Direct rooting and hardening of tea microshoots in the field

Micropropagation of tea (Camellia sinensis (L.) O. Kuntze) has been widely attempted but commercial exploitation of this method is limited by heavy losses during the hardening procedures. In the present study, optimization of time of harvesting (spring and early summer) of microshoots, shoot size, soil pH (4.0–6.4), plant growth regulator treatment (IBA; 500 mg l^-1, 30 min) CO₂ (9.09/10×10⁻⁵ mol l^-1 to 10.22/10×10^-5 mol l^-1 and 20/11×10⁻⁵ mol l^-1 to 80/13×10⁻⁷ mol l^-1) enrichment and light (15 μ mol m^-2 s^-1) conditions in specially designed hardening chambers, made a significant impact on the percent of success for hardening. Following the standardized procedure, up to 71.6% root induction and 73% survival could be achieved. Successful field transfer was also accomplished. This revised version was published online in June 2006 with corrections to the Cover Date.     

Toxicity of anionic and cationic surfactant to Acinetobacter junii in pure culture

The harmful effects of surfactants to the environment are well known. We were interested in investigating their potential toxicity in a pure culture of Acinetobacter junii, a phosphate (P)-accumulating bacterium. Results showed a high acute toxicity of sodium dodecyl sulfate (SDS) and hexadecyltrimethylammonium bromide (HDTMA) against A. junii. The estimated EC₅₀ values of the HDTMA for the inhibition of CFUs in the pure culture of A. junii was 3.27 ± 1.12 × 10⁻⁷ mol L⁻¹ and for the inhibition of the P-uptake rates 2.47 ± 0.51 × 10⁻⁶ mol L⁻¹. For SDS, estimated EC₅₀ values for the inhibition of CFUs in the pure culture of A. junii was 5.00 ± 2.95 × 10⁻⁶ mol L⁻¹ and for the inhibition of the P-uptake rates 3.33 ± 0.96 × 10⁻⁴ mol L⁻¹. The obtained EC₅₀ values in the standardised yeast toxicity test using Saccharomyces cerevisiae were 3.03 ± 0.38 × 10⁻⁴ and 4.33 ± 0.32 × 10⁻⁵ mol L⁻¹ for SDS and HDTMA, respectively. These results emphasized the need to control concentrations of surfactants entering the activated sludge system. The negative effects of these toxicants could greatly decrease populations of P-accumulating bacteria, as well as eukaryotic organisms, inhabiting activated sludge systems, which in turn could result in the decrease of the system efficiency.     

Characterisation of acidic sites of Pseudomonas biomass capable of binding protons and cadmium and removal of cadmium via biosorption

Summary The ability of Pseudomonas aeruginosa to accumulate Cd(II) ions from wastewater industries was experimentally investigated and mathematically modelled. From the potentiometric titration and non-ideal competitive analysis (NICA) model, it was found that the biomass contains three acidic sites. The values of proton binding (pK _i =1.66±3.26×10⁻³, 1.92±1.63×10⁻⁴ and 2.16±3.79×10⁻⁴) and binding constant of cadmium metal ions (pK _M1=1.99±2.45×10⁻³ and pK _M2=1.67±4.08×10⁻³) on the whole surface of biomass showed that protonated functional groups and biosorption of Cd(II) ions could be attributed to a monodentate binding to one acidic site, mainly the carboxylic group. From the isothermal sorption experimental data and Langmuir model, it was also found that the value of Langmuir equilibrium (pK _f) constant is 2.04±2.1×10⁻⁵ suggesting that the carboxyl group is the main active binding site. In addition, results showed that the maximum cadmium capacity (q _max) and affinity of biomass towards cadmium metal ions (b) at pH 5.1 and 20 min were 96.5±0.06 mg/g and 3.40×10⁻³± 2.10×10⁻³, respectively. Finally, interfering metal ions such as Pb(II), Cu(II), Cr(III), Zn(II), Fe(II), Mn(II), Ca(II) and Mg(II) inhibited Cd(II) uptake. Comparing the biosorption of Cd(II) by various Pseudomonas isolates from contaminated environment samples (soil and sewage treatment plant) showed that maximum capacities and equilibrium times were different, indicating that there was a discrepancy in the chemical composition between biomasses of different strains.     

The effect of substituted amides of pyrazine-2-carboxylic acids on flavonolignan production in <Emphasis Type="Italic">Silybum marianum</Emphasis> culture <Emphasis Type="Italic">in vitro</Emphasis>

In vitro plant tissue and cell cultures were used to study herbicide effects on growth, selected metabolic activities and other phenomena. The effect of abiotic elicitors, two newly synthesized substituted amides of pyrazine-2-carboxylic acids (synthesized at the Department of Pharmaceutical Chemistry and Drug Control, School of Pharmacy in Hradec Kralove), on the flavonolignan accumulation in callus and suspension culture of Silybum marianum (L.) Gaertn. was investigated. The compounds markedly influenced production of flavonolignans in an in vitro culture. Particularly after the elicitation with a solution of compound 3-methylamide 5-tert-butylpyrazine-2-carboxylic acid at a concentration of 3.71×10⁻⁷ mol 1⁻¹ and within 72 h of elicitation, an increase in flavonolignan production by 893 % in suspension culture versus control took place. The flavonolignan accumulation in callus culture after the elicitation with a solution of 5-brom-2-hydroxyphenylamide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid was also increased by about 1039% (24 hour elicitation and concentration of 2.59×10⁻⁴ mol 1⁻¹).     

Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos ofPicea abies (Norway spruce)

Summary Embryogenic callus was initiated from radicles of mature embryos removed from imbibed seeds (24 h). Embryogenic and other nonembryogenic types of callus proliferated on a modified half-strength Murashige-Skoog medium (MS) basal medium (BM) supplemented withmyo-inositol, casein hydrolysate (CH), L-glutamine (gln) and growth regulators kinetin (KN), N⁶-benzyladenine (BAP) each (20×10⁻⁶ M), 2,4-dichlorophenoxyacetic acid (2,4-D) (50×10⁻⁶ M) Embryogenic callus bearing suspensor-like cells in a mucilaginous gel matrix was isolated and maintained by subculture every 10 to 12 days on BM with KN, BAP each (2×10⁻⁶ M) and 2,4-D (5×10⁻⁶ M). Somatic embryos developed spontaneously from the callus on this medium at 23±1° C. Closer examination revealed that numerous polyembryonic clusters, comprised of elongated cells (suspensors) and small dense cells with large nuclei (somatic embryos), occurred in the viscous gel. When this enriched embryonal-suspensor mass was subcultured to low 2,4-D (1×10⁻⁶ M), globular embryos developed by 40 to 60 days. Upon transfer to a liquid medium without growth regulators, the embryos elongated and developed cotyledons and shoots with needles. Plantlet development was completed by 30 days in a basal medium without CH, gln and growth regulators. The total culture time was 150 days. Approximately 40±10 embryos were formed from 500 mg of initial callus. Somatic embryogenesis became aberrant if embryos remained attached to the callus mass and were not subcultured within 10 to 12 days according to the described protocol. Somatic embryos were encapsulated in an alginate gel and stored at 4° C for nearly two months without visible adverse effects on viability. Editor's Statement This paper presents advances in the in vitro regeneration of a commercially useful plant species from stored seeds. In addition, data is presented on short-term storage of the plantlets, and long-term proliferation of the embryonal mass in vitro.     

Toxicity of dodecylpyridinium and cetylpyridinium clorides against phosphate-accumulating bacterium

The antibacterial effect of cationic surfactants against the pure culture of phosphate (P)-accumulating bacterium Acinetobacter junii was investigated. The estimated EC₅₀ values of the N-dodecylpyridinium chloride (DPC) for growth inhibition was 1.4±0.5 × 10⁻⁶ mol L⁻¹ and for the inhibition of the P-uptake rates 7.3±2.6 × 10⁻⁵ mol L⁻¹. The estimated EC₅₀ values of the N-cetylpyridinium chloride (CPC) for growth inhibition was 4.9±1.3 × 10⁻⁷ mol L⁻¹ and for the inhibition of the P-uptake rates 7.7±2.9 × 10⁻⁶ mol L⁻¹. This suggests the importance of controlling the amounts of cationic surfactants in influent of the wastewater treatment systems in order to avoid the possible failure of the biological P removal from wastewaters.     

Effect of new rotenoid glycoside from the fruits of Amorpha fruticosa LINNE on the growth of human immune cells

A new compound, rotenoid isoflavone glycoside named, 6′-O-β-d-glucopyranosyl-12a-hydroxydalpanol was isolated from the methanolic (MeOH) fruit extract of Amorpha fruticosa LINNE by means of multi-stage column chromatography. Immuno-modulatory activities of this new glycoside were compared with the partitioned fractions of Amorpha fruticosa LINNE. Both of the fractions and purified single compound showed a 19% relatively low cytotoxicity at a maximum concentration of 1.0 g/L in a cultivated normal human lung cell line (HEL299). The purified single compound showed less cytotoxicity than the crude extracts, possibly because residual toxicants were eliminated during purification processes. Cell growth of human T cells was increased by about 15% by adding 0.5 g/L of the fractions compared to the control. Specific production rates of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) from T cell were higher as 1.16 × 10⁻⁴ and 1.86 × 10⁻⁴ pg/cell, respectively, in the purified compound, compared to 1.38 × 10⁻⁴ and 2.22 × 10⁻⁴ pg/cell, respectively, by adding 0.5 g/L of the dichloromethane fraction. Natural killer cell-92MI (NK-92MI) growth supplemented with the supernatant of human T cell was up to 19% higher with the dichloromethane fraction compared with a new single compound at a concentration of 0.5 g/L. Overall, the dichloromethane fraction showed relatively higher immuno-modulatory activities compared with a new single compound, probably due to the synergic effect given by other substances existing in the fractions.     

Physiological differences in the growth of <Emphasis Type="Italic">Sargassum horneri</Emphasis> between the germling and adult stages

The effects of temperature, irradiance, and daylength on Sargassum horneri growth were examined at the germling and adult stages to discern their physiological differences. Temperature–irradiance (10, 15, 20, 25, 30°C × 20, 40, 80 μmol photons m⁻²s⁻¹) and daylength (8, 12, 16, 24 h) experiments were carried out. The germlings and blades of S. horneri grew over a wide range of temperatures (10–25°C), irradiances (20–80 μmol photons m⁻²s⁻¹), and daylengths (8–24 h). At the optimal growth conditions, the relative growth rates (RGR) of the germlings were 21% day⁻¹ (25°C, 20 μmol photons m⁻²s⁻¹) and 13% day⁻¹ (8 h daylength). In contrast, the RGRs of the blade weights were 4% day⁻¹ (15°C, 20 μmol photons m⁻²s⁻¹) and 5% day⁻¹ (12 h daylength). Negative growth rates were found at 20 μmol photons m⁻²s⁻¹ of 20°C and 25°C treatments after 12 days. This phenomenon coincides with the necrosis of S. horneri blades in field populations. In conclusion, we found physiological differences between S. horneri germlings and adults with respect to daylength and temperature optima. The growth of S. horneri germlings could be enhanced at 25°C, 20 μmol photons m⁻²s⁻¹, and 8 h daylength for construction of Sargassum beds and restoration of barren areas.     

Use of clove oil to anaesthetize freshwater amphipods

The use of clove oil as a potential anaesthetic for freshwater amphipods was examined at 20 °C. Individuals of Gammarus minus, a common species in southern Illinois, USA, spanning the entire body size range (4.3–14.3 mm), were used to test four anaesthetic concentrations varying from 1.48 × 10⁻⁴ ml ml⁻¹ to 5.9 × 10⁻⁴ ml ml⁻¹. Small-bodied individuals (mean size = 5.4 mm ± 0.27SE) were used to test additional concentrations, up to 14.7 × 10⁻⁴ ml ml⁻¹, a 10-fold span, to identify potential lethal concentrations. At the lowest concentration, time to anaesthesia and recovery was constant at all body sizes. For the three next higher concentrations, time to anaesthesia decreased with increasing concentration while recovery time increased. Activity of amphipods was not affected by the ethanol carrier. In addition, activity did not differ between amphipods that had recovered from anaesthesia and unexposed amphipods. At clove oil concentrations of 8.84 × 10⁻⁴ ml ml⁻¹ and 14.7 × 10⁻⁴ ml ml⁻¹, mortality was 7 and 40%, respectively, indicating, that 5.9 × 10⁻⁴ ml ml⁻¹ was a safe working concentration. No mortality was observed with Gammarus acherondytes, a federally endangered cave amphipod on which the protocol with 80 μl of stock was used in the field. The method enabled us to obtain information on the endangered amphipod which normally would have required the sacrifice of individuals. Thus, research can continue on species for which population numbers are low and for which basic information is needed to formulate meaningful recovery plans.     

Bacteriostatic effects of cerium-humic acid complex

The bacteriostatic potency of the cerium-humic acid complex was evaluated by experimental measurement of this complex interaction with E. coli, Bacillus pyocyaneus, Staphylococcus aureus, Leuconostoc and Streptococcus faecalis, and by comparison bacteriostatic effects with the cerium-citrate complex. The experimental results indicated that the cerium-humic acid complex strongly inhibited growth of all five bacterial strains, and its diameter of bacteriostatic circles were more than 30 mm. The minimal bacteria-inhibiting concentration were 1×10⁻³, 2×10⁻³ and 1×10⁻² mol/L for E. coli and Bacillus pyocyaneus, Staphylococcus aureus, and Leuconostoc and Streptococcus faecalis individually, and the measured minimal bactericidal concentrations were 2×10⁻³ and 1×10⁻² mol/L for Bacillus pyocyaneus, E. coli, and Leuconostoc. To kill Staphylococcus aureus and Streptococcus faecalis, the concentration had to be more than 1×10⁻² mol/L. On the contrary, we found that cerium-citrate complex did not inhibit the growth of the above five bacteria, but stimulated bacterial growth. The completely different bacteriostatic results of two cerium complexes may hint that the association and chemical properties of the two complexes were different.     

Impact of commonly used agrochemicals on bacterial diversity in cultivated soils

The effects of three selected agrochemicals on bacterial diversity in cultivated soil have been studied. The selected agrochemicals are Cerox (an insecticide), Ceresate and Paraquat (both herbicides). The effect on bacterial population was studied by looking at the total heterotrophic bacteria presence and the effect of the agrochemicals on some selected soil microbes. The soil type used was loamy with pH of 6.0–7.0. The soil was placed in opaque pots and bambara bean (Vigna subterranean) seeds cultivated in them. The agrochemicals were applied two weeks after germination of seeds at concentrations based on manufacturer’s recommendation. Plant growth was assessed by weekly measurement of plant height, foliage appearance and number of nodules formed after one month. The results indicated that the diversity index (Di) among the bacteria populations in untreated soil and that of Cerox-treated soils were high with mean diversity index above 0.95. Mean Di for Ceresate-treated soil was 0.88, and that for Paraquattreated soil was 0.85 indicating low bacterial populations in these treatment-type soils. The study also showed that application of the agrochemicals caused reduction in the number of total heterotrophic bacteria population sizes in the soil. Ceresate caused 82.50% reduction in bacteria number from a mean of 40 × 10⁵ cfu g⁻¹ of soil sample to 70 × 10⁴ cfu g⁻¹. Paraquat-treated soil showed 92.86% reduction, from a mean of 56 × 10⁵ cfu g⁻¹ to 40 × 10⁴ cfu g⁻¹. Application of Cerox to the soil did not have any remarkable reduction in bacterial population number. Total viable cell count studies using Congo red yeast-extract mannitol agar indicated reduction in the number of Rhizobium spp. after application of the agrochemicals. Mean number of Rhizobium population numbers per gram of soil was 180 × 10⁴ for the untreated soil. Cerox-treated soil recorded mean number of 138 × 10⁴ rhizobial cfu g⁻¹ of soil, a 23.33% reduction. Ceresate- and Paraquat-treated soils recorded 20 × 10⁴ and 12 × 10⁴ cfu g⁻¹ of soil, respectively, representing 88.89% and 93.33% reduction in Rhizobium population numbers. Correspondingly, the mean number of nodules per plant was 44 for the growth in untreated soil, 30 for the plant in the Cerox-treated soil, 8 for the plant in Paraquat-treated soil and 3 for the plant in Ceresate-treated soil. The study has confirmed detrimental effect of insecticide on bacterial populations in the soil. Total heterotrophic counts, rhizobial counts as well as the number of nodules of all samples taken from the chemically treated soils were all low as compared to values obtained for the untreated soil. However, the effect of the insecticide was minimal in all cases as compared to the effects of the herbicides on the soil fauna. Indiscriminate use of agrochemicals on farms can therefore affect soil flora and subsequently food production.     

Genetic transformation of the medicinal plant <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated method

A high-frequency and simple procedure for Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Salvia miltiorrhiza was developed. Leaf discs were pre-cultured on MS medium supplemented with 6.6 μmol l⁻¹ BAP and 0.5 μmol l⁻¹ NAA for one day, then co-cultured with A. tumefaciens strain EHA105 harboring the plasmid pCAMBIA 2301 for three days on the same medium. Regenerated buds were obtained on selection medium (co-culture medium supplemented with 60 mg l⁻¹ kanamycin and 200 mg l⁻¹ cefotaxime) after two cycles’ culture of 10 days each and then transferred to fresh MS medium with 60 mg l⁻¹ kanamycin for rooting. Fifteen days later, the rooted plantlets were obtained and then successfully transplanted to soil. The transgenic nature of the regenerated plants was confirmed by PCR, Southern hybridization analysis and GUS histochemical assay. Averagely, 1.1 independent verified transgenics per explant plated were obtained through this protocol. Adopting this procedure, positive transformed plants could be obtained within 2–3 months from mature seeds germination to transplant to soil, and more than 1,000 transgenic plants with several engineered constructs encoding different genes of interest were produced in our lab in the past two years.     

Plant regeneration from callus culture of a Paphiopedilum hybrid

Totipotent calli of a Paphiopedilum hybrid (Paphiopedilum callosum ‘Oakhi’ × Paph. lawrenceanum ‘Tradition’) were induced from seed-derived protocorms on a 1/2 strength Murashige–Skoog medium plus 1–10 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–1 mg l⁻¹ 1-phenyl-3-(1.2.3-thiadiazol-5-yl)urea (TDZ). These calli grew well when subcultured on the same medium, but proliferated more on 1/2 MS medium plus 5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ TDZ. Calli developed further along a route of production of protocorm-like bodies and eventually formed plantlets that could be transplanted to pots and grew well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii (Cyanophyta)

The effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii were determined. Of the three temperatures tested, 10, 25 and 35°C, the maximal geosmin concentration and geosmin productivity were yielded at 10°C, while the highest chl a production was observed at 25°C. In the studies on light intensity, the maximal geosmin concentration and geosmin productivity were observed at 10 μmol m⁻² s⁻¹, while the highest chl a production was at 20 μmol m⁻² s⁻¹. It was suggested that more geosmin was synthesized with lower chl a demand. Meanwhile, the relative amounts of extra- and intracellular geosmin were investigated. Under optimum growth conditions (20 μmol m⁻² s⁻¹, 25°C; BG-11 medium), the amounts of extracellular geosmin increased as the growth progressed and reached the maximum in the stationary phase, while the intracellular geosmin reached its maximum value in the late exponential phase, and then began to decline. However, under the low temperature (10°C) or light (10 μmol m⁻² s⁻¹) conditions, more intracellular geosmin was synthesized and mainly accumulated in the cells. The proportions of extracellular geosmin were high, to 33.33 and 32.27%, respectively, during the stationary phase at 35°C and 20 μmol m⁻² s⁻¹. It was indicated that low temperature or light could stimulate geosmin production and favor the accumulation of geosmin in cells, while more intracellular geosmin may be released into the medium at higher temperatures or optimum light intensity.     

Production of plants resistant to <Emphasis Type="Italic">Alternaria carthami</Emphasis> via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 10⁵ spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min⁻¹ mg⁻¹ protein) from organogenesis and 47% (0.23 μmol min⁻¹ mg⁻¹ protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg⁻¹ protein) and 19.5% (145 U mg⁻¹ protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

Cell growth and nutritive value of the tropical benthic diatom, <Emphasis Type="Italic">Amphora</Emphasis> sp., at varying levels of nutrients and light intensity,and different culture locations

Two series of experiments were conducted to determine suitable growth factors for the mass propagation of the local algal isolate Amphora sp. A higher growth rate of 0.2 doubling (μ) day⁻¹ was attained at a lower photosynthetic photon flux density (PPFD; 11.4 μmol photon m⁻²s⁻¹) compared to cultures exposed to higher levels of PPFD (16.1 μmol photon m⁻²s⁻¹, −0.1 μ day ⁻¹; 31.3 μmol photon m⁻²s⁻¹, 0.0 μ day⁻¹). Cultures located inside the laboratory had a significantly higher cell density (133 × 10⁴ cells cm⁻²) and growth rate (0.3 μ day⁻¹) compared to those located outdoors (100 × 10⁴ cells cm⁻², 0.2 μ day⁻¹). A comparison of nutrient medium across two locations showed that lipid content was significantly higher in cultures enriched with F/2MTM (macronutrients + trace metals) and F/2MV (macronutrients + vitamins). Saturated fatty acids were also present in high concentrations in cultures enriched with F/2M (macronutrients only). Significantly higher amounts of saturated fatty acids were observed in cultures located outdoors (33.1%) compared to those located indoors (26.6%). The protein, carbohydrates and n-6 fatty acid content of Amphora sp. were influenced by the location and enrichment of the cultures. This study has identified growth conditions for mass culture of Amphora sp. and determined biochemical composition under those culture conditions. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Auxin synthesis by the higher fungus Lentinus edodes (Berk.) sing in the presence of low concentrations of indole compounds

The auxin formation in a submerged culture of the xylotrophic basidiomycete Lentinus edodes (Berk.) Sing (Lentinula edodes (Berk.) Pegler) (shiitake) is studied. Biologically active substances of an indole nature are identified, “the effect of small doses” of which lies in not only the stimulation of growth of the mycelium (indole-3-acetic acid, 2 × 10⁻⁷–2 × 10⁻⁴ g/l), but also in the induction of tryptophan-independent paths of auxin biosynthesis. The above-mentioned path is realized in the presence of exogenous indole (1 × 10⁻³–1 × 10⁻⁴ g/l), as well as while inducing the biosynthesis of indole-3-acetic acid by its microadditives (1 × 10⁻⁵−1 × 10⁻⁸ g/l), and is accompanied by the formation of anthranilic acid (up to 1.5 mg/l). Induction of the generative development stage of shiitake by indole derivatives is revealed. It was found that among the studied compounds only indoleacetamide at a concentration of an order of ×10⁻⁴ g/l in the culture fluid of L. edodes had a pronounced stimulatory effect on the formation of shiitake’s brown mycelial film.     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Toxicity assessment of paraverine hydrochloride and papaverine-derived metabolites in primary cultures of rat hepatocytes

Summary The present study was undertaken to assess and compare the toxic effects of papaverine hydrochloride and its metabolites. Primary cell cultures of rat hepatocytes were treated with papavarine (papaver), 3′-O-desmethyl (3′-OH), 4′-O-desmethyl (4′-OH), and 6-O-desmethyl (6-OH) papaverine at 1×10⁻⁵, 1×10⁻⁴, and 1×10⁻³ M for 4,8, 12, and 24-h periods. Cell injury was determined by: a) cell viability using the trypan blue exclusion test; b) cytosolic enzyme leakage of lactate dehydrogenase and aspartate aminotransferase; c) morphologic alterations; and d) lactate: pyruvate (L:P) ratios. Cell cultures showed concentration-and time-dependent responses. For example, a decrease in cell viability and an increase in enzyme leakage were observed after cell treatment with 1×10⁻⁴ and 1×10⁻³ M papaver for 8 h; 1×10⁻³ M 6-OH papaverine for 8 h and 1×10⁻⁴ M for 24 h; and 1×10⁻³ M 4′-OH papaverine for 24 h (P<0.05). Furthermore, changes in morphology correlated to cell viability and enzyme release in those cultures treated with papaver, 4′-OH and 6-OH papaverine. Some of these changes included size deformation, cell detachment from the dishes, and cell necrosis. On the other hand, an increase in L:P ratios (P<0.05) was detected with papaver as early as 8 h with 1×10⁻⁴ and 1×10⁻³ M and 12 h with 1×10⁻⁵ M; 6-OH showed an increase, in L:P ratios at 8 h with 1×10⁻³ M and 12 h with 1×10⁻⁴ M; these changes were evident with 4′-OH at 12 h with 1×10⁻³ M. In contrast, cells treated with 3′-OH papaverine did not show significant damage with any time period and concentration used in this study. The results of this study indicate that papaverine-derived metabolites are less cytotoxic than its parent compound, papaver. The toxicity was ranked as follows: papaver>6-OH>4′-OH>−3′-OH. This work was supported in part by grant ES04200-02 from the National Institute of Environmental Health Sciences, Bethesda, MD. Presented in part at the fall ASPET meeting in Salt Lake City, August, 1989. Daniel Acosta is a Burroughs Wellcome Scholar in Toxicology.