditions (16 h photoperiod). This protocol was successfully applied to three alder genotypes, with recovery rates higher than 50 %.     

Quantifying Salmonella Population Dynamics in Water and Biofilms

Members of the bacterial genus Salmonella are recognized worldwide as major zoonotic pathogens often found to persist in non-enteric environments including heterogeneous aquatic biofilms. In this study, Salmonella isolates that had been detected repeatedly over time in aquatic biofilms at different sites in Spring Lake, San Marcos, Texas, were identified as serovars Give, Thompson, Newport and -:z10:z39. Pathogenicity results from feeding studies with the nematode Caenorhabditis elegans as host confirmed that these strains were pathogenic, with Salmonella-fed C. elegans dying faster (mean survival time between 3 and 4 days) than controls, i.e., Escherichia coli-fed C. elegans (mean survival time of 9.5 days). Cells of these isolates inoculated into water at a density of up to 10⁶?ml^?1 water declined numerically by 3 orders of magnitude within 2 days, reaching the detection limit of our quantitative polymerase chain reaction (qPCR)-based quantification technique (i.e., 10³ cells ml^?1). Similar patterns were obtained for cells in heterogeneous aquatic biofilms developed on tiles and originally free of Salmonella that were kept in the inoculated water. Cell numbers increased during the first days to more than 10⁷ cells cm^?2, and then declined over time. Ten-fold higher cell numbers of Salmonella inoculated into water or into biofilm resulted in similar patterns of population dynamics, though cells in biofilms remained detectable with numbers around 10⁴ cells cm^?2 after 4 weeks. Independent of detectability by qPCR, samples of all treatments harbored viable salmonellae that resembled the inoculated isolates after 4 weeks of incubation. These results demonstrate that pathogenic salmonellae were isolated from heterogeneous aquatic biofilms and that they could persist and stay viable in such biofilms in high numbers for some time.     

Fatty acid composition and biological activities of Isochrysis galbana T-ISO,Tetraselmis sp. and Scenedesmus sp.: possible application in the pharmaceutical and functional food industries

Organic and water extracts of Isochrysis galbana T-ISO (=Tisochrysis lutea), Tetraselmis sp. and Scenedesmus sp. were evaluated for their antioxidant activity, acetylcholinesterase (AChE) inhibition, cytotoxicity against tumour cell lines, and fatty acids and total phenolic content (TPC). I. galbana T-ISO had the highest TPC (3.18 mg GAE g^?1) and radical scavenging activity, with an IC₅₀ value of 1.9 mg mL^?1 on the acetone extract. The extracts exhibited a higher ability to chelate Fe²⁺ than Cu²⁺, and the maximum Fe²⁺ chelating capacity was observed in the hexane extract of Scenedesmus sp. (IC₅₀=0.73 mg mL^?1) and Scenedesmus sp. (IC₅₀?=?0.73 mg mL^?1). The highest ability to inhibit AChE was observed in the water and ether extracts of Scenedesmus sp., with IC₅₀ values of 0.11 and 0.15 mg mL^?1, respectively, and in the water extract of I. galbana (IC₅₀?=?0.16 mg mL^?1). The acetone extract of I. galbana T-ISO significantly reduced the viability of human hepatic carcinoma HepG2 cells (IC₅₀?=?81.3 μg mL^?1) as compared to the non-tumour murine stromal S17 cell line, and displayed a selectivity index of 3.1 at the highest concentration tested (125 μg mL^?1). All species presented a highly unsaturated fatty acids profile. Results suggest that these microalgae, particularly I. galbana T-ISO, could be a source of biomolecules for the pharmaceutical industry and the production of functional food ingredients and can be considered as an advantageous alternative to several currently produced microalgae.     

Cloning,Heterologous Expression,Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k _cat of 189.4 s^?1 and K _m of 28.26 mM while M12 GOx had k _cat of 352.0 s^?1 and K _m of 13.33 mM for glucose at pH 5.5. Specificity constants k _cat/K _m of wt (6.70 mM^?1 s^?1) and M12 GOx (26.7 mM^?1 s^?1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.