Determination of 5-S-cysteinyldopa in plasma and urine using a fully automated solid-phase extraction–high-performance liquid chromatographic method for an improvement of specificity and sensitivity of this prognostic marker of malignant melanoma

5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S- -cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l⁻¹ and 25 to 5000 μg l⁻¹ for plasma and urine samples, respectively, a limit of detection of 0.17 μg l⁻¹, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons.     

Determination of

Summary This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l^−1 2,4-dichlorophenoxyacetic acid and 0.5–2.0 mg l^−1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0–2.0 mg l^−1 α-naphthaleneacetic acid (NAA) and 0.5–2.0 mg l^−1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l^−1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl^−1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.     

Callus induction and high-frequency plant regeneration from hypocotyl and cotyledon explants of Arctium lappa L.

Summary This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l^−1 2,4-dichlorophenoxyacetic acid and 0.5–2.0 mg l^−1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0–2.0 mg l^−1 α-naphthaleneacetic acid (NAA) and 0.5–2.0 mg l^−1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l^−1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl^−1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.     

Effects of Growth Regulators on Direct Flowering of Isolated Ginseng Buds <Emphasis Type="Italic">in vitro</Emphasis>

An in vitro method for obtaining gingseng inflorescences directly from explants of gingseng (Panax ginseng) is reported. Isolated shoot-buds of somatic embryo-derived plantlets ginseng were used as explants and incubated in B5 medium supplemented with 1 mg l⁻¹ benzyladenine (BA) and 1 mg l⁻¹ gibberellic acid (GA₃). About 15% of the buds flowered directly without developing vegetative organs. Cytokinin was found to be the key factor for inducing these isolated buds to proliferate and flower, but both these processes also occurred when benzyladenine (BA) was replaced by thidiazuron (TDZ). The optimal concentration of TDZ for obtaining the best ratios of bud proliferation and total flowering was 0.1 mg l⁻¹, while the highest number of vegetative shoots was obtained in medium supplemented with 1 mg l⁻¹ GA₃ and 0.1 mg l⁻¹ TDZ. The explant elongated abnormally in the presence of 10 mg l⁻¹ GA₃. Although a low concentration (1 mg l⁻¹) of NAA increased the bud proliferation ratio in the medium supplemented with 0.1 mg l⁻¹ TDZ and 1 mg l⁻¹ GA₃, a high concentration (5 mg l⁻¹) of NAA reduced the bud proliferation ratio and inhibited the flowering.     

Sulfalene concentrations in plasma and blood cells of Plasmodium falciparum malaria cases after treatment with metakelfin using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method using acetonitrile–methanol–1 M perchloric acid–water (25:9:0.8:95, v/v/v) at a flow-rate of 1.0 ml min⁻¹ on LiChrospher 100 RP 18 column (250×4 mm; 5 μm) with UV (254 nm) detection has been developed for the determination of sulfalene in plasma and blood cells after oral administration of the antimalarial drug metakelfin. Calibration curves were linear in the range 0.5–100 μg ml⁻¹. The limit of quantification was 50 ng ml⁻¹. Within-day and day-to-day coefficients of variation averaged 3.84 and 5.31%, respectively. Mean extraction recoveries of sulfalene from plasma and blood cells were 87.21 and 84.65%, respectively. Mean concentrations of sulfalene in plasma of P. falciparum cases on days 2, 7 and 15 were 44.58, 14.90 and 1.70 μg ml⁻¹, respectively; in blood cells concentrations of sulfalene were 7.77, 3.25 and 0.75 μg ml⁻¹, respectively, after oral treatment with two tablets (1000 mg) of metakelfin. Significant difference was recorded on day 2 for sulfalene concentration in blood cells of healthy and P. falciparum cases (t=9.49; P<0.001).     

Development of haploid asparagus embryos from liquid cultures of anther-derived calli is enhanced by ancymidol

Summary The effect of ancymidol concentration on the development of haploid asparagus embryos was determined. Liquid cultures from anther-derived calli were grown for three weeks in MS medium plus 1.0 mg l^–1 2,4-D, 0.1 mg l^–1 NAA, 0.2 mg l^–1 kinetin, 800 mg l^–1 glutamine, 500 mg l^–1 casein hydrolysate, 2% sucrose and 0.0–1.0 mg l^–1 ancymidol. Cell clumps (224–500 m) were plated on solid embryo maturation medium (MS medium plus 3% sucrose, 0.1 mg l^–1 NAA, 0.5 mg l^–1 kinetin and 0.0–1.0 mg l^–1 ancymidol) and grown for eight weeks. Ancymidol enhanced embryo maturation and germination and was more critical in the solid than liquid medium. Total embryo number did not vary among most treatments. The best response was observed when ancymidol concentrations were 0.1 and 0.5 mg l^–1 in the liquid and solid media, respectively; two-thirds of the embryos produced were bipolar and 35% of bipolar embryos germinated. Seven to 82% of plants recovered from different ancymidol treatments were haploid; the others were diploid, triploid or chimeric for ploidy level.Abbreviations NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962)     

Induction of umbelliferone in sweet potato cell suspension culture using mercuric chloride

HgCl₂ was used at up to 10 mg l^–1 as an elicitor of phytoalexins in sweet potato (Ipomoea batatas (L.) Lam. cv Centennial) cell suspension cultures. Maximum stimulation of a coumarin compound was after one day of exposure using 1 mg HgCl₂ l^–1. The compound was identified by HPLC and GC-MS analyses as 7-hydroxycoumarin (umbelliferone).     

Clonal propagation of Enset (Ensete superbum (Roxb.) Cheesman) through shoot tip cultures

Summary The morphogenetic potential of the shoot tip explants ofEnsete superbum (Roxb.) Cheesman, a wild relative of the cultivated bananas, was investigated and an effective clonal propagation method devised. Shoot tip explants grown in modified MS medium containing 1.5 mg l^–1 BAP and 1 mg l^–1 KIN developed corms which on transfer to medium containing 3 mg l^–1 IBA and 1.5 mg l^–1 BAP, regenerated a large number of shoots from the surface of the corm, the origin of which was traced to single hypodermal cells. Shoots were rooted on a half-strength MS medium salts containing 3 mg l^–1 IBA and 0.1 mg l^–1 BAP. The rooted plantlets were hardened and planted in the field where the plants looked normal.     

Enzymatic pre-hydrolysis applied to the anaerobic treatment of effluents from poultry slaughterhouses

A pool of hydrolases with 21.4 U g⁻¹ lipase activity was produced through solid-state fermentation of the fungus Penicillium restrictum in waste from the Orbignya oleifera (babassu) oil processing industry. Enzymatic hydrolysis and anaerobic biodegradability tests were conducted on poultry slaughterhouse effluents with varying oil and grease contents (150–1200 mg l⁻¹) and solid enzymatic pool concentrations (0.1–1.0% w/v). Enhanced anaerobic treatment efficiency relative to raw effluent was achieved when a 0.1% concentration of enzymatic pool was used in the pre-hydrolysis stage with 1200 mg oil and grease l⁻¹ (chemical oxygen demand (COD) removal efficiency of 85% vs. 53% and biogas production of 175 ml vs. 37 ml after 4 d).     

Isolation and characterization of a white rot fungus <Emphasis Type="Italic">Bjerkandera</Emphasis> sp. strain capable of oxidizing phenanthrene

Strain BOL13 was selected from 18 fungal strains isolated from an oil-spill contaminated site in Oruro, Bolivia. It was identified as a basidiomycete with high homology to Bjerkandera. The fungus degraded 100 mg phenanthrene l⁻¹ at 0.17 mg l⁻¹ d⁻¹ at 30 °C at pH 7. During phenanthrene degradation, a maximum manganese peroxidase activity of 100–120 U l⁻¹ was measured after 10 days of incubation. The ability of Bjerkandera sp. to produce lignin-modifying enzymes and to oxidize phenanthrene under various pH and temperature conditions was confirmed.     

Immobilization of -glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and -glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized -glucose oxidase membrane was 0.34 units cm⁻² and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized -glucose oxidase membrane was 1.6 × 10⁻³ mol l⁻¹ and that of free enzyme was 4.8 × 10⁻² mol l⁻¹. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized -glucose oxidase membrane. The enzyme electrode responded linearly to -glucose over the concentration 0–1000 mg dl⁻¹ within 10 s. When the enzyme electrode was applied to the determination of -glucose in human serum, within day precision (CV) was 1.29% for -glucose concentration with a mean value of 106.8 mg dl⁻¹. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized -glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of -glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Efficient Plant Regeneration System From Leaf Discs of Zonal (Pelargonium x hortorum) and two scented (P. capitatum and P. graveolens) Geraniums

An efficient and rapid plant regeneration system was established for zonal and scented geraniums using leaf discs as explants. Several explants, medium and culture conditions were studied to optimize shoot induction. Leaf discs taken from 4–5 weeks old in vitro grown plants, whatever the genotype, were more effective for shoot regeneration than those taken from greenhouse grown plants. Darkness proved to be a stimulating factor for shoot regeneration and the combination between NAA and two cytokinins gave the best results. Direct shoot regeneration (100%) was obtained from leaf discs of P. capitatum on half-strength MS medium supplemented with 0.5 mg l⁻¹ NAA in combination with 1 mg l⁻¹ of BAP and zeatin in darkness (11.4 shoots per explant). In the same medium and culture conditions, all P. graveolens leaf discs also exhibited direct shoot regeneration (7.3 shoots per explant). For P. x hortorum, 100% of leaf discs underwent shoot regeneration on a MS medium supplemented with 0.2 mg l⁻¹ NAA in combination with 0.5 mg l⁻¹ of BAP and zeatin in darkness (8.8 shoots per explant) or under low light conditions with 0.2 mg l⁻¹ NAA and 1 mg l⁻¹ of BAP and zeatin (7.5 shoots per explant). For this species, the best results for shoot elongation were obtained on half-strength MS medium gelled with Phytagel 0.3% (v/v). Whatever the genotype, all shoots rooted readily when transferred to diluted MS medium (MS/2) containing 1 mg l⁻¹ IAA. Acclimatized plants grew normally and flowered in greenhouse conditions. Flow cytometry analysis made on leaves of acclimatized plants revealed that all the scented geranium plants are similar to mother plants while 71% of P. x hortorum plants which showed strong growth were tetraploid.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Efficient GA3-assisted plant regeneration from cell suspensions of three grape genotypes via somatic embryogenesis

Petioles from in vitro grown plants of interspecific grapevine hybrids cvs `Bianca', `Podarok Magaracha' and `Intervitis Magaracha' were cultured on solid NN medium supplemented with 2,4-D and BA at various concentrations. The callus developed was cultured in liquid NN medium supplemented with 0.5 mg l<sup>–1</sup> BA to induce formation of somatic embryos. Somatic embryos of globular and heart-stage developed in suspensions of `Podarok Magaracha' and `Intervitis Magaracha'. In contrast, `Bianca' did not undergo embryogenesis beyond globular stage. This made it necessary to perform subculture of the suspensions to HTE liquid medium supplemented with 0.2 mg l^–1 BA for the development of globular embryos into heart stage. Heart-stage embryos developed into torpedo-stage after subculturing suspensions of all three cultivars to liquid HTE medium supplemented with 0.1 mg l^–1 IAA and 30 mg l^–1 sodium hummate. Torpedo-stage embryo suspensions were subcultured in liquid HTE medium supplemented with 0.5 mg l^–1 BA, 0.5 mg l^–1 GA₃ and 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA. After 12 days of incubation, plantlets were cultured on solid M2MS medium: without growth regulators and with 0.5 mg l^–1 BA. Plantlets that developed in liquid HTE media with 0.5 mg l^–1 GA₃ or 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA produced 82–90% shoots on solid M2MS medium with 0.5 mg l^–1 BA after 50 days of culture.     

Effects of nutritional factors on the formation of Anthraquinones in callus cultures of Rheum ribes

Rheum ribes L. (Polygonaceae) is the source of one of the most important crude drugs in Asiatic regions .The medicinal character of rhubarb is due to its anthraquinone content . Different parts of sterile seedlings were cultured on MS medium to study the generation of callus. The explants were cultured with different ranges of plant growth regulators and the best range of plant growth regulators for generation of callus was IBA (1 mg l^–1) and BA (1 mg l^–1). The content of anthraquinones were determined by HPLC . The concentration of sucrose, vitamins and Myo-inositol and ratio of NO₃ to NH₄ in the medium was changed and growth rate and content of 2 anthraquinones was determined . The growth rate of callus declined with increased rate of secondary metabolites production. Myo-inositol 100 mg l^–1 in the medium increased anthraquinone content and in medium that had NO₃/NH₄:1/1 the maximum content of anthraquinone was obtained.     

Determination of phytic acid by gas chromatography–mass spectroscopy: application to biological samples

A GC–MS method is reported for the determination of phytic acid based on purification by anion-exchange chromatography, enzymatic hydrolysis of phytic acid to myo-inositol and derivation to trimethylsilyl derivative, with scyllo-inositol as an internal standard. Analytical features of the method are: limit of detection 9 μg l⁻¹ phytic acid, linear working range 18–500 μg l⁻¹ phytic acid, and coefficient of variation 1.9%. The method has been successfully applied to a variety of biological samples: various rat organs (kidney, liver, brain and bone), human plasma and urine and kidney stones. A comparative study of sample treatments, including deproteization, lipid extraction and the presence of a chelator, is also reported. Phytic acid amounts found in rat organs ranged from 1.07 g kg⁻¹ for bone to 32.0 g kg⁻¹ for brain. Phytic acid in human plasma was of the order of 0.14 mg l⁻¹. In kidney stones, phytic acid was found in calcium containing stones.     

A description of ammonium content of output waters from trout farms in relation to stocking density and flow rates

Daily levels of ammonium content in the outflow water of an intensive trout farm and of a mountain pond stocked with rainbow trout were monitored. Output water was sampled for 30 days and analysed every 30 min by an automatic ion selective electrode system (Applikon ADI 2013); input water was also monitored. The ammonium output level was influenced more by atmospheric events than nitrogen excretion by fish. Observed data confirmed an overall ammonium excretion model previously estimated in both laboratory and field conditions. The high water flow, that characterises the intensive trout farm where the observations were made induced a high dilution of metabolites. Consequently, if the peaks of ammonium output did not reach values of 0.35–0.40 mg l⁻¹ the environmental impact was limited and not easily detected. Our results allow us to affirm that the optimal level of water flow rate for effective zeolite-operated filtration is around 10.3 l t⁻¹ s⁻¹.     

Synthesis of -Dopa by Escherichia intermedia cells immobilized in a carrageenan gel

Escherichia intermedia cells were immobilized by entrapment in a carrageenan gel and used for -DOPA synthesis from catechol, pyruvate, and ammonia. A preparation containing 75 mg of cell per gram of gel retained 60–65% of its original activity. The effect of substrate concentrations on the initial rate of -DOPA synthesis was very similar for free and immobilized cells, and substrate inhibition was observed for the three substrates. In batch reactors, up to 7.8 g l⁻¹ of -DOPA was obtained in 20 h (productivity 0.39 g l⁻¹ h⁻¹). Cells immobilized in a carrageenan gel showed higher -DOPA synthesis, in both initial rates conditions and batch reactors, than cells immobilized in a polyacrylamide gel.     

Determination of zearalenone and its metabolites α- and β-zearalenol in beer samples by high-performance liquid chromatography–tandem mass spectrometry

A fast, robust and sensitive LC–MS–MS method for the determination of zearalenone (ZON) and its metabolites α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in beer samples is described. Sample preparation was performed by direct RP-18 solid-phase extraction of undiluted beer samples followed by selective determination of analytes by LC–MS–MS applying an atmospheric pressure chemical ionization (APCI) interface. Using the negative ion mode limits of determination of 0.03–0.06 μg l⁻¹ beer and limits of quantification of 0.07–0.15 μg l⁻¹ beer were achieved, which was distinctly more sensitive than in the positive ion mode. Twenty-three beer samples from different countries, produced from different grains and under different brewing conditions, were investigated by this method, but only in one sample could β-ZOL and ZON be detected. Independently of the type of beer, relative standard deviations between 2.1% and 3.3%, a linear working range of 0.15 μg l⁻¹ to 500 μg l⁻¹ beer and recovery rates around 100% could be achieved when zearalanone (ZAN) was used as internal standard.     

Rapid micropropagation of Woodfordia fruticosa (L.) Kurz (Lythraceae), a rare medicinal plant

A rapid propagation method comprising initiation of in vitro shoot tip culture from field-grown flowering plants and reculture of the nodal segments of regenerated shoots in Schenk and Hildebrandt (1972) medium was developed for Woodfordia fruticosa (L.) Kurz., a rare medicinal shrub. A medium supplement of 6-benzylaminopurine (0.2 mg.l^–1) induced high frequency (88%) development of axillary shoot buds (3.2) in 4–5 weeks. Subculture of the explants with multiple new shoots in fresh medium for 30 days yielded an even larger number (9.7) of shoots. Highest multiplication (26–35 shoots) was recorded when using culture initiation media with 0.5 mg.l^–1 each of BAP and NAA followed by subculture in 0.2 mg.l^–1 BAP. The shoot multiplication rate was further accelerated by reculturing 0.4–0.6 cm nodal segments of regenerated shoots in media with 1.0 mg.l^–1 BAP. Shoot cuttings (3.5–7.0 cm) were rooted in 0.2 mg.l^–1 IAA. Regenerated plants displayed uniform morphological, growth and flowering characteristics.Abbreviations BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA indole-3-butyric acid - SH Schenk and Hildebrandt (1972) medium