Sensitive isocratic liquid chromatographic assay for the determination of 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin in plasma and tissue with electrochemical detection

A simple extraction procedure and a sensitive high-performance liquid chromatographic (HPLC) method are described for the determination of the photodynamic therapeutic agent 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in plasma and tumour tissue. Reversed-phase high-performance liquid chromatography was performed on a C₁₈ column (70×4.6 mm I.D.) with a mobile phase of 0.01 M potassium dihydrogenphosphate buffer, pH 2.5-acetonitrile (55:45, v/v) and a coulometric detection (+0.80 V). The mean recoveries of mTHPC in the concentration ranges (5–2000 and 10–1000 ng/ml) were 90 and 89% for plasma and tumour samples, respectively. The procedure for plasma and tissue preparation involved solvent precipitation using methanol combined with ammonia solution and dimethyl sulphoxide (4, 0.2, 0.1, v/v/v) and (2, 0.1, 0.1, v/v/v) for plasma and tissue, respectively. For mTHPC at concentrations ranging from 5 to 2000 ng/ml, the within-day relative standard deviations, based on triplicate determinations were less than 8% and the between-day relative standard deviations calculated by performing extraction procedure of plasma samples on three different days ranged from 3 to 18%. This highly sensitive method, 5 and 10 ng/ml for plasma and tissue respectively, was applied successfully to the determination of mTHPC in mouse tumours for pharmacokinetic studies.     

Bioconversion of butyronitrile to butyramide using whole cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of β-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10°C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10°C for 6 h.     

Fermentative production of P(3HB-co-3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method

The feeding of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Alcaligenes eutrophus ATCC17697 was optimized using a fed-batch culture system. The concentration of propionic acid was maintained at 3 g l^–1 as growth was inhibited by propionic acid in the broth. A pH-stat substrate feeding system was used in which propionic acid was fed automatically to maintain a pH of the culture broth at 7.0. By feeding a substrate solution containing 20% (w/v) propionic acid, 4.9% (w/v) ammonia water [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10] in cell growth phase, the concentration of propionic acid in the broth was maintained at 3 g l^–1 giving a specific growth rate of 0.4 h^–1. To promote P(3HB-co-3HV) production, two stage fed-batch culture which consisted of the stage for the cell growth and the stage for the P(3HB-co-3HV) accumulation was carried out. When the substrate solution whose C/N molar ratio was 50 was fed in P(3HB-co-3HV) accumulation phase, the cell concentration and the P(3HB-co-3HV) content in the cells reached 64 g l^–1 and 58% (w/w) in 55.5 h, respectively.     

Cellulolytic enzymes of a thermotolerantAspergillus terreus strain and their action on cellulosic substrates

A thermotolerant fungal strainAspergillus terreus produced high activities of cellulolytic enzymes when grown in shake flasks for 8 days at 40°C or 14 days at 28°C in medium containing 2.5% (w/v) cellulose powder and 1% (w/v) wheat bran. There was little difference between the final activities of endo-(1,4)--glucanase (ca. 14.4 U/ml); filter paper activity (ca. 1.3 U/ml) and -glucosidase (ca. 10 U/ml). Endoglucanase had maximum activity at 60°C and pH 3.8; the other two enzymes were optimal at 60°C and pH 4.8. The maximum hydrolysis of different cellulosic substrates (about 50%) was obtained within 48 h when 1.1 U/ml of filter paper cellulase activity were employed to saccharify 100 mg alkali-treated cotton, filter paper, bagasse, and rice straw at 50°C and pH 4.8. The major end-product, glucose, was produced from all substrates, with traces of cellobiose and other larger oligosaccharides being present in rice straw hydrolysates.     

西瓜花叶病毒诱导对不同抗性美洲南瓜防卫基因表达的影响

为了明确防卫基因PAL与美洲南瓜抗西瓜花叶病毒(watermelon mosaic virus,WMV)之间的关系,通过室内接种和实时荧光定量PCR技术,测定了WMV侵染对不同抗性美洲南瓜体内防卫基因PAL表达的影响。结果表明:(1)室内测定显示,抗病品种GBRV-8发病率和病情指数(15.6%和14.2)显著低于感病品种‘光板’(91.1%和65.9)。(2)实时荧光定量PCR表明,接种WMV后不同抗感品种不同组织部位PAL基因相对表达量随着接种时间增加,整体呈现出先增加后降低的趋势,而且不同组织部位PAL基因相对表达量总体呈现出叶片较高,叶柄和茎秆次之。(3)接种后5个品种不同组织部位PAL基因相对表达量与对照相比均存在显著差异,且抗病和中抗品种不同组织部位PAL基因相对表达量显著高于感病品种,尤其抗病品种GBRV-8不同组织部位PAL基因相对表达量最高,感病品种光板最低。研究认为,防卫基因PAL表达量与美洲南瓜品种抗病毒病强弱密切相关。     

Optimizing conditions for poly(β-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> LC1 in batch culture with sucrose as carbon source

Halomonas boliviensis LC1 is able to accumulate poly(β-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l⁻¹ CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l⁻¹ and 14 g l⁻¹, respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l⁻¹ in 17 h but reduced the PHB content in the cells to 52 wt.%.     

Assessment of the essential oil composition of Tornabenea annua, Tornabenea insularis and Tornabenea tenuissima fruits from Cape Verde Islands

The essential oils of Tornabenea annua, Tornabenea insularis and Tornabenea tenuissima herbarium or in vivo fruits, collected in Fogo, Santiago, Santo Antão, São Nicolau and São Vicente Islands, from Cape Verde archipelago, or from plants grown in Lisbon, Portugal, were isolated by hydrodistillation and analysed by GC and GC–MS. The yellowish oils were obtained in variable average yields, lower in herbarium samples [0.05% (v/w)] and higher from in vivo samples [1.3% (v/w)]. Whereas T. annua fruits' oils were all dominated by myristicin (92–100%), most of the T. insularis fruit samples' oils were elemicin rich (82–90%). No clear information could be obtained for T. tenuissima fruits' oils as each of the samples gave different chemical composition. Cluster analysis of the essential oil composition from the fruits' samples studied, confirmed these chemical differences.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

Cellulase production and activity in a species ofCladosporium

ACladosporium species produced large amounts of cellulase enzyme components when grown in shake-culture with medium containing carboxymethylcellulose. There was significantly less activity when Avicel, filter paper or cotton were used as substrates. KNO₃ was better than NH₄Cl or urea for the production of cellulase. Tween 80 at 0.1% (w/v) increased the production of cellulase by 1.5 to 4.5-fold. All the cellulase components were optimally active in the assay at pH 5.0 and 60°C.     

The effect of co-cultivation and selection parameters on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese soybean varieties

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l⁻¹) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l⁻¹ hygromycin for 10 days, 5 mg l⁻¹ hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l⁻¹ hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.     

Measurement of particle diameter of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> microcapsule by spray drying and analysis on its microstructure

Lactobacillus acidophilus, as a probiotic, is widely used in many functional food products. Microencapsulation not only increases the survival rate of L. acidophilus during storage and extends the shelf-life of its products, but also optimal size microcapsule makes L. acidophilus have an excellent dispersability in final products. In this paper, L. acidophilus was microencapsulated using spray drying (inlet air temperature of 170°C; outlet air temperature of 85–90°C). The wall materials used in this study were β-cyclodextrin and acacia gum in the proportion of 9:1 (w/w), and microcapsules were prepared at four levels of wall materials (15, 20, 25 and 30% [w/v]) with a core material concentration of 6% (v/v). The microcapsule diameters were measured by Malvern’s Mastersizer-2000 particle size analyzer. The results showed that the particle diameters of microcapsule were mostly within 6.607 μm and 60.256 μm and varied with 2.884–120.226 μm (the standard smaller microcapsule designated as <350 μm). Through comparison of microcapsule size and uniformity with different concentration of wall materials, we concluded that the optimal concentration of wall material was 20% (w/v), which gave microcapsule with a relatively uniform size (averaging 22.153 μm), and the number of surviving encapsulated L. acidophilus was 1.50 × 10⁹ c.f.u./ml. After 8 weeks storage at 4°C, the live bacterial number was above 10⁷ c.f.u./ml, compared with unencapsulated L. acidophilus, 10⁴–10⁵ c.f.u./ml. Through the observation of scanning electron microscopy, we found that the shapes of microcapsule were round and oval, and L. acidophilus cells located in the centre of microcapsule.     

Micropropagation of Chokecherry and Pincherry cultivars

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of chokecherry (Prunus virginiana L.), `Garrington', and pincherry (P. pensylvanica L.f), `Mary Liss' and `Jumping Pound', were examined using various combinations of growth regulators. Dormant winter buds were used as explants. MSMO medium supplemented with 0.49 μM IBA and either 4.44 or 8.87 μM BA was found to be optimal for culture initiation of both species and cultivars. GA₃ (28.89 μM) significantly reduced (p=0.0001) the number of successfully established cultures. BA concentrations 8.87–12.82 μM gave optimal shoot proliferation in chokecherry and 4.44 μM BA in both cultivars of pincherry. Auxin treatments were required for ex vitro rooting of approximately 10 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (84%) was obtained with IBA/NAA (9.80/2.69 μM). A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%) mixture, was also effective (75%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Verticillium lecanii Spore Formulation Using UV Protectant and Wetting Agent and the Biocontrol of Cotton Aphids

Verticillium lecanii spores (10⁸ spores ml⁻¹) suspended in 1% (w/v) montmorillonite SCPX-1374 and 1% (w/v) of the wetting agent, EM-APW#2, which is a polyoxyethylene, had approx. 80% survival after exposure to UV-C for 30 min and about 93% after exposure to UV-B for 6 h. In greenhouse testing, cotton aphid densities increased 14-fold over their initial density in 15 d without spore application. However, initial cotton aphid densities were decreased by 60% of the initial level when plants were treated with the spore formulation.     

Laboratory evaluation of deet and permethrin impregnated wide-mesh netting against mosquitoes

Behaviour of two species of mosquitoes was studied by releasing them into a tunnel of cages in which either deet or permethrin impregnated wide-mesh cotton or nylon netting was stretched across the tunnel between the mosquito entry point and an animal bait.Di-ethyl toluamide (deet) was found to be more effective in repelling Culex quinquefasciatus say when pieces of impregnated thick cotton netting were placed at a distance from the bait rather than close to the bait. Anopheles gambiae Giles was repelled at lower deet dosages than was Cx. quinquefasciatus. One impregnation remained effective for 7 weeks when the netting was impregnated with 25 ml per m² held in still air in a room. When it was held in a plastic bag the shelf life was at least one year.Permethrin impregnated nylon netting of 4 mm or 6 mm mesh-size had a strong toxic and excitorepellent effect on An. gambiae. However, the effects were much weaker when the mesh size was 13 mm. Permethrin impregnated 8 mm-mesh thick, cotton netting offered very good protection against mosquitoes for at least 30 weeks. There was no marked difference in effectiveness between doses of 0.2 or 0.5 g/m².

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Résumé Le comportement de deux espèces de moustiques a été examiné en les lâchant dans un tunnel formé par 6 cages successives dans lesquelles étaient tendus, entre l'entrée et un animal proie, des filets de coton ou de nylon imprégnés de deet ou de permethrine. La répulsion par le deet (di-éthyl toluamide) s'est révélé plus efficace contre Culex quinquefasciatus avec les filets de coton tendus à une certaine distance de la proie plutôt qu'à proximité. Anopheles gambiae a été repoussé par des doses plus faibles de deet que C. quinquefasciatus. Une imprégnation du filet avec 25 ml/m² est resté efficace pendant 7 semaines dans une pièce où l'air était calme. Conservé dans un emballage plastique la durée de conservation a été au moins d'un an.La permethrine imprégnant un filet de nylon de maille 4 à 6 mm a présenté un effet toxique et repulsif-excitant très fort contre A. gambiae. L'effet a été beaucoup plus faible lorsque les mailles étaient de 18 mm. La permethrine, imprégnant un filet de coton de maille 8 mm, a fourni une très bonne protection contre les moustiques pendant au moins 30 semaines. Il n'y a pas eu de différences significatives entre des doses de 0,2 et 0,5 g/m².

     

Production of Deuterated Lutein by Chlorella protothecoides and its Detection by Mass Spectrometric Methods

Chlorella protothecoides, a lutein-producing microalga, was grown aerobically in a mineral medium prepared with 70% (v/v) deuterated water. HPLC/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MS) analysis revealed 58% replacement of hydrogen by deuterium atoms as indicated by the molecular mass cluster at around m/z 599. The rapidly growing microalga had much higher levels (58%) of deuterium substitution relative to previously reported (9–15%) natural sources of lutein.     

High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of a new oral thrombin inhibitor (compound I) in the blood of rats and dogs. The analyte was deproteinized with a 1.5 volume of methanol and a 0.5 volume of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a mixture of acetonitrile and 0.2% triethylamine of pH 2.3 (31:69, v/v) with a flow-rate of 1.0 ml/min at UV 231 nm. The retention time of compound I was approximately 9.3 min. The calibration curve was linear over the concentration range of 0.05–100 mg/l for rat blood (r²>0.9995, n=6) and dog blood (r²>0.9993, n=6). The limit of quantitation was 0.05 mg/l for both bloods using a 100-μl sample. For the 5 concentrations (0.05, 0.1, 1, 10, and 100 mg/l), the within-day recovery (n=4) and precision (n=4) were 98.1–104.1% and 1.5–6.8% for rat blood and 95.4–105.7% and 1.4–5.3% for dog blood, respectively. The between-day recovery (n=6) and precision (n=6) were 99.8–105.3% and 3.7–12.6% for rat blood and 87.5–107.1% and 2.9–15.3% for dog blood, respectively. The absolute recoveries were 82.4–93.3%. No interferences from endogenous substances were observed. In conclusion, the presented simple, sensitive, and reproducible HPLC method proved and was used successfully for the determination of compound I in the preclinical pharmacokinetics.     

Intergeneric protoplast fusion between Kluyveromyces and Saccharomyces cerevisiae – to produce sorbitol from Jerusalem artichokes

The construction of inulin-assimilating and sorbitol-producing fusants was achieved by intergeneric protoplast fusion between Kluyveromyces sp. Y-85 and Saccharomyces cerevisiae E-15. The cells of parental strains were pretreated with 0.1% EDTA (w/v) and 2-mercaptoethanol (0.1%, v/v) and then exposed to 2.0% (w/v) Zymolase at 30 °C for 30–40 min. The optimized fusion condition demonstrated that with the presence of 30% (w/v) polyethylene glycol 6000 (PEG-6000) and 10 mM CaCl₂ for 30 min, the fusion frequency reached 2.64 fusants/10⁶ parental cells. The fusants were screened by different characters between two parental strains and further identified by DNA contents, inulinase activity and sorbitol productivity. One of the genetically stable fusants, Strain F27, reached a maximal sorbitol production of 4.87 g/100 ml under optimal fermentation condition.     

Disease forecasting model for newly emerging bacterial seed and boll rot of cotton disease and its vector (Dysdercus cingulatus)

Bacterial seed and boll rot disease is a newly emerging threat to the cotton growers. Disease prediction model was devised to predict the disease progression impacted by the vector (Dysdercus cingulatus) and environmental variables (maximum air temperature, minimum air temperature, relative humidity and rainfall) on four varieties to minimise its losses and disease management cost. Impact of a-biotic environmental variables (maximum and minimum air temperature, relative humidity and rainfall) was assessed on bacterial seed and rot of cotton disease and its vector (D. cingulatus) on FH-941, FH-942, MNH-886 and FH-114 cotton varieties. Maximum red cotton bug population was assessed at 29–31 °C maximum temperature and at 15–17 °C minimum temperature. Disease severity was noticed maximum when maximum and minimum temperature was measured at 28–29 °C and 13–14.5 °C, respectively. Vector population was maximum when relative humidity and rainfall were 63–66% and 1.50–2.5 mm, respectively. Relative humidity at 66–68% and 0.5–1.5 mm rainfall favoured disease development. With increase in number of bugs, increase in disease severity was noted, maximum disease severity 45–48% noticed when 7–8 bugs were recorded. Red cotton bug (Dysdercus cingulatus) population prediction model was devised based on a-biotic factors (maximum and minimum air temperature, relative humidity and rainfall) on four cotton varieties. Disease forecasting model was developed based on biotic (D. cingulatus) and a-biotic factors. A close resemblance between observed and the predicted red cotton bugs and disease severity was seen.     

A comparative electrophoretic study of the seed albumins fromSophora microphylla andPisum sativum (Leguminosae)

The albumin proteins from seed ofSophora microphylla Ait. and from cotyledons ofPisum sativum L. (cv. Greenfeast) have been analysed electrophoretically using a range of gels of varied pore size. Plots of mobility [as 100 log₁₀ (R _f × 100)] vs.acrylamide content of gel indicate that very few of the albumins fromS. microphylla are homologous with albumins fromP. sativum. Despite the diverse compositions of the two fractions, their amino acid analyses were surprisingly similar.