A gene cloning system for the aranciamycin producer strain Streptomyces echinatus Lv 22

Streptomyces echinatus Lv 22 (=DSM 40730) produces an anthracycline antibiotic aranciamycin. Development of DNA transfer using conjugation from Escherichia coli into this strain is described. Various replicative plasmids (pKC1139, pKC1218E, pSOK1O1, pCHZ101) as well as actinophage (φC31- and VWB-based vectors pSET152 and pVWB, respectively, were transferred from E. coli ET12567 (pUB307) into the S. echinatus at a frequency ranging from 2.4 × 10^?3 to 1.6 × 10^?4. The transconjugants did not differ from wild type in their ability to produce aranciamycin and morphological features. There is one attB site for pSET152 and pVWB integrative plasmids in the S. echinatus chromosome. Developed DNA transfer system was used for expression of heterologous regulatory genes in S. echinatus cells. Expression of relA gene of ppGpp synthetase increased antibiotic production in S. echinatus. The absA2 gene of S. ghanaensis appears to play a negative role in the control of aranciamycin biosynthesis. Additional copies of absA2 leads to inhibition of aranciamycin’s production. absB and afsS had no effect on aranciamycin biosynthesis as well as on the morphological features of S. echinatus. Obtained results indicate efficiency of the developed system for gene cloning in S. echinatus.     

Internal dose assessment of <Superscript>210</Superscript>Po using biokinetic modeling and urinary excretion measurement

The mysterious death of Mr. Alexander Litvinenko who was most possibly poisoned by Polonium-210 (²¹⁰Po) in November 2006 in London attracted the attention of the public to the kinetics, dosimetry and the risk of this high radiotoxic isotope in the human body. In the present paper, the urinary excretion of seven persons who were possibly exposed to traces of ²¹⁰Po was monitored. The values measured in the GSF Radioanalytical Laboratory are in the range of natural background concentration. To assess the effective dose received by those persons, the time-dependence of the organ equivalent dose and the effective dose after acute ingestion and inhalation of ²¹⁰Po were calculated using the biokinetic model for polonium (Po) recommended by the International Commission on Radiological Protection (ICRP) and the one recently published by Leggett and Eckerman (L&E). The daily urinary excretion to effective dose conversion factors for ingestion and inhalation were evaluated based on the ICRP and L&E models for members of the public. The ingestion (inhalation) effective dose per unit intake integrated over one day is 1.7 × 10⁻⁸ (1.4 × 10⁻⁷) Sv Bq⁻¹, 2.0 × 10⁻⁷ (9.6 × 10⁻⁷) Sv Bq⁻¹ over 10 days, 5.2 × 10⁻⁷ (2.0 × 10⁻⁶) Sv Bq⁻¹ over 30 days and 1.0 × 10⁻⁶ (3.0 × 10⁻⁶) Sv Bq⁻¹ over 100 days. The daily urinary excretions after acute ingestion (inhalation) of 1 Bq of ²¹⁰Po are 1.1 × 10⁻³ (1.0 × 10⁻⁴) on day 1, 2.0 × 10⁻³ (1.9 × 10⁻⁴) on day 10, 1.3 × 10⁻³ (1.7 × 10⁻⁴) on day 30 and 3.6 × 10⁻⁴ (8.3 × 10⁻⁵) Bq d⁻¹ on day 100, respectively. The resulting committed effective doses range from 2.1 × 10⁻³ to 1.7 × 10⁻² mSv by an assumption of ingestion and from 5.5 × 10⁻² to 4.5 × 10⁻¹ mSv by inhalation. For the case of Mr. Litvinenko, the mean organ absorbed dose as a function of time was calculated using both the above stated models. The red bone marrow, the kidneys and the liver were considered as the critical organs. Assuming a value of lethal absorbed dose of 5 Gy to the bone marrow, 6 Gy to the kidneys and 8 Gy to the liver, the amount of ²¹⁰Po which Mr. Litvinenko might have ingested is therefore estimated to range from 27 to 1,408 MBq, i.e 0.2–8.5 μg, depending on the modality of intake and on different assumptions about blood absorption.     

Biochemical Studies on Streptomyces sioyaensis

Non-proliferating mycelium of Streptomyces sioyaensis was shown to form siomycin in phosphate buffer without addition of an energy source or precursors. This increase of siomycin in phosphate buffer was suppressed by glucose, acetate, l-cysteine, casamino acid, metals (Fe⁺⁺, Cu⁺⁺), various metabolic inhibitors, and antibiotics (chloramphenicol, erythromycin), whereas it was promoted by yeast extract, beef extract, l-isoleucine, Mg, etc.

The mechanism of the inhibitory effect of glucose on siomycin formation was investigated. Although glucose suppressed siomycin formation, it was the best carbon source for Streptomyces sioyaensis and vigorously metabolized to keto acids and other metabolites. Glucose suppressed siomycin formation by promoting cellular metabolism and mycelial growth. Siomycin formation was not only different from but also competitive to mycelial growth (cellular protein synthesis).     

Carob pulp as raw material for production of the biocontrol agent <Emphasis Type="Italic">P. agglomerans</Emphasis> PBC-1

Large-scale production has been the major obstacle to the success of many biopesticides. The spreading of microbial biocontrol agents against postharvest disease, as a safe and environmentally friendly alternative to synthetic fungicides, is quite dependent on their industrial mass production from low-cost raw materials. Considerable interest has been shown in using agricultural waste products and by-products from food industry as nitrogen and carbon sources. In this work, carob pulp aqueous extracts were used as carbon source in the production of the biocontrol agent Pantoea agglomerans PBC-1. Optimal sugar extraction was achieved at a solid/liquid ratio of 1:10 (w/v), at 25°C, for 1 h. Batch experiments were performed in shake flasks, at different concentrations and in stirred reactors at two initial inoculums concentrations, 10⁶ and 10⁷ cfu ml⁻¹. The initial sugar concentration of 5 g l⁻¹ allowed rapid growth (0.16 h⁻¹) and high biomass productivity (0.28 g l⁻¹ h⁻¹) and was chosen as the value for use in stirred reactor experiments. After 22 and 32 h of fermentation the viable population reached was 3.2 × 10⁹ and 6.2 × 10⁹ cfu ml⁻¹ in the fermenter inoculated at 10⁶ cfu ml⁻¹ and 2.7 × 10⁹ and 6.7 × 10⁹ cfu ml⁻¹ in the bioreactor inoculated at 10⁷ cfu ml⁻¹. A 78% reduction of the pathogen incidence was achieved with PBC-1 at 1 × 10⁸ cfu ml⁻¹, grown in medium with carob extracts, on artificially wounded apples stored after 7 days at 25°C against P. expansum.     

High frequency transformation of the industrial erythromycin-producing bacterium Saccharopolyspora erythraea

The DNA transformation in the industrial erythromycin-producing Saccharopolyspora erythraea was investigated as standard protoplast transformation methods are ineffective. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies from 0.05 × 10⁻⁸ to 7.2 × 10⁻⁸ exconjugants generated per recipient. Electroporation-mediated methodologies were also established. More than 10⁵ transformants were acquired per μg DNA. The proposed protocol provides an alternative route for the introduction of DNA into industrial strains.     

Evaluation of inert and organic carriers for <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation

Growth and sporulation of Verticillium lecanii on inert and organic carriers (sugar-cane bagasse, corncob, rice straw, polyurethane foam and activated carbon) in a solid-state fermentation process was studied. Sugar-cane bagasse and polyurethane foam produced 10¹⁰ spores g⁻¹ dry carrier whereas corncob, rice straw, and activated carbon yielded, respectively 8 × 10⁹, 4 × 10⁹, and 3 × 10⁸ spores g⁻¹. Chitinase activity of the conidia was in the following order: sugar-cane bagasse (3.3 U mg⁻¹) > wheat bran (3.0 U mg⁻¹) > polyurethane foam (2.7 U mg⁻¹). There was no significant difference (2.5–2.7 U mg⁻¹) in the proteinase activity among the conidia from the three cultures. Scanning electron microscopy shows that aerial mycelium freely penetrated into the internal area of polyurethane foam. Sugar-cane bagasse provided enough area for vegetative hyphae to attach. Of the carriers analyzed, polyurethane foams and sugar-cane bagasse were the best carriers for V. lecanii growth and spore production.     

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.     

Plant regeneration and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of two elite aspen hybrid clones from <Emphasis Type="Italic">in vitro</Emphasis> leaf tissues

Summary An efficient regeneration and transformation system was developed for two elite aspen hybrid clones (Populus canescens × P. grandidentada and P. tremuloides × P. davidiana). Callus was induced from in vitro leaf explants on modified Murashige and Skoog medium (MSA) and woody plant medium (WPM) containing four different combinations of cytokinins and auxins. Callus tissues regenerated into shoots on WPM medium supplemented with 2.0 mgl⁻¹ (9.12 μM) zeatin or 0.01 mgl⁻¹ (0.045 μM) thidiazuron. P. canescens × P. grandidentata exhibited the higher callus and shoot production. In vitro leaf explants from the two hybrid clones were cocultivated with Agrobacterium tumefaciens strain EHA105 harboring the binary Ti plasmid pBI121 carrying the uidA gene encoding for β-glucuronidase (GUS) and the npt II gene encoding for neomycin phosphotransferase II. Transformation was confirmed by GUS assays, polymerase chain reaction, and Southern blot analyses. Agrobacterium concentration, acetosyringone, and pH of the cocultivation medium were evaluated for enhancing transformation efficiency with the clone P. canescens × P. grandidentata.     

Characterization of a recombinant endo-type alginate lyase (Alg7D) from <Emphasis Type="Italic">Saccharophagus degradans</Emphasis>

A gene, alg7D, from Saccharophagus degradans, coding for a putative alginate lyase belonging to the family of polysaccharide lyase-7, was overexpressed in Escherichia coli. The properties of the recombinant Alg7D were characterized. The enzyme endolytically depolymerized alginate by β-elimination into oligo-alginates with degrees of polymerization of 2–5. Its activity was maximal at 50°C and pH 7 and was slightly increased in the presence of Na⁺. The K _M , V _max , k _cat , and k _cat /K _M values were: 3 mg ml⁻¹, 6.2 U mg⁻¹, 1.9 × 10⁻² s⁻¹, and 6.3 × 10⁻³ mg⁻¹ ml s⁻¹, respectively.     

Migratory history and habitat use by New Zealand freshwater eels <Emphasis Type="Italic">Anguilla dieffenbachii</Emphasis> and <Emphasis Type="Italic">A. australis</Emphasis>, as revealed by otolith microchemistry

 The migratory history of Anguilla dieffenbachii and A. australis, collected from a coastal lake of New Zealand, was examined using analysis of strontium (Sr) and calcium (Ca) concentrations. Line analysis of Sr : Ca ratios along the life history transect of each otolith showed a peak (Ca. 16–20 × 10⁻³) between the core and elver mark, which corresponded to the period of their leptocephalus and early glass eel stages in the ocean. The mean Sr : Ca ratios from the elver mark to the otolith edge indicated that eels had different migratory histories, which included freshwater residency in some eels (average Sr : Ca ratios, 1.7 × 10⁻³–2.4 × 10⁻³) but not in others (average Sr : Ca ratios, 3.1 × 10⁻³–6.5 × 10⁻³). These findings suggest that New Zealand freshwater eels have a flexible migration strategy and an ability to adapt to various habitats and salinities. Received: November 25, 2002 / Revised: January 17, 2003 / Accepted: January 17, 2003     

Transfer of megaplasmid pKB1 from the rubber-degrading bacterium <Emphasis Type="Italic">Gordonia westfalica</Emphasis> strain Kb1 to related bacteria and its modification

Because engineering of the 101.016-bp megaplasmid pKB1 of Gordonia westfalica Kb1 failed due to the absence of an effective transfer system, pKB1 was transferred by conjugation from G. westfalica Kb1 to a kanamycin-resistant mutant of Rhodococcus opacus PD630 at a frequency of about 6.2 × 10⁻⁸ events per recipient cell. Furthermore, pKB1 was transferred to G. polyisoprenivorans strains VH2 and Y2K and to Mycobacterium smegmatis by electroporation at frequencies of 5.5 × 10³, 1.9 × 10³, and 8.3 × 10² transformants per microgram plasmid DNA. The pKB1-encoded cadmium resistance gene cadA was used for selection in these experiments. Recombinant pKB1-containing G. polyisoprenivorans VH2 and M. smegmatis were then used to engineer pKB1. A kanamycin resistance cassette was inserted into the pKB1-encoded cadA gene, ligated to suicide plasmid pBBR1MCS-5, and the resulting plasmid was electroporated into plasmid-harboring strains. Homologous recombination between cadA on suicide plasmid and the respective sequence in pKB1 led to its integration into pKB1. Thus, two selection markers were accommodated in pKB1 to monitor plasmid transfer into Gordonia and related taxa for analysis of genes essential for rubber degradation and others. In this study, two transfer methods for large plasmids and strategies for engineering of pKB1 were successfully applied, thereby, extending the tool box for Gordonia.     

Spring phytoplankton assemblages in the Southern Ocean between Australia and Antarctica

Variations of phytoplankton assemblages were studied in November–December 2001, in surface waters of the Southern Ocean along a transect between the Sub-Antarctic Zone (SAZ) and the Seasonal Ice Zone (SIZ; 46.9°–64.9°S; 142°–143°E; CLIVAR-SR3 cruise). Two regions had characteristic but different phytoplankton assemblages. Nanoflagellates(<20 μm) and pico-plankton (∼2 μm) occurred in similar concentrations along the transect, but were dominant in the SAZ, Sub-Antarctic Front (SAF), Polar Front Zone (PFZ) and the Inter-Polar Front Zone (IPFZ), (46.9°–56.9°S). Along the entire transect their average cell numbers in the upper 70 m of water column, varied from 3 × 10⁵ to 1.1 × 10⁶ cells l⁻¹. Larger cells (>20 μm), diatoms and dinoflagellates, were more abundant in the Antarctic Zone-South (AZ-S) and the SIZ, (60.9°–64.9°S). In AZ-S and SIZ diatoms ranged between 2.7 × 10⁵ and 1.2 × 10⁶ cells l⁻¹, dinoflagellates from 3.1 × 10⁴ to 1.02 × 10⁵ cells l⁻¹. A diatom bloom was in progress in the AZ-S showing a peak of 1.8 × 10⁶ cells l⁻¹. Diatoms were dominated by Pseudo-nitzschia spp., Fragilariopsis spp., and Chaetoceros spp. Pseudo-nitzschia spp. outnumbered other diatoms in the AZ-S. Fragilaropsis spp. were most numerous in the SIZ. Dinoflagellates contained autotrophs (e.g. Prorocentrum) and heterotrophs (Gyrodinium/Gymnodinium, Protoperidinium). Diatoms and dinoflagellates contributed most to the cellular carbon: 11–25 and 17–124 μg C l⁻¹, respectively. Small cells dominated in the northern region characterized by the lowest N-uptake and new production of the transect. Larger diatom cells were prevalent in the southern area with higher values of N-uptake and new production. Diatom and nanoflagellate cellular carbon contents were highly correlated with one another, with primary production, and productivity related parameters. They contributed up to 75% to the total autotrophic C biomass. Diatom carbon content was significantly correlated to nitrate uptake and particle export, but not to ammonium uptake, while flagellate carbon was well correlated to ammonium uptake, but not to export. Diatoms have contributed highly to particle export along the latitudinal transect, while flagellates played a minor role in the export.     

Mycelial cultivation of <Emphasis Type="Italic">Phellinus linteus</Emphasis> using cheese-processing waste and optimization of bioconversion conditions

A medicinal mushroom, Phellinus linteus, was successfully cultivated using a cheese-processing waste, whey, and the optimal bioconversion conditions for the maximum mycelial growth rate was also estimated through solid-state cultivation experiments. Response surface analysis with a face-centered design (center point replication = 5) was applied to statistically approximate the simultaneous effects of the three variables, i.e., substrate concentration (10–30 g lactose l⁻¹), temperature (20–30°C), and pH (4–6), on the mycelial growth rate of P. linteus. The following is a partial cubic model where η is the mycelial growth rate (K _r ) and x _k is the corresponding variable term (k = substrate concentration, temperature, and pH in order): η = −23.8 + 8.67 × 10⁻² x ₁ + 1.48x ₂ + 1.77x ₃ + 8.00 × 10⁻⁴ x ₁ x ₂ + 7.25 × 10⁻² x ₁ x ₃ + 5.13 × 10⁻² x ₂ x ₃ −1.28 × 10⁻² x ₁² –3.18 × 10⁻² x ₂². −2.64 × 10⁻¹ x ₃² −3.28 × 10⁻³ x ₁ x ₂ x ₃ + 4.68 × 10⁻⁴ x ₁² x ₂. The produced response surface model proved to be significant (r ² > 0.99, P-value <0.0001, coefficient of variation <5%) to describe the explored space. Temperature was found to be the most significant factor of dominant effects on the mycelial growth rate, and other variables such as temperature², pH, pH², and (substrate concentration² × temperature) also showed significant effects on the model output. The maximum mycelial growth rate was predicted to be 2.80 mm d⁻¹ at 29.7 g lactose l⁻¹, 26.2°C, and pH 5. Our results proved a good potential of whey to serve as an alternative growth medium for cultivating P. linteus mycelia. This may provide another potential for managing this nutrient-rich waste in a cost-effective way.     

Effects of H2O2 on the growth, secretion, and metabolism of hybridoma cells in culture

Summary The effect of low concentrations of hydrogen peroxide (H₂O₂) (5 × 10⁻⁷−9.5 × 10⁻⁷ M) on cell growth and antibody production was investigated with murine hybridoma cells (Mark 3 and anti-hPL) in culture. Cell growth, measured by flow cytometry with morphological parameters, was significantly stimulated by H₂O₂ (8 × 10⁻⁷ M) but H₂O₂ concentration of 7 × 10⁻⁶ M and above increased cell death. H₂O₂ stimulation of antibody production was nonsignificant. The metabolism of cells treated with 8 × 10⁻⁷ or 1 × 10⁻⁵ M H₂O₂ was similar to that of the control in terms of glucose and glutamine consumption, lactate and ammonia production, and amino acid concentrations in the medium. The concentrations of lactate dehydrogenase, a marker of cell death, in test and control cells were similar. However, concentrations of intracellular free radicals measured by flow cytometry with dihydrorhodamine 123 (DHR 123) and dichlorofluorescein diacetate (DCFH-DA) as fluorochromes were different. The reactive oxygen species content of cells in 8 × 10⁻⁷ M H₂O₂ was similar to that of the controls, but there was a sudden, marked production of superoxide anions (detected with DHR 123) and H₂O₂ or peroxides (detected with DCFH-DA) by cells incubated with 1 × 10⁻⁵ M H₂O₂ which increased with increasing H₂O₂ until cell death.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Body and scale growth of wild Atlantic salmon smolts during seaward emigration

The relationship between scale and body growth for emigrating Atlantic salmon, Salmo salar, smolts was previously not understood and therefore was examined in this study using mark-recapture techniques. The size of smolts at time of recapture was significantly greater than when marked (P = 0.0002). The growth in length of smolts emigrating 5 km over an average of 20 days was 7.7 ± 6.1 mm per day. Instantaneous somatic growth (G _body) ranged from 7.0 × 10⁻⁴ to 5.1 × 10⁻³ (mean = 2.7 × 10⁻³ ± 1.3 × 10⁻³). The mean number of plus growth circuli present per scale was significantly greater for smolts when recaptured compared to when marked (P = 0.0014). The instantaneous growth rate of scales (G _scale) ranged from 1.4 × 10⁻³ to 11.5 × 10⁻³ (mean = 6.6 × 10⁻³ ± 4.3 × 10⁻³). The relationship between body size and scale radius showed positive allometry rather than isometry. The relationship of G _scale with G _body showed positive allometry indicating that scales grew at a slightly faster rate than the body during the emigratory period.     

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.     

Effect of enzyme concentrations on protoplast isolation and protoplast culture of <Emphasis Type="Italic">Spathiphyllum</Emphasis> and <Emphasis Type="Italic">Anthurium</Emphasis>

Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 10⁶ protoplasts g⁻¹ and 1 × 10⁵ protoplasts g⁻¹ could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl₂·2H₂O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm⁻¹ alternating current for 60 s and subsequent generation of two pulses of 4500 V cm⁻¹ direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.     

Improving Toxicity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Contains the <Emphasis Type="Italic">cry8Ca</Emphasis> Gene Specific to <Emphasis Type="Italic">Anomala corpulenta</Emphasis> Larvae

The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein, with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain of B. thuringiensis (HD-73⁻), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC₅₀) of the two mutants were 0.2334 × 10⁸ and 0.2591 × 10⁸ colony-forming units g⁻¹, considerably lower than the LC₅₀ of the wild-type strain HBF-1 (0.9583 × 10⁸ CFU g⁻¹) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 10⁸ CFU g⁻¹).     

Determination of shelf life of <Emphasis Type="Italic">Spirulina platensis</Emphasis> (MI<Subscript>2</Subscript>) grown in the Philippines

Gamma linolenic acid (GLA) degradation in Spirulina followed first-order reaction kinetics. At an accelerated temperature range of 45 to 55°C, the degradation rate constants (k _r) of GLA obtained were 4.0 × 10⁻² to 8.8 × 10⁻² day⁻¹. The energy of activation (E _a) was 16.53 kcal mol⁻¹, and the Q₁₀ was 2.22. Based on 20% GLA degradation, the shelf life of sun-dried Spirulina at 30°C is 263 days or 8.6 months using the Arrhenius plot, and 258 days or 8.5 months using the Q ₁₀ approach. Presented at the 6th Meeting of the Asia Pacific Society of Applied Phycology, Manila, Philippines.