The influence of cytokinins on proliferation and polyphenol accumulation in shoot cultures of Scutellaria altissima L.

S. altissima shoots were cultivated on MS (Murashige and Skoog) solid medium supplemented with IAA (indole-3-acetic acid, 0.57 μM) and various concentrations of cytokinin: zeatin, kinetin, BAP (6-benzylaminopurine) (1, 2, 4, 8 μM) or TDZ (thidiazuron) (0.2, 0.5, 1 μM). The effects on shoot proliferation and their accumulation of pharmacologically valuable phenolic compounds (baicalin, wogonoside, luteolin, luteolin-7-O-glucoside, verbascoside) were evaluated. Ultra-high performance liquid chromatography (UHPLC) was used for quantitative analysis of the compounds accumulated in the plant biomass. The metabolites were identified by comparing their retention time, UV spectra and mass spectra with those of the standard compounds and published data. The highest metabolite contents were recorded in shoots treated with TDZ at a concentration of 1 μM; under these conditions, the total level of all evaluated flavones expressed as the sum of baicalin, wogonoside, luteolin and luteolin-7-O-glucoside (17.35 mg/g dry wt) was more than twice that of those grown on MS cytokinin-free medium (7.55 mg/g dry wt). Verbascoside accumulation was also stimulated by 1 μM TDZ; its level was about six times higher than that found on the control medium (6.2 mg/g dry wt vs. 1.03 mg/g dry wt). The highest number of shoots (5.5–6.4 per explant within five weeks) was achieved with 2–8 μM BAP.     

Cyanobacterial biomass as N-supplement to agro-waste for hyper-production of laccase from Pleurotus ostreatus in solid state fermentation

The potential application of dry biomass of a cyanobacterium Anacystis nidulans as a supplement in SSF for the production of laccase from Pleurotus ostreatus was evaluated. Experiments were carried out in solid culture using groundnut shell as a basic substrate supplemented with four independent nitrogen sources (ammonium sulphate, urea, yeast extract and dry powder of cyanobacteria). All the four supplements enhanced the enzyme yield, and yeast extract showed precedence over inorganic nitrogenous sources. However, when dry biomass of A. nidulans was used as an additive to groundnut shell (agricultural residues), it supported maximum cell growth (56.83 ± 5.56 mg/g dry substrate) and laccase production (49.21 ± 4.89 U/g dry substrate). Addition of 1 mM copper salt in the medium containing groundnut shell supplemented with yeast extract gave laccase activity of 32.64 ± 3.4 U/g dry substrate. When dry powder of cyanobacterial biomass was used as N-supplement, laccase production enhanced to 65.42 ± 6.48 U/g dry substrate. In addition to the enhancement to enzyme production inhibitory effects of high concentrations of copper was also diminished in the medium having dry cyanobacterial biomass. This study, forms the first report on the potential application of cyanobacterial biomass as an additive for production of laccase by Pleurotus ostraetus MTCC 1804 in solid state fermentation and has relevance in scale-up production of this fungal enzyme of commercial significance.     

Increased production of FAEEs for biodiesel with lipase enhanced Saccharomyces cerevisiae

In this study, we expressed lipase 2 from Candida sp. 99-125 in Saccharomyces cerevisiae, and tried direct biodiesel production. Driven by 3-phosphoglycerate kinase promoter, Lip2 showed high expression level in cytoplasm. SDS-PAGE analysis confirmed the successful lipase expression with a 40 kDa molecular weight. The enzyme assay indicated that lipase 2 had a specific activity of 12.12 μmol/min/mg toward p-nitrophenyl palmitate. Gas chromatography showed that the main fatty acids of S. cerevisiae lipids were palmitoleic acid (31.79%) and oleic acid (29.84%). By three-step addition of 4% ethanol to culture broth, the yield of fatty acid ethyl esters by recombinant S. cerevisiae reached 11.4 mg/g dry cell weight. This work proposed a novel pathway for S. cerevisiae that could be applied for producing biodiesel directly.     

Effect of iron and aeration on superoxide dismutase and catalase activity of PHB-producing Azotobacter chroococcum

The effect of aeration level and iron concentration on Azotobacter chroococcum 23 growth, PHB accumulation and antioxidative enzyme activities was investigated in shake flask experiments. Biomass yield and carbon source conversation coefficients increased in the presence of iron in the growth medium and under decreased aeration. The highest biomass production was observed for the culture grown in a medium with 36 μM of initial iron concentration and moderate aeration level. The highest PHB accumulation level (70–72% from cell dry weight) under our experimental conditions was observed at decreased aeration in the growth medium with 180 μM of initial iron concentration. Results obtained prove that both aeration level and iron supply have a marked influence on the activity of SOD and catalase. Bearing in mind the necessity of iron for the synthesis of both enzymes, only catalase showed a specific dependence on the intracellular iron accumulation level.     

Haematological and biochemical parameters and tissue accumulations of cadmium in Oreochromis niloticus exposed to various concentrations of cadmium chloride

Oreochromis niloticus, weighing 36.45 ± 1.12 g were exposed to 10%, 20% and 30% of the LC₅₀ of CdCl₂ which represents treatments (T1)1.68, (T2)3.36 and (T3)5.03 mg/l, respectively, for a period of 10, 20 and 30 days. It was found that, compared to a control group reading of 0.19 ± 0.03 μg/g dry weight, accumulation of Cd in the gills was significantly (p < 0.05) increased in samples ranging between 7.64 ± 0.86 and 61.73 ± 0.82 μg/g dry weight from T1 at 10 days to T3 at 30 days. The accumulation of Cd in the liver, meanwhile, was also observed to significantly increase (p < 0.05) with increasing time and concentrations with results ranging between 3.21 ± 0.12 and 181.61 ± 1.32 compared to the control group results of 0.29 ± 0.04 μg/g dry weight. Although muscles exhibited lower levels of accumulation than the gills and liver they still showed the same pattern of increase compared to the control group, with a significant difference ranging between 0.32 ± 0.02 and 2.16 ± 0.08 compared to the control group results of 0.03 ± 0.001 μg/g dry weight. Also, haematological parameters such as red blood cells (RBCs), haemoglobin (Hb) and haematocrit (Hct) were reduced in fish exposed to Cd at all periods, with significant differences (p < 0.05). Plasma glucose concentration showed a significant increase. Total protein levels of fish showed a significant reduction (p > 0.05) for all exposed treatments. Also, the total lipid level increased significantly as fish were exposed to increasing cadmium concentrations, compared to control fish. Finally, the activities of aspartate aminotransferase (AST IU/l) and alanine aminotransferase (ALT IU/l) showed a significant increase (p < 0.05) with increasing time and concentrations.     

The effect of glucose and maltose concentrations on pyruvate and ascorbate production,antioxidant enzyme activities and LPO levels in Fusarium equiseti

Changes in pyruvate and ascorbate production and antioxidant enzyme activities together with lipid peroxidation levels in Fusarium equiseti were investigated in relation to changes in the concentrations of glucose and maltose as carbon sources in the range of 5–25 g/l in Armstrong Fusarium Medium (AFM). The highest pyruvate concentration obtained at 20 g/l maltose was 67.5 ± 0.69 μg/ml while ascorbic acid reached a maximum value at 25 g/l glucose of 1866±26.1 μg/ml The maximum superoxide dismutas (SOD) activities related to increased pyruvate production were determined in AFM medium containing 20 g/l glucose as 41.49±0.65 and maltose as 61.12±0.8 IU/mg. Catalase (CAT) activity variations showed coherence with SOD activity in a medium containing maltose and reached 219.11±2.8 IU/mg while they were decreased with increasing glucose concentration. glutathione peroxidase (GSH-Px) activities in F. equiseti did not change significantly with glucose and maltose concentration and were determined to be 1.21±0.22 and 1.67±0.15 IU/mg, respectively. Minimum lipid peroxidation levels for each carbon source were determined in both 20 g/l maltose and glucose concentrations as 0.9 and 1.62 nmol MDA/g wet weight.     

Effect of serum albumin supplementation on in vitro capacitation and fertilization of caprine oocytes

The aim of the investigation was to study the effect of purity and the type of serum albumin on in vitro fertilization (IVF) and cleavage rate of in vitro matured goat oocytes. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulfate per ml. A total of 2509 cumulus oocyte complexes (COCs) were collected from 1313 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (5 μg/ml) and estradiol-17β (1 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO₂ in an incubator under humidified air for 27 h. After 27 h of in vitro maturation (IVM), oocytes were denuded, washed and randomly divided into 4 groups. Group 1 consisted of in vitro matured oocytes (n = 627) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml crystalline bovine serum albumin (BSA) fraction V and 10 μg/ml heparin. Group 2 was comprised of in vitro matured oocytes (n = 470), co-incubated with sperm in a 50 μl drop of TALP medium containing 3 mg/ml crystalline BSA fraction V, 10% estrous goat serum and 10 μg/ml heparin. Group 3 was comprised of in vitro matured oocytes (n = 489) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml fatty acid free BSA and 10 μg/ml heparin. Group 4 consisted of in vitro matured oocytes (n = 422) co-incubated with sperm in a 50 μl drop of TALP medium containing 20% estrous goat serum and 10 μg/ml heparin. After 18 h of co-incubation, the oocyte–sperm mixture was washed in the culture medium 15–20 times and cultured in 50 μl EDM. Cleavage of the in vitro fertilized oocytes were recorded 48 h post-insemination under an inverted phase contrast microscope. The average oocyte recovery rate/ovary and maturation rate was 1.91% and 80.03%, respectively. The cleavage rate in Group 1, Group 2, Group 3 and Group 4 was 1.59%, 8.93%, 11.86% and 35.30%, respectively. It could be concluded that the use of fatty acid free albumin resulted in a significantly higher (P < 0.05) cleavage rate, compared to unmodified albumin, and the supplementation of 20% estrous goat serum in the fertilization medium, significantly (P < 0.05) increased the cleavage rate of in vitro matured goat oocytes, compared to defatted albumin.     

Bioprocess engineering to produce 10-hydroxystearic acid from oleic acid by recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Microbial hydroxylation of long chain fatty acids has been extensively investigated. However, biotransformation productivity remains below ca. 1.0 g/g cell dry weight (CDW)/h under process conditions. In the present study, a highly efficient microbial hydroxylation process to convert oleic acid into 10-hydroxystearic acid was developed. A recombinant Escherichia coli expressing ohyA, the gene encoding oleate hydratase of Stenotrophomonas maltophilia, was used as the biocatalyst. Investigation of the ohyA expression and biotransformation conditions (e.g., inducer concentration, gene expression period before initiating biotransformation, mixing condition of reaction medium) enabled 10-hydroxystearic acid to accumulate to a final concentration of approximately 46 g/L in the culture medium. The specific product formation rate and product yield reached approximately 2.0 g/g CDW/h (i.e., 110 U/g CDW) and 91%, respectively. The specific product formation rate was more than 3-fold higher than those of a bioprocess using wild type Stenotrophomonas sp. cells. Additionally, the product of the whole-cell biotransformation was recovered at a yield of 70.9% and a purity of 99.7% via solvent fraction crystallization at low temperature. These results will contribute to developing a biological process for hydroxylation of oleic acid.     

Resistance exercise modulates lipid plasma profile and cytokine content in the adipose tissue of tumour-bearing rats

Cancer cachexia is a multifactorial syndrome characterised by progressive weight loss, frequently accompanied by anorexia, sarcopenia, and chronic systemic inflammation. The white adipose tissue is markedly affected by cachexia and contributes to this syndrome throught the secretion of pro-inflammatory factors which reach the adjacent tissues and the circulation. A nonpharmacologic intervention that may attenuate cancer cachexia is chronic physical activity, but the effect of resistance training upon adipose tissue inflammation in cachexia has never been examined. For that purpose we designed a protocol in which animals were randomly assigned to a control group (CT, n = 7), a Tumour bearing group (TB, n = 7), a Resistance Trained group (RT, n = 7) and a Resistance Trained tumour bearing group (RTTB, n = 7). Trained rats climbed a vertical ladder with an extra load attached to the tail, representing 75–90% of total body mass, 3 times per week, for 8 weeks. In the 6th week of resistance training, tumour cells (3 × 10⁷ Walker 256 carcinosarcoma) were inoculated in the tumour groups. Body, adipose tissue, muscle and tumour mass was determined, as well a blood biochemical parameters, and the hormone and cytokine profile assessed. The glycogen content of the liver and muscle was measured. IL-10, IL-6 and TNF-α protein expression was evaluated in the mesenteric adipose tissue (MEAT) examined. Resistance training increased by 9% body weight gain in RTTB (final weight 310.8 ± 9.8 g), when compared with TB (final weight 288.3 ± 4.9 g). LDL-c levels were decreased in RTTB (0.28 ± 0.9 mmol/L) by 43% when compared with TB (0.57 ± 0.1 mmol/L). HDL-c levels were increased in RTTB (1.31 ± 0.12 mmol/L) by 15% in regard to CT (1.13 ± 0.7 mmol/L) and 22% as compared with TB (1.07 ± 0.07 mmol/L). RTTB testosterone levels (577 ± 131 ng/mL) were 55% higher when compared with CT (254 ± 41.3 ng/mL) and 63% higher when compared with TB (221 ± 23.1 ng/mL). Adiponectin levels were augmented in RT (23 μg/mL) by 43% when compared with TB (11 μg/mL). Protein expression of IL-6 was increased 38% in TB MEAT (5.95 pg/μg), as compared with CT (3.64 pg/μg) and 50% compared with RTTB (2.91 pg/μg). Similar results with respect to TNF-α TB (7.18 pg/μg) were observed: 39% and 46%, higher protein expression in comparison with CT (4.63 pg/μg) and RTTB (3.8 pg/μg), respectively. IL-10 protein expression was found to be increased in TB (4.4 pg/μg) and RTTB (3.2 pg/μg) 50% and 47%, respectively, in comparison with CT (1.2 pu/μg). The IL-10/TNF-α ratio was higher in RTTB in relation to all others experimental groups. The results show a robust effect of resistance exercise training in preventing important symptoms of cancer cachexia, thus strongly suggesting it may appear as an alternative to endurance exercise as a non-pharmacological therapy in the management of this syndrome.     

Evaluation of single cell oil from Aureobasidium pullulans var. melanogenum P10 isolated from mangrove ecosystems for biodiesel production

In this study, the yeast strain P10 which was identified to be a member of Aureobasidium pullulans var. melanogenum isolated from the mangrove ecosystems was found to be able to accumulate high content of oil in its cells. After optimization of the medium for lipid production and cell growth by the yeast strain P10, it was found that 8.0 g of glucose per 100 ml, 0.02 g of yeast extract per 100 ml, 0.02 g of ammonium sulfate per 100 ml, pH 6.0 in the medium were the most suitable for lipid production. During 10-l fermentation, a titer was 66.3 g oil per 100 g of cell dry weight, cell mass was 1.3 g per 100 ml, a yield was 0.11 g of oil per g of consumed sugar and a productivity was 0.0009 g of oil per g of consumed sugar per h within 120 h. At the same time, only 0.07 g of reducing sugar per 100 ml was left in the fermented medium. The compositions of the fatty acids produced were C_16:0 (26.7%), C_16:1(1.7%), C_18:0 (6.1%), C_18:1 (44.5%), and C_18:2 (21.0%). The biodiesel produced from the extracted lipid could be burnt well.     

Characterisation of novel angiotensin-I-converting enzyme inhibitory tripeptide,Gly-Val-Arg derived from mycelium of Pleurotus pulmonarius

It has been shown that fraction D₆ from Pleurotus pulmonarius has the potential to inhibit ACE. After this discovery, additional studies were conducted to obtain peptides from that fraction, as ACE inhibitors. By size exclusion chromatography, single peak was resolved and termed as Psec. The IC₅₀ of Psec was assessed to be 4.50 μg/mL, which was 2.5 times lower than that of D₆. When Psec was resolved by SDS-PAGE, three bands with estimated molecular weight of 63 kDa, 55 kDa and 11 kDa were observed. The protein bands were subjected to MALDI-Tof MS/MS for protein identification. By using the BIOPEP database for predicting in silico digestion of gastrointestinal (GI) enzymes, four stable tripeptides with ACE inhibitor potential resulting from GI enzyme digestion were identified, namely GVR, VVR, NPR, and VVL. The IC₅₀ was estimated to be 55 μg/mL, 93 μg/mL, 110 μg/mL and >250 μg/mL individually. Based on a Lineweaver-Burk plot, tripeptide GVR was determined to be a competitive inhibitor and this was confirmed by molecular docking analysis. At 100 mg/kg of body weight (bw), the tripeptide GVR reduced SBP 33.5 mm Hg in SHRs. The results suggested that this tripeptide is potentially beneficial as an antihypertensive agent.     

Continuous Cr(VI) removal by Scenedesmus incrassatulus in an airlift photobioreactor

Cr(VI) removal by Scenedesmus incrassatulus was characterized in a continuous culture system using a split-cylinder internal-loop airlift photobioreactor fed continuously with a synthetic effluent containing 1.0 mg Cr(VI) l^?1 at dilution rate (D) of 0.3 d^?1. At steady state, there was a small increase (6%) on the dry biomass (DB) concentration of Cr(VI)-treated cultures compared with the control culture. 1.0 mg Cr(VI) l^?1 reduced the photosynthetic pigments content and altered the cellular morphology, the gain in dry weight was not affected. At steady state, Cr(VI) removal efficiency was 43.5 ± 1.0% and Cr(VI) uptake was 1.7 ± 0.1 mg Cr(VI) g^?1 DB. The system reached a specific metal removal rate of 458 μg Cr(VI) g^?1 DB d^?1, and a volumetric removal rate of 132 μg Cr(VI) l^?1 d^?1.     

Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50 °C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187 g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170 U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli.     

Simplified feeding strategies for fed-batch cultivation of Kluyveromyces marxianus in cheese whey

The present work reports the effect of simple feeding strategies to obtain high-cell-density cultures of Kluyveromyces marxianus maximizing β-galactosidase productivity using cheese whey as basic medium. Linear and exponential feeding strategies, with feeding times of 20, 25 and 35 h, and three different feeding media concentrations (140 g/L, 210 g/L, and 280 g/L lactose concentration), were tested. Final biomass concentration reached 35 g cells dry weight/L and our results showed that continuous lactose addition to culture were able to produce high specific enzyme activities, consequently improving volumetric activities of β-galactosidase when compared to batch cultivations. The best fed-batch strategy, which was the feeding of three-fold lactose concentration in the cheese whey-medium during 25 h, resulted in β-galactosidase productivity of 291 U/L h, representing an increase of more than 50% compared to batch cultivations.     

Comparison of the profiles of non-glycosylated triterpenoids from leaves of plants of selected species of genus Dioscorea

Remarkable qualitative and quantitative differences in non-glycosylated triterpenoid profiles of twelve Dioscorea spp. leaves were demonstrated with the use of GC–MS/FID analysis. The total content of tetracyclic triterpenoids and their esters ranged from 397 μg/g of dry leaf weight in D. bulbifera to 762 μg/g d.w. in D. discolor and 777 μg/g d.w. in D. alata. Three main phytosterols, i.e. campesterol (1), sitosterol (2) and stigmasterol (3) were found in extracts from all analyzed species in total amount ranging from 316 μg/g in D. bulbifera to 676 μg/g of dry leaf weight in D. hispida, with either sitosterol (2) or stigmasterol (3) as predominant in the profile. Extracts from D. hispida and D. purpurea leaves were distinguished from the others by particularly high amount of campesterol (1). In the majority of the species, except for D. caucasica, other tetracyclic triterpenoids were found: cycloartanol (4), 24-methylenecycloartanol (5) and cycloeucalenol (6). Less common steroids, stigmastan-3-en-6β-ol (7) and ergosta-7,22-dien-3β-ol (8) were detected in D. japonica. The significant content (992 μg/g) of pentacyclic triterpenoids of ursane, oleanane, taraxastane and taraxerene (friedooleanane)-type carbon skeletons, i.e. α-amyrin (9), β-amyrin (10), taraxasterol (11) and taraxerol (12), respectively, was found in D. caucasica. The obtained results supplement the knowledge of biochemical diversity of Dioscorea genus.     

Antibiotic resistance profiling and phenotyping of Aeromonas species isolated from aquatic sources

This study aimed to investigate antibiotics resistance pattern and phenotyping of Aeromonas species isolated from different aquatic sources in Melaka, Malaysia. A total of 53 Aeromonas species were isolated from the following sources: sediment (n = 13), bivalve (n = 10), sea cucumber (n = 16) and sea water (n = 14) and resistance to 12 antibiotics – Tetracycline (30 μg), Kanamycin (30 μg), Oxytetracycline (30 μg), Ampicillin (10 μg), Streptomycin (10 μg), Gentamicin (10 μg), Sulphamethoxazole (25 μg), Nalixidic acid (30 μg), Trimethoprim (1.25 μg), Novobiocin (5 μg), Penicilin (10 μg) and Chloramphenicol (10 μg) was tested. The results obtained from this study reveal multi drug resistance pattern among the isolates. All the isolates were completely resistant to Ampicillin, Novobiocin, Sulphamethoxazole and Trimethoprim, respectively but susceptible to Tetracycline (100%), Kanamycin (5.7%), Gentamicin (5.7%) and Oxytetracycline (24.5%). Antibiotics phenotyping of the bacteria revealed 21 different phenotypes among the isolates.     

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Guanosine 5′-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5′-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue.     

Liquid chromatography-tandem mass spectrometry method for determination of the pyridinium aldoxime 4-PAO in brain,liver, lung,and kidney

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and applied to the in vitro determination of 4-[(hydroxyimino)methyl]-1-octylpyridinium cation (4-PAO), which can penetrate the blood–brain barrier and reactivate acetylcholinesterase (AChE) inhibited by alkylphosphonate in the brain, liver, lung, and kidney. The limit of detection (LOD) was 0.235 μg cation/g wet weight, and the quantification range and linearity of the calibration curve extended over a range of 0.470–941 μg cation/g wet weight. For the proof of applicability, when 4-PAO was administrated intravenously via the rat tail vein at 10% LD₅₀, we were able to quantify the 4-PAO concentration in the tissues: brain 7.60 ± 1.32 μg cation/g wet weight (mean ± SD, n = 5), liver 26.8 ± 2.82 μg cation/g, lung 76.4 ± 24.9 μg cation/g, and kidney 638 ± 266 μg cation/g. In addition, the methods for 4-[(hydroxyimino)methyl]-1-decylpyridinium bromide (4-PAD) and 4-[(hydroxyimino)methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) were partly validated referring to the findings of the 4-PAO full validation. Thus, the LC-MS/MS method described in this study can be useful for quantification of pyridinium aldoxime methiodide (PAM)-type oximes in biological samples.     

Saxitoxin monitoring in three species of Florida puffer fish

Beginning in April 2002, three species of Florida puffer fish from around the state of Florida, USA were monitored for the presence of saxitoxin (STX). In total, 873 southern (Sphoeroides nephelus), 171 checkered (S. testudineus), and 53 bandtail (S. spengleri) puffer fish were collected between 2002 and 2006 from eight regions: Jacksonville, the Indian River Lagoon, Tequesta, the Florida Keys, Charlotte Harbor, Tampa Bay, Cedar Key, and Apalachicola. Emphasis was placed on collecting specimens from the Indian River Lagoon (IRL), where recreational harvesting of puffer fish led to 28 cases of saxitoxin puffer fish poisoning (SPFP) between January 2002 and May 2004. Southern puffer fish from the northern IRL routinely contained the highest concentrations of STX, with average levels in the skin of 1787 μg STXequiv./100 g tissue. Elevated concentrations were also found in the muscle (1102 μg STXequiv./100 g), gut contents (539 μg STXequiv./100 g), gonads (654 μg STXequiv./100 g), and liver (214 μg STXequiv./100 g). Lower, yet significant (above the action limit of 80 μg STXequiv./100 g tissue), concentrations of STX were also detected in the skin (599 μg STXequiv./100 g), muscle (233 μg STXequiv./100 g), gut contents (197 μg STXequiv./100 g), and gonads (239 μg STXequiv./100 g) of southern puffer fish from Tequesta in the southern IRL, as well as in the gonads (122 μg STXequiv./100 g) of Jacksonville southern puffer fish and the skin (265 μg STXequiv./100 g) of Tampa Bay southern puffer fish. STX concentrations above the action limit were also found in the skin of bandtail puffer fish from the IRL (620 μg STXequiv./100 g), Tequesta (374 μg STXequiv./100 g), and the Florida Keys (230 μg STXequiv./100 g). Checkered puffer fish collected from the IRL, Tequesta, and the Florida Keys on average were nontoxic, containing STX levels below the action limit in all tissues.