In vitro studies of DNA damage and repair mechanisms induced by BNCT in a poorly differentiated thyroid carcinoma cell line

Boron neutron capture therapy (BNCT) for aggressive tumors is based on nuclear reaction [¹⁰B (n, α) ⁷Li]. Previously, we demonstrated that BNCT could be applied for the treatment of undifferentiated thyroid carcinoma. The aim of the present study was to describe the DNA damage pattern and the repair pathways that are activated by BNCT in thyroid cells. We analyzed γH2AX foci and the expression of Ku70, Rad51 and Rad54, main effector enzymes of non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, respectively, in thyroid follicular carcinoma cells. The studied groups were: (1) C [no irradiation], (2) gamma [⁶⁰Co source], (3) N [neutron beam alone], (4) BNCT [neutron beam plus 10 µg ¹⁰B/ml of boronphenylalanine (¹⁰BPA)]. The total absorbed dose was always 3 Gy. The results showed that the number of nuclear γH2AX foci was higher in the gamma group than in the N and BNCT groups (30 min–24 h) (p?<?0.001). However, the focus size was significantly larger in BNCT compared to other groups (p?<?0.01). The analysis of repair enzymes showed a significant increase in Rad51 and Rad54 mRNA at 4 and 6 h, respectively; in both N and BNCT groups and the expression of Ku70 did not show significant differences between groups. These findings are consistent with an activation of HRR mechanism in thyroid cells. A melanoma cell line showed different DNA damage pattern and activation of both repair pathways. These results will allow us to evaluate different blocking points, to potentiate the damage induced by BNCT.     

The effectiveness of the high-LET radiations from the boron neutron capture [<Superscript>10</Superscript>B(n,α)<Superscript>7</Superscript>Li] reaction determined for induction of chromosome aberrations and apoptosis in lymphocytes of human blood samples

Provided that a selective accumulation of ¹⁰B-containing compounds is introduced in tumor cells, following irradiation by thermal neutrons produces high-LET alpha-particles (⁴He) and recoiling lithium-7 (⁷Li) nuclei emitted during the capture of thermalized neutrons (0.025 eV) from ¹⁰B. To estimate the biological effectiveness of this boron neutron capture [¹⁰B(n,α)⁷Li] reaction, the chromosome aberration assay and the flow cytometry apoptosis assay were applied. At the presence of the clinically used compounds BSH (sodium borocaptate) and BPA (p-boronophenylalanine), human lymphocytes were irradiated by sub-thermal neutrons. For analyzing chromosome aberrations, human lymphocytes were exposed to thermally equivalent neutron fluences of 1.82 × 10¹¹ cm^?2 or 7.30 × 10¹¹ cm^?2 (corresponding to thermal neutron doses of 0.062 and 0.248 Gy, respectively) in the presence of 0, 10, 20, and 30 ppm of BSH or BPA. Since the kerma coefficient of blood increased by 0.864 × 10^?12 Gy cm² per 10 ppm of ¹⁰B, the kerma coefficients in blood increase from 0.34 × 10^?12 cm² (blood without BSH or BPA) up to 2.93 × 10^?12 Gy cm² in the presence of 30 ppm of ¹⁰B. For the ¹⁰B(n, α)⁷Li reaction, linear dose–response relations for dicentrics with coefficients α = 0.0546 ± 0.0081 Gy^?1 for BSH and α = 0.0654 ± 0.0075 Gy^?1 for BPA were obtained at 0.062 Gy as well as α = 0.0985 ± 0.0284 Gy^?1 for BSH and α = 0.1293 ± 0.0419 Gy^?1 for BPA at 0.248 Gy. At both doses, the corresponding ¹⁰B(n, α)⁷Li reactions from BSH and BPA are not significantly different. A linear dose–response relation for dicentrics also was obtained for the induction of apoptosis by the ¹⁰B(n, α)⁷Li reaction at 0.248 Gy. The linear coefficients α = 0.0249 ± 0.0119 Gy^?1 for BSH and α = 0.0334 ± 0.0064 Gy^?1 for BPA are not significantly different. Independently of the applied thermal neutron doses of 0.062 Gy or 0.248 Gy, the ¹⁰B(n, α)⁷Li reaction from 30 ppm BSH or BPA induced an apparent RBE of about 2.2 for the production of dicentrics as compared to exposure to thermal neutrons alone. Since the apparent RBE value is defined as the product of the RBE of a thermal neutron dose alone times a boron localization factor which depends on the concentration of a ¹⁰B-containing compound, this localization factor determines the biological effectiveness of the ¹⁰B(n, α)⁷Li reaction.     

Determination of the thermal and epithermal neutron sensitivities of an LBO chamber

An LBO (Li₂B₄O₇) walled ionization chamber was designed to monitor the epithermal neutron fluence in boron neutron capture therapy clinical irradiation. The thermal and epithermal neutron sensitivities of the device were evaluated using accelerator neutrons from the ⁹Be(d, n) reaction at a deuteron energy of 4 MeV (4 MeV d-Be neutrons). The response of the chamber in terms of the electric charge induced in the LBO chamber was compared with the thermal and epithermal neutron fluences measured using the gold-foil activation method. The thermal and epithermal neutron sensitivities obtained were expressed in units of pC cm², i.e., from the chamber response divided by neutron fluence (cm^?2). The measured LBO chamber sensitivities were 2.23 × 10^?7 ± 0.34 × 10^?7 (pC cm²) for thermal neutrons and 2.00 × 10^?5 ± 0.12 × 10^?5 (pC cm²) for epithermal neutrons. This shows that the LBO chamber is sufficiently sensitive to epithermal neutrons to be useful for epithermal neutron monitoring in BNCT irradiation.     

Micronucleus assay in bovine lymphocytes after exposure to bisphenol A in vitro

Bisphenol A (BPA) [2,2-bis-(4-hydroxyphenyl) propane] is an important industrial agent, made by combining acetone and phenol, that is used extensively as a monomer in the production of polycarbonate plastics and as a precursor of epoxy resins. Micronucleus assays have served as an index of cytogenetic damage in in vivo and in vitro studies. We studied the genotoxic and cytotoxic effects of BPA on bovine peripheral lymphocytes in vitro. Lymphocyte cultures from two donors were exposed to four different concentrations of BPA (1?×?10^?4, 1?×?10^?5, 1?×?10^?6, and 1?×?10^?7 mol.L^?1) for 48 h. The highest concentration of BPA (1?×?10^?4 mol.L^?1) resulted in a significant increase in the number of micronuclei in comparison with the negative control (67.50?±?2.121/1,000 binucleated cells versus 36.0?±?5.657/1,000 binucleated cells in the DMSO control, P??=??0.018). BPA did not affect the nuclear division index at any treatment concentrations. The present results thus demonstrate a significant genotoxic effect by BPA on bovine peripheral lymphocytes in vitro, only at the highest concentration.     

Rapid and efficient degradation of bisphenol A by chloroperoxidase from <Emphasis Type="Italic">Caldariomyces fumago</Emphasis>: product analysis and ecotoxicity evaluation of the degraded solution

Objectives

To degrade enzymatically bisphenol A (BPA) that causes serious environmental concerns and is difficult to be degraded by chemical or physical methods.

Results

BPA (150 mg l^?1) was completely degraded by chloroperoxidase (CPO)/H₂O₂ within 7 min at room temperature, atmospheric pressure with the enzyme at 6 μg CPO ml^?1. The degradation products were identified by HPLC–MS, which suggested involvement of multiple steps. Enzymatic treatment followed by existing bioremediation technologies (activated sludge) enhanced removal of COD from 9 to 54 %. Using an ecotoxicity evaluation with Chlorella pyrenoidosa, the degradation products had a lower toxicity than BPA.

Conclusion

BPA can be degraded rapidly and efficiently under mild conditions with chloroperoxidase at 6 μg ml^?1. The degradation products had a lower toxicity than BPA.

     

Biodistribution of sodium borocaptate (BSH) for boron neutron capture therapy (BNCT) in an oral cancer model

Boron neutron capture therapy (BNCT) is based on selective accumulation of ¹⁰B carriers in tumor followed by neutron irradiation. We previously proved the therapeutic success of BNCT mediated by the boron compounds boronophenylalanine and sodium decahydrodecaborate (GB-10) in the hamster cheek pouch oral cancer model. Based on the clinical relevance of the boron carrier sodium borocaptate (BSH) and the knowledge that the most effective way to optimize BNCT is to improve tumor boron targeting, the specific aim of this study was to perform biodistribution studies of BSH in the hamster cheek pouch oral cancer model and evaluate the feasibility of BNCT mediated by BSH at nuclear reactor RA-3. The general aim of these studies is to contribute to the knowledge of BNCT radiobiology and optimize BNCT for head and neck cancer. Sodium borocaptate (50 mg ¹⁰B/kg) was administered to tumor-bearing hamsters. Groups of 3–5 animals were killed humanely at nine time-points, 3–12 h post-administration. Samples of blood, tumor, precancerous pouch tissue, normal pouch tissue and other clinically relevant normal tissues were processed for boron measurement by optic emission spectroscopy. Tumor boron concentration peaked to therapeutically useful boron concentration values of 24–35 ppm. The boron concentration ratio tumor/normal pouch tissue ranged from 1.1 to 1.8. Pharmacokinetic curves showed that the optimum interval between BSH administration and neutron irradiation was 7–11 h. It is concluded that BNCT mediated by BSH at nuclear reactor RA-3 would be feasible.     

Grazing impact on the cyanobacterium Microcystis aeruginosa by the heterotrophic flagellate Collodictyon triciliatum in an experimental pond

We estimated the grazing impact of the heterotrophic flagellate Collodictyon triciliatum on the harmful, bloom-forming cyanobacterium Microcystis aeruginosa in an experimental pond during a Microcystis bloom from summer to winter in 2010. For these experiments, we calculated the grazing rates from the digestion rate of C. triciliatum and its food vacuole contents. During the study period, M. aeruginosa exhibited one bloom event with a maximum density of 1.1 × 10⁵ cells ml^?1. The cell density of C. triciliatum fluctuated from below the detection limit to 291 cells ml^?1. The number of M. aeruginosa cells ingested by C. triciliatum food vacuoles ranged between 0.4 and 10.8 cells flagellate^?1, and the digestion rate of C. triciliatum at 25 °C was 0.73 % cell contents min^?1. The grazing rate of C. triciliatum on the M. aeruginosa prey was 0.2–6.9 cells flagellate^?1 h^?1, and its grazing impact was 0.0–25.3 % standing stock day^?1. The functional response of C. triciliatum to the M. aeruginosa prey followed the Michaelis–Menten model of significance (r ² = 0.873, p < 0.001) in our experimental systems, in which the prey concentration varied from 1.0 × 10⁴ to 2.1 × 10⁶ cells ml^?1. The maximum grazing rate was 6.2 prey cells grazer^?1 h^?1, and the half-saturation constant was 1.2 × 10⁵ cells ml^?1. We present evidence that C. triciliatum grazing explained the remarkable decrease in M. aeruginosa cell density in the pond. The present study is the first demonstration of the high potential of protistan grazing on M. aeruginosa to reduce cyanobacterial blooms.     

Production of cyclodextrin glycosyltransferase by immobilized <Emphasis Type="Italic">Bacillus</Emphasis> sp. on chitosan matrix

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol–sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml^?1 (36 h), 47.50 U ml^?1 (36 h) and 68.36 U ml^?1 (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml^?1 (18 h) on cassava, 79.17 U ml^?1 (12 h) on potato and 55.37 U ml^?1 (in 6 h and max 77.75 U ml^?1 in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.     

A rapid knockdown effect of Penicillium citrinum for control of the mosquito Culex quinquefasciatus in Thailand

Twenty local isolates of entomopathogenic fungi were determined for control of the larvae and adults of Culex quinquefasciatus. In a laboratory experiment, a Penicillium sp. CM-010 caused 100 % mortality of third-instar larvae within 2 h using a conidial suspension of 1 × 10⁶ conidia ml^?1. Its LC₅₀ was 3 × 10⁵ conidia ml^?1, and the lethal time (LT₅₀) was 1.06 h. Cloning and sequencing of its internal transcribed spacer region indicated that this Penicillium species is Penicillium citrinum (100 % identity in 434 bp). Mortality of the adult was highest with Aspergillus flavus CM-011 followed with Metarhizium anisopliae CKM-048 from 1 × 10⁹ conidia ml^?1. P. citrinum CM-010 at 1 × 10⁶ conidia ml^?1 killed 100 % larvae within 2 h while Bacillus thuringiensis var. israelensis at 5 ITU ml^?1 required 24 h. This P. citrinum CM-010 also greatly reduced survival of C. quinquefasciatus larvae in an unreplicated field test. Light and transmission electron micrographs showed that the fungal conidia were ingested by the larvae and deposited in the gut. The metabolite patulin was produced by P. citrinum CM-010 instead of citrinin.     

Enhanced Melanogenesis Induced by Tyrosinase Gene-Transfer Increases Boron-Uptake and Killing Effect of Boron Neutron Capture Therapy for Amelanotic Melanoma

Specific and powerful cancer killing effect for melanoma by boron neutron capture therapy (BNCT) using DOPA analogue, ¹⁰B-p-boronophenylalanine (¹⁰B-BPA), has been established, but amelanotic melanoma is insufficiently responsive to ¹⁰B-BPA BNCT in comparison with actively melanin-producing melanoma. Although the accumulation mechanism of ¹⁰B-BPA within melanoma was not established, we have recently obtained findings suggesting that melanin monomers, key intermediates for melanin polymer formation, play a critical role in ¹⁰B-BPA accumulation. In addition, there are some kinds of human amelanotic melanomas, such as MEL2A, in which expression of tyrosinase is repressed or lacking though tyrosinase-related protein (TRP)-l and TRP-2 are well expressed. Thus, by using a similarly tyrosinase-lacking mouse amelanotic melanoma cell line, A1059, we constructed TA1059 cells by transfecting human tyrosinase-cDNA into these cells. TA1059 cells acquired higher DOPA-oxidase and DOPAchrome tautomerase activity as well as eumelanin content at even higher levels than those of B16F10 cells. TA1059 cells showed about 2.5 times higher ^p-boronophenylalanine (BPA) uptake than A1059 cells in culture. In animal experiments, by using these cell lines, tumor growth of TA1059 was significantly suppressed by ¹⁰B-BPA BNCT as compared with A1059. These findings indicate that the induction of active melanin biosynthesis by melanogenic gene-transfer effectively improves the treatment of amelanotic melanoma by BNCT.     

Optimizing modes of inoculation of <Emphasis Type="Italic">Rhipicephalus</Emphasis> ticks (Acari: Ixodidae) with a mitosporic entomopathogenic fungus in the laboratory

The process of strain selection is an important step in the development of insect pathogens for biological control. Bioassays were conducted in the laboratory to evaluate the efficacy of different methods of inoculation using Rhipicephalus pulchellus Gerstäcker (Acari: Ixodidae) as a model. Initially, an oil-based formulation of Metarhizium anisopliae (Metsch.) Sorok. (Ascomycota: Hypocreales) titred at 10⁹ conidia ml^?1 was applied to R. pulchellus adults using a Burgerjon spray tower or a microapplicator. Inoculation by microapplicator yielded poor results (25.0% tick mortality) compared to Burgerjon’s spray tower (52.3% tick mortality), although the mean number of fungal conidia on R. pulchellus adults was lower (1.5 × 10⁴ ± 1.1 × 10³ conidia ml^?1) after spraying by Burgerjon’s spray tower compared to 1 × 10⁶ conidia ml^?1 obtained with the microapplicator. Thus, inoculation by Burgerjon’s spray tower was selected for further investigations. Different modes of inoculation were tested and included direct spray of inoculum on the tick and substrate (SS), direct spray on the substrate and tick followed by transfer of the tick to clean uncontaminated Petri dish (SP) or indirect inoculation of ticks through substrate (SW). The LC₅₀ values following contamination of nymphs (LC₅₀ = 1.4 × 10⁷ conidia ml^?1) and adults (LC₅₀ = 6.7 × 10⁷ conidia ml^?1) in SS were significantly lower compared to SP; nymphs (LC₅₀ = 5.7 × 10⁸ conidia ml^?1) and adults (LC₅₀ = 5.3 × 10⁹ conidia ml^?1) and SW; nymphs (LC₅₀ = 5 × 10⁸ conidia ml^?1). Although the LC₅₀ value in SS was the lowest, it recorded the highest tick mortality among control ticks (24.2% at 2 weeks post-treatment) and (23.3% at 3 weeks post-treatment) in nymphs and adults respectively compared to SP (2.5 and 5.8%, respectively) and SW (0.0 and 0.0). Results show that among the modes of inoculation tested, SP was the most appropriate for inoculating R. pulchellus adults. SW and SP were identified as appropriate techniques for infecting the R. pulchellus nymphs with conidia formulated in oil.     

Nisin A and Polymyxin B as Synergistic Inhibitors of Gram-positive and Gram-negative Bacteria

Nisin A and polymyxin B were tested alone and in combination in order to test their antagonism against Listeria innocua HPB13 and Escherichia coli RR1, respectively. While the combination of both antibacterial substances was synergistically active against both target bacteria, nisin A alone did not show any inhibition of E. coli RR1. The nisin A/polymyxin B combination at 1.56/2.5 μg ml^?1 caused lysis of about 35.86 ± 0.35 and 73.36 ± 0.14% of L. innocua HPB13 cells after 3 and 18 h, respectively. Polymyxin B at 0.12 μg ml^?1 and nisin A/polymyxin B at 4.64/0.12 μg ml^?1 decreased the numbers of viable E. coli RR1 cells by about 0.23 and 0.65 log₁₀ CFU ml^?1, respectively, compared to the control. Our data suggest that the concentration of nisin A required for the effective control of pathogenic strains Listeria spp. could be lowered considerably by combination with polymyxin B. The use of lower concentrations of nisin A or polymyxin B should slow the emergence of bacterial populations resistant to these agents.     

Expression of the acidic-subunit of amarantin, carrying the antihypertensive biopeptides VY, in cell suspension cultures of Nicotiana tabacum NT1

A modified version of amarantin, main seed storage protein of Amaranthus hypochondriacus, carrying four antihypertensive biopeptides Val-Tyr into the acidic-subunit of the protein, was expressed in cell suspension cultures of Nicotiana tabacum L. NT1. Cell growth and viability kinetics were assessed to determine optimal conditions for genetic transformation via Agrobacterium tumefaciens. Selection of putative transgenic calli was conducted using 25 μg ml^?1 hygromycin. Presence of the transgene was confirmed using histological glucuronidase assay and PCR analysis. Accumulation and expression of the recombinant protein was detected using Western blot analysis. Protein hydrolysate of transgenic calli showed high levels of inhibition of the angiotensin converting enzyme, with an IC₅₀ value of 3.5 μg ml^?1. This was 10-fold lower than that of protein extracts of wild-type cells, with an IC₅₀ of 29.0 μg ml^?1.     

Endophytic actinomycetes isolated from Aquilaria crassna Pierre ex Lec and screening of plant growth promoters production

A total of 10 endophytic actinomycete strains were successfully isolated from healthy shoots and roots of Aquilaria crassna Pierre ex Lec (eaglewood). Analysis of 16S rDNA sequencing of those isolates showed that they belong to members of the genera Streptomyces (2 isolates), Nonomuraea (1 isolate), Actinomadura (1 isolate), Pseudonocardia (1 isolate) and Nocardia (3 isolates). The remaining 2 isolates were unidentified. All of isolates produced the amount of indole-3-acetic acid (IAA) and ammonia ranging between 9.85 ± 0.31 to 15.14 ± 0.22 μg ml^?1 and 2 to 60 mg ml^?1, respectively. Among 10 isolates tested, the amount of hydroxamate-type siderophore produced by 2 isolates was undetectable. While the remaining 8 isolates produced the amount of hydroxamate-type ranging between 3.21 ± 0.12 and 39.30 ± 0.40 μg ml^?1. Also, catechols-type siderophore produced by 9 isolates was undetectable. Actinomadura glauciflava is only one isolate that produced catechols-type 4.12 ± 0.90 μg ml^?1. In addition, 10 endophytic actinomycetes showed protease activity ranging from undetectable to 8.16 ± 0.15 unit ml^?1. Genetic relatedness amongst these isolates was determined base on Random amplified polymorphic DNA (RAPD) and Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC PCR). Both methodologies generated specific patterns corresponding to particular genotypes. RAPD fingerprinting proved to be slightly more discriminatory than ERIC PCR. This study is the first published report that actinomycetes can be isolated as endophytes within this plant. It is also the first published report that endophytic actinomycetes are capable of producing IAA and siderophores.     

Expression and evaluation of enzymes required for the hydrolysis of galactomannan

The cost-effective production of bioethanol from lignocellulose requires the complete conversion of plant biomass, which contains up to 30 % mannan. To ensure utilisation of galactomannan during consolidated bioprocessing, heterologous production of mannan-degrading enzymes in fungal hosts was explored. The Aspergillus aculeatus endo-β-mannanase (Man1) and Talaromyces emersonii α-galactosidase (Agal) genes were expressed in Saccharomyces cerevisiae Y294, and the Aspergillus niger β-mannosidase (cMndA) and synthetic Cellvibrio mixtus β-mannosidase (Man5A) genes in A. niger. Maximum enzyme activity for Man1 (374 nkat ml^?1, pH 5.47), Agal (135 nkat ml^?1, pH 2.37), cMndA (12 nkat ml^?1, pH 3.40) and Man5A (8 nkat ml^?1, pH 3.40) was observed between 60 and 70 °C. Co-expression of the Man1 and Agal genes in S. cerevisiae Y294[Agal-Man1] reduced the extracellular activity relative to individual expression of the respective genes. However, the combined action of crude Man1, Agal and Man5A enzyme preparations significantly decreased the viscosity of galactomannan in locust bean gum, confirming hydrolysis thereof. Furthermore, when complemented with exogenous Man5A, S. cerevisiae Y294[Agal-Man1] produced 56 % of the theoretical ethanol yield, corresponding to a 66 % carbohydrate conversion, on 5 g l^?1 mannose and 10 g l^?1 locust bean gum.     

Iodine from bacterial iodide oxidization by Roseovarius spp. inhibits the growth of other bacteria

Microbial activities in brine, seawater, or estuarine mud are involved in iodine cycle. To investigate the effects of the microbiologically induced iodine on other bacteria in the environment, a total of 13 bacteria that potentially participated in the iodide-oxidizing process were isolated from water or biofilm at a location containing 131 μg ml^?1 iodide. Three distinct strains were further identified as Roseovarius spp. based on 16 S rRNA gene sequences after being distinguished by restriction fragment length polymorphism analysis. Morphological characteristics of these three Roseovarius spp. varied considerably across and within strains. Iodine production increased with Roseovarius spp. growth when cultured in Marine Broth with 200 μg ml^?1 iodide (I^?). When 10⁶ CFU/ml Escherichia coli, Pseudomonas aeruginosa, and Bacillus pumilus were exposed to various concentrations of molecular iodine (I₂), the minimum inhibitory concentrations (MICs) were 0.5, 1.0, and 1.0 μg ml^?1, respectively. However, fivefold increases in the MICs for Roseovarius spp. were obtained. In co-cultured Roseovarius sp. IOB-7 and E. coli in Marine Broth containing iodide (I^?), the molecular iodine concentration was estimated to be 0.76 μg ml^?1 after 24 h and less than 50 % of E. coli was viable compared to that co-cultured without iodide. The growth inhibition of E. coli was also observed in co-cultures with the two other Roseovarius spp. strains when the molecular iodine concentration was assumed to be 0.52 μg ml^?1.     

Winter bloom of picoeukaryotes in Hungarian shallow turbid soda pans and the role of light and temperature

The seasonal dynamics of picophytoplankton communities in shallow turbid alkaline pans in Hungary was studied between July 2006 and May 2007. Similarly to other aquatic environments in the temperate zone, dominance of picocyanobacteria was observed in summer and that of picoeukaryotes in winter. The mild winter in 2006–2007, with midday water temperatures of 5–10°C, resulted in large winter phytoplankton blooms (maximum chlorophyll a concentration 800 μg l^?1) in the shallow pans. The phytoplankton was composed of single-celled picoeukaryotes and had a maximum of 108 × 10⁶ cells ml^?1 in Büdös-szék pan, 50 × 10⁶ cells ml^?1 in Kelemen-szék pan in April 2007, and 47 × 10⁶ cells ml^?1 in Zab-szék pan in March 2007. In order to explain the winter dominance of picoeukaryotes, we isolated picoeukaryotic and picocyanobacterial strains and determined the temperature and light dependence of their photosynthesis. Under temperatures <15°C, the photosynthetic activity of the picoeukaryotic strain was higher and their light utilization was better than those of the picocyanobacterial strain. The results indicate that low temperature and light intensity in winter provide a competitive advantage to picoeukaryotes, while higher temperatures and light intensity are more favorable for picocyanobacteria.     

Boron neutron capture synovectomy (BNCS) as a potential therapy for rheumatoid arthritis: boron biodistribution study in a model of antigen-induced arthritis in rabbits

Boron neutron capture synovectomy (BNCS) is explored for the treatment of rheumatoid arthritis (RA). The aim of the present study was to perform boron biodistribution studies in a model of antigen-induced arthritis (AIA) in female New Zealand rabbits, with the boron carriers boronophenylalanine (BPA) and sodium decahydrodecaborate (GB-10) to assess the potential feasibility of BNCS for RA. Rabbits in chronic phase of AIA were used for biodistribution studies employing the following protocols: intra-articular (ia) (a) BPA-f 0.14 M (0.7 mg ¹⁰B), (b) GB-10 (5 mg ¹⁰B), (c) GB-10 (50 mg ¹⁰B) and intravenous (iv), (d) BPA-f 0.14 M (15.5 mg ¹⁰B/kg), (e) GB-10 (50 mg ¹⁰B/kg), and (f) BPA-f (15.5 mg ¹⁰B/kg) + GB-10 (50 mg ¹⁰B/kg). At different post-administration times (13–85 min for ia and 3 h for iv), samples of blood, pathological synovium (target tissue), cartilage, tendon, muscle, and skin were taken for boron measurement by inductively coupled plasma mass spectrometry. The intra-articular administration protocols at <40 min post-administration both for BPA-f and GB-10, and intravenous administration protocols for GB-10 and [GB-10 + BPA-f] exhibited therapeutically useful boron concentrations (>20 ppm) in the pathological synovium. Dosimetric estimations suggest that BNCS would be able to achieve a therapeutically useful dose in pathological synovium without exceeding the radiotolerance of normal tissues in the treatment volume, employing boron carriers approved for use in humans. Radiobiological in vivo studies will be necessary to determine the actual therapeutic efficacy of BNCS to treat RA in an experimental model.     

Effects of reduced seawater pH on fertilisation, embryogenesis and larval development in the Antarctic seastar Odontaster validus

The effects of ocean acidification will be pronounced in high-latitude marine communities, although little is known on how reproduction in free-spawning polar invertebrates will respond. Using the circum-Antarctic sea star Odontaster validus, we examined fertilisation, larval survival and development under a controlled seawater treatment (temperature = ?0.5 °C, pH 8.1, pCO₂(aq) = 326.6 μatm, TA = 2,274.2 μmol kg soln^?1), two near-future pH treatments (pH 7.8 and 7.6) and an extreme treatment (pH 7.0). At a sperm concentration of 3.5 × 10⁵ sperm ml^?1, percentage of fertilisation was 98–90 % across a pH 8.1–7.0 range. At near-future pH ranges (pH 7.8 and 7.6), fertilisation was not significantly lower than in the control pH 8.1 except at the lowest sperm concentration (2.2 × 10³ sperm ml^?1) where fertilisation was reduced to 60 and 61 % in pH 7.6 and 7.8, respectively. Larval survival was not significantly affected by a decrease in pH of 0.3 units, but at pH 7.6 survival was significantly reduced. This difference was apparent at 9 days, and at the end of the experiment at 58 days, survival was 55 % compared with 85 % in the ambient treatment. Near-future changes to pH yielded smaller larvae, a result of both subtle differences in their morphology and slowed development rates, while larvae at pH 7.0 showed evidence of abnormal development. O. validus fertilisation and larval success declines in seawater pH conditions expected in coastal Antarctica over the coming decades, although the responses observed are within the range observed in warmer-water echinoderms.     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.