Kinetics of laccase-catalysed TEMPO oxidation

The oxidation of TEMPO (2,2,6,6-tetramethyl-piperidine-1-oxyl radical) has been studied in the presence of recombinant laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) from Polyporus pinsitus (rPpL), Myceliophthora thermophila (rMtL), Coprinus cinereus (rCcL) and Rhizoctonia solani (rRsL) in buffer solution pH 4.5–7.3 and at 25 °C. At pH 5.5 the oxidation constant calculated from the initial rate of TEMPO oxidation was 1.7 × 10⁴, 1.4 × 10³, 7.8 × 10² and 5.2 × 10² M⁻¹ s⁻¹ for rPpL, rRsL, rCcL and rMtL, respectively. The maximal activity of rPpL-catalysed TEMPO oxidation was at pH 5.0. The pK_a obtained in neutral pH range was 6.2. The reactivity of laccases is in a good agreement with laccases copper type I redox potential.

TEMPO oxidation rate increased 541 times in the presence of 10-(3-propylsulfonate) phenoxazine (PSPX). The model of synergistic TEMPO and PSPX oxidation was proposed. Experimentally obtained rate constants for rPpL-catalysed PSPX oxidation were in a good agreement with those calculated from the synergistic model, therefore confirming the feasibility of the model. The acceleration of TEMPO oxidation with high reactive laccase substrates opens new possibilities for TEMPO application as a mediator.     

Radical-scavenging activity of butylated hydroxytoluene (BHT) and its metabolites

To clarify the radical-scavenging activity of butylated hydroxytoluene (BHT), a food additive, stoichiometric factors (n) and inhibition rate constants (k_inh) were determined for 2,6-di-tert-butyl-4-methylphenol (BHT) and its metabolites 2,6-di-tert-butyl-p-benzoquinone (BHT-Q), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHA-CHO) and 3,5-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadiene-1-one (BHT-OOH). Values of n and k_inh were determined from differential scanning calorimetry (DSC) monitoring of the polymerization of methyl methacrylate (MMA) initiated by 2,2′-azobis(isobutyronitrile) (AIBN) or benzoyl peroxide (BPO) at 70 °C in the presence or absence of antioxidants (BHT-related compounds). The n values declined in the order BHT (1–2) > BHT-CHO, BHT-OOH (0.1–0.3) > BHT-Q (0). The n value for BHT with AIBN was approximately 1.0, suggesting dimerization of BHT. The k_inh values declined in the order BHT-Q ((3.5–4.6)×10⁴ M⁻¹ s⁻¹) > BHT-OOH (0.7–1.9×10⁴ M⁻¹ s⁻¹) > BHT-CHO ((0.4–1.7)×10⁴ M⁻¹ s⁻¹) > BHT ((0.1–0.2)×10⁴ M⁻¹ s⁻¹). The k_inh for metabolites was greater than that for the parent BHT. Growing MMA radicals initiated by BPO were suppressed much more efficiently by BHT or BHT-Q compared with those initiated by AIBN. BHT was effective as a chain-breaking antioxidant.     

Enhancement of growth and gymnodimine production by the marine dinoflagellate, Karenia selliformis

Because of its novel bioactive properties the production of gymnodimine for use as a pharmaceutical precursor has aroused interest. The dinoflagellate, Karenia selliformis produces gymnodimine when grown in bulk culture using GP + selenium medium but the growth rates (μ) and levels of gymnodimine are low (μ, 0.05 days⁻¹; gymnodimine 250 μg L⁻¹ max). We describe the effects of organic acid additions (acetate, glycolate, alanine and glutamate additions and combinations of these) in enhancing growth and gymnodimine production in axenic cultures. The most effective organic acid combinations in decreasing order were: glycolate/alanine > acetate > glycolate. Glycolate/alanine optimised gymnodimine production by prolonging growth (maximum cell yield, 1.76 × 10⁵ cells mL⁻¹; gymnodimine, 1260 μg L⁻¹; growth rate (μ), 0.2 days⁻¹) compared to the control (growth maximum cell yield, 7.8 × 10⁴ cells mL⁻¹; gymnodimine, 780 μg L⁻¹; μ, 0.17 days⁻¹). Acetate enhanced gymnodimine by stimulating growth rate (μ, 0.23 days⁻¹) and the large concentration of gymnodimine per cell (16 pg cell⁻¹ cf. 9.8 pg cell⁻¹ for the control) suggests a role for this compound in gymnodimine biosynthesis. Amending culture media with Mn²⁺ additions resulted in slightly decreased growth in control cultures and increased the gymnodimine while in glycolate/alanine cultures growth was stimulated but gymnodimine production decreased. The results suggest that the organic acid can enhance gymnodimine production by either enhancing growth maximum or the biosynthetic pathway.     

Human fibroblast culture on a crosslinked dermal porcine collagen matrix

The use of a novel porcine-derived collagen biomaterial as a dermal tissue engineering matrix was examined. The matrix is derived from porcine dermis, and is processed to retain the native collagen (Type 1) and elastin structure. Human primary fibroblasts were cultured on the matrix to examine its potential for creating a dermal replacement. Attachment of fibroblasts on the collagen was compared to tissue culture plastic and PET membranes. Cell proliferation was assessed using the MTT assay and DAPI staining. For seeding densities of 5×10⁴ and 1×10⁵ cells cm⁻², PET and plastic demonstrated >95% attachment of seeded numbers after 3 h. The collagen matrix reached levels >80% after 3–4 h with no influence of the seeding density. Matrix samples with perforating pores of 40 μm diameter were also studied. After 216 h culture in static culture, with media replacement every 3 days, the final cell numbers reached 2.1×10⁵ (perforated) and 2.0×10⁵ cells cm⁻² (unperforated). In comparison fibroblast culture in a perfusion bioreactor, with continuous media replacement, reached 2.3×10⁵ (unperforated) and 2.5×10⁵ cells cm⁻² (perforated) after 216 h.     

Graft copolymerization of acrylic acid onto guar gum initiated by vanadium (V)–mercaptosuccinic acid redox pair

Guar gum has been modified by graft copolymerization with acrylic acid in aqueous medium using vanadium (V)–mercaptosuccinic acid redox system. The optimum reaction conditions affording maximum grafting ratio, efficiency, add on and conversion have been determined. The grafting parameters have been found to increase with increase in vanadium (V) concentration upto 1.0 × 10⁻² mol dm⁻³, but these parameters decrease on further increasing the vanadium (V) concentration. On increasing the mercaptosuccinic acid concentration from 1.0 × 10⁻² to 4.0 × 10⁻² mol dm⁻³ grafting ratio, efficiency and add on increase up to 2.0 × 10⁻² mol dm⁻³ but decrease with further increase in mercaptosuccinic acid concentration. On varying the acrylic acid concentration from 5.0 × 10⁻² to 30.0 × 10⁻² mol dm⁻³, maximum grafting ratio, efficiency and add on have been obtained at 20.0 × 10⁻² mol dm⁻³. The grafting ratio, add on and conversion increase, on increasing the H⁺ ion concentration from 1.5 × 10⁻¹ to 6.0 × 10⁻¹ mol dm⁻³. On increasing the guar gum concentration the grafting parameters increase. The grafting ratio, add on and conversion have been found to increase with time period while efficiency started decreasing after 120 min. It has been observed that %G increases on increasing the temperature up to 35 °C. The graft copolymer has been characterized by IR spectroscopy and thermogravimetric analysis.     

Catalysis of plastocyanin electron self-exchange by redox-inert multivalent cations

Electron self-exchange in solutions of the ‘blue’ copper protein plastocyanin is catalysed by the redox-inert multivalent cations Mg²⁺ or Co(NH₃)³⁺₆. Measurements of specific ¹H-NMR line broadening with 50% reduced solutions in the presence of these cations show that electron exchange proceeds through encounters of cation-protein complexes which dissociate at high ionic strength. In the presence of 8mM (5 equivalents/total protein) Co(NH₃)³⁺₆, with 10 mM cacodylate (pH^*6.0) as background electrolyte, the bimolecular rate constant at 25°C is 7 × 10⁴ M⁻¹·s⁻¹. For comparison, the ‘electrostatically screened’ rate constant measured in 0.1 M KCl in the absence of added multivalent cations is ˜ 4 × 10³ M¹·s⁻¹.

Plastocyanin Electron self-exchange NMR Protein-protein interaction Multivalent cation Blue copper protein     

Synthesis of graft copolymer (k-carrageenan-g-N,N-dimethylacrylamide) and studies of metal ion uptake, swelling capacity and flocculation properties

Graft copolymer of k-carrageenan and N,N-dimethylacrylamide has been synthesized by free radical polymerization using peroxymonosulphate/glycolic acid redox pair in an inert atmosphere. The grafting parameters i.e. grafting ratio, add on and efficiency decrease with increase in concentration of k-carrageenan from 0.6 to 1.4 g dm⁻³ and hydrogen ion from 3 × 10⁻³ to 7 × 10⁻³ mol dm⁻³, but these grafting parameters increase with increase in concentration of N,N-dimethylacrylamide from 16 × 10⁻² to 32 × 10⁻² mol dm⁻³, and peroxymonosulphate from 0.8 × 10⁻² to 2.4 × 10⁻² mol dm⁻³. The metal ion sorption, swelling behaviour and flocculation properties have been studied. The intrinsic viscosity of pure and grafted samples has been measured by using Ubbelohde capillary viscometer. Flocculation capability of k-carrageenan and k-carrageenan-g-N,N-dimethylacrylamide for both coking and non-coking coals has been studied for the treatment of coal mine waste water. The graft copolymer has been characterized by Infrared (IR) spectroscopy and thermogravimetric analysis.     

Purification and characterization of a Na/K dependent alginate lyase from turban shell gut Vibrio sp. YKW-34

An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The k_cat/K_m value for alginate (heterotype) was 1.7 × 10⁶ s⁻¹ M⁻¹. The enzyme activity was completely lost by dialysis and restored by addition of Na⁺ or K⁺. The optimal activity exhibited in 0.1 M of Na⁺ or K⁺. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA).     

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

The role of the heme propionate groups in determining the electron transfer and electrostatic properties of myoglobin have been studied by thermodynamic, kinetic, and spectroscopic studies of horse heart myoglobin in which the heme propionate groups are esterified. Spectroelectrochemical analysis has established that the E_m,7 of dimethylester heme-substituted Mb (DME-Mb) (E_m,7 = 100.2(2) mV vs. NHE (Normal Hydrogen Electrode) (25 °C) is increased  40 mV relative to that of the native protein with ΔH° = −12.9(2) kcal/mol and ΔS° = −51.0(8) cal/mol/deg (pH 7.0, μ = 0.1 M (phosphate)). The second order rate constant for reduction of DME-metMb by Fe(EDTA)²⁻ is increased  > 400-fold relative to that for reduction of native metMb to a value of 1.34(2) × 10³ M⁻¹ s⁻¹ with ΔS^‡ = −13(1) cal/mol/deg and ΔH^‡ = 9.2(3) (pH 7.0, μ = 0.1 M (phosphate)). Analysis of the pH dependences of the reduction potential and rate constant for reduction by Fe(EDTA)²⁻ demonstrates that heme propionate esterification introduces significant changes into the electrostatic interactions in myoglobin. These changes are also manifested by differences in the pH dependences of the ¹H NMR spectra of native and DME-metMb that reveal shifts in pK_a values for specific His residues as the result of heme propionate esterification. In sum, the current results establish that heme propionate esterification not only affects the electron transfer properties of myoglobin but also influences the titration behavior of specific His residues.     

Bench-scale fermentation of Trichoderma viride on wastewater sludge: Rheology, lytic enzymes and biocontrol activity

Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94 × 10⁷ CFU mL⁻¹ at 96 h) was obtained at 30 g L⁻¹ suspended solids. The maximum lytic enzyme activities were achieved around 12–30 h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96 h of fermentation at 30 g L⁻¹ suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290 SBU μL⁻¹ at 30 g L⁻¹ suspended solids concentration at the end of fermentation (96 h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35 g L⁻¹ compared to 30 and 25 g L⁻¹. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k_La at 35 g L⁻¹ did not increase in comparison to 30 g L⁻¹ due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent.     

Epinephrine quantification in pharmaceutical formulations utilizing plant tissue biosensors

A plant tissue biosensor associated with flow injection analysis is proposed to determine epinephrine in pharmaceutical samples. The polyphenol oxidase enzymes present in the fibers of a palm tree fruits (Livistona chinensis), catalyses the oxidation of epinephrine to epinephrinequinone as a primary product. This product is then electrochemically reduced (at −0.10 V versus Ag/AgCl_sat) on the biosensor surface and the resulting current is used for the quantification of epinephrine. The biosensor provides a linear response for epinephrine in the concentration range from 5.0 × 10⁻⁵ to 3.5 × 10⁻⁴ mol l⁻¹. The limit of detection estimated for this interval was 1.5 × 10⁻⁵ mol l⁻¹ and the correlation coefficient of 0.998, working under a flow rate of 2.0 ml min⁻¹ and using a sample loop of 100 μl. The repeatability (R.S.D. for 10 consecutive determinations of a 3.0 × 10⁻⁴ mol l⁻¹ epinephrine solution) was 3.1%. The results obtained by the method here proposed were compared with the official UV spectrophotometric procedure and also using a plant tissue reactor. The responses obtained with the proposed strategies were in good agreement with both ways of analyses, whereas the values obtained by the official spectrophotometric method was strongly affected by benzoic acid, present in the formulation of pharmaceutical product utilized for inhalation. Such favorable results obtained with the carbon paste biosensor or utilizing the bioreactor, joined with the simplicity of its preparation turns these procedures very attractive for epinephrine quantification in pharmaceutical products.     

Binding of genistein to human serum albumin demonstrated using tryptophan fluorescence quenching

Genistein is an isoflavone and phytoestrogen that is a potent inhibitor of cell proliferation and angiogenesis. This study was designed to investigate the binding of genistein to human serum albumin (HSA) under physiological conditions with drug concentrations in the range of 6.7 × 10⁻⁶ to 2.0 × 10⁻⁵ mol L⁻¹ and HSA concentration at 1.5 × 10⁻⁶ mol L⁻¹. Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to determine the binding mode, the binding constant and the protein structure changes in the presence of genistein in aqueous solution. Changes in the CD spectra and FT-IR spectra were observed upon ligand binding, and the degree of tryptophan fluorescence quenching change did significantly in the complexes. These data have proved the change in protein secondary structure accompanying ligand binding. The change in tryptophan fluorescence intensity was used to determine the binding constants. The thermodynamic parameters, the enthalpy change (ΔH) and the entropy change (ΔS) were calculated to be −22.24 kJ mol⁻¹and 19.60 J mol⁻¹ K⁻¹ according to the van’t Hoff equation, which indicated that hydrophobic and electrostatic interactions play the main role in the binding of genistein to HSA.     

SEC of mono-carboxymethyl cellulose (CMC) in a wide range of pH; Mark–Houwink constants

A method for determination of carboxymethyl cellulose (CMC) molecular weight (M_W) and chemical heterogeneity (degree of oxidation (DO)) using a bi-detector HPSEC (UV-detector online with refractometer) has been developed. It has been found that the use of 0.5 N NaOH or 0.4 M acetate buffer as the eluent ensures CMC separation according to M_W. It has been revealed that the universal calibration for the polyelectrolyte CMC and the neutral polymer dextran is valid under the conditions applied. The Mark–Houwink equations for CMC in 0.5 N NaOH and 0.4 M acetate buffer have been estimated to be [η]=5.37×10⁻⁴ M_W^0.73 and [η] =2.24×10⁻⁴ M_W^0.83 dl g⁻¹, respectively. The equation log K=1.64−4.00 ml g⁻¹ for CMC has been estimated. An approach for determining DO from adsorption at 290 or 313 nm has been developed.     

Direct electrochemistry of cytochrome c at ordered macroporous active carbon electrode

Three-dimensionally (3D) ordered macroporous active carbon has been fabricated and used as electrode substrate for the direct electrochemistry of horse heart cytochrome c (Cyt c). The Cyt c immobilized on the surface of the ordered macroporous active carbon shows a pair of well-defined and nearly reversible redox waves at the formal potential of −0.033 V in pH 6.8 phosphate buffer solution. The interaction between Cyt c and the 3D macroporous active carbon makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods have been used to investigate the interaction between Cyt c and the porous active carbon. The immobilized Cyt c maintains its biological activity, and shows a surface controlled electrode process with the electron-transfer rate constant (k_s) of 17.6 s⁻¹ and the charge-transfer coefficient (a) of 0.52, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). A potential application of the Cyt c-immobilized porous carbon electrode as a biosensor to monitor H₂O₂ has been investigated. The steady-state current response increases linearly with H₂O₂ concentration from 2.0 × 10⁻⁵ to 2.4 × 10⁻⁴ mol l⁻¹. The detection limit (3σ) for determination of H₂O₂ has been found to be 1.46 × 10⁻⁵ mol l⁻¹.     

Electrochemical impedance spectroscopy for study of aptamer-thrombin interfacial interactions

A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin. The signal enhancement was achieved by using gold nanoparticles (GNPs), which was electrodeposited onto a glassy carbon electrode (GCE), as a platform for the immobilization of the thiolated aptamer. In the measurement of thrombin, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12 nM to 30 nM. The association and dissociation rate constants of the immobilized aptamer–thrombin complex were 6.7 × 10³ M⁻¹ s⁻¹ and 1.0 × 10⁻⁴ s⁻¹, respectively. The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed. This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein. This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.     

Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues

It is well accepted that estradiol (E₂) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E₁S) ‘via sulfatase’ is a much more likely precursor for E₂ than is androstenedione ‘via aromatase’. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E₂ was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at –80 °C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45–75 mg) were incubated in 20 mM Tris–HCl, pH 7.2 with physiological concentrations of [³H]-E₁S (5 × 10⁻⁹ M) alone or in the presence of E₂ (5 × 10⁻⁵ to 5 × 10⁻⁷ M) during 30 min or 3 h. E₁S, E₁ and E₂ were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E₁ was 3.20 ± 0.15 and 0.42 ± 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 ± 1.8 and 3.5 ± 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 × 10⁻⁷ M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 × 10⁻⁵ M after 30 min incubation. The values were 24% and 18% for 5 × 10⁻⁷ M and 49% and 42% for 5 × 10⁻⁵ M, respectively after 3 h incubation. It was observed that [³H]-E₁S is only converted to [³H]-E₁ and not to [³H]-E₂ in normal or cancerous breast tissues, which suggests a low or no 17β-hydroxysteroid dehydrogenase (17β-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone.     

Alexandrium catenella and Alexandrium minutum blooms in the Mediterranean Sea: Toward the identification of ecological niches

Annual recurrent blooms of the toxic dinoflagellates Alexandrium catenella and Alexandrium minutum were detected from 2000 to 2003 in harbours along the Catalan coast. The interrelation study between the occurrence of the blooms and specific external conditions at the study sites demonstrated that different factors are required for the bloom of each Alexandrium species. Concentrations higher than 10⁵ cells l⁻¹ of A. catenella were only detected in Tarragona harbour. These blooms were associated with water surface temperature between 21 and 25 °C and salinities of around 34 psu or higher than 37 psu. A. minutum appeared widely spread along the Catalan coast, though the most intensive and recurrent blooms of this species were observed in Arenys de Mar harbour. Concentrations of millions of cells per litre of A. minutum were associated with water temperatures below 14 °C and salinities of around 34–36 psu. A. minutum cell densities showed a positive significant correlation with NO₃ but a negative correlation with NH₄. On the other hand, A. catenella blooms dominated when both NO₃ and NH₄ levels were high. The prevailing inorganic nitrogen form (NO₃ vs. NH₄) could explain why these two species rarely coincide in the same harbours. Accumulation of cysts in the sediment was found to be an important potential factor for the recurrence of these species. The 4.3 × 10³ A. catenella cysts cm⁻³ of wet sediment in Tarragona harbour and the 3.02 × 10³ A. minutum cysts cm⁻³ of wet sediment in Vilanova harbour were the highest concentrations observed from the cyst study. Confined waters such as harbours play an important role as reservoirs for the accumulation of cysts and vegetative cells, which contributes to the expansion of these dinoflagellates in the region. However, the particular environmental conditions are also decisive factors of bloom intensity.     

The dinoflagellate Alexandrium minutum in Cape Town harbour (South Africa): Bloom characteristics, phylogenetic analysis and toxin composition

A novel bloom of Alexandrium minutum occurred in an inner basin of the Cape Town harbour from November 2003 to February 2004. Cellular concentrations reached a maximum of 1.4 × 10⁸ cells l⁻¹ during the mid-December period with corresponding chlorophyll a concentrations of 243 mg m⁻³. Primary productivity measurements conducted during the latter part of the bloom revealed a maximum assimilation number of 11.17 mg C mg Chl a⁻¹ h⁻¹ during the middle of the day. Productivity during this post-peak period was sustained largely by the reduced nitrogen species NH₄ and urea (96%) as measured using ¹⁵N tracer techniques. The large subunit ribosomal DNA sequence of A. minutum isolates from Cape Town harbour was identical to conspecifics collected in Western Europe and in Australia. The composition of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP) was limited to gonyautoxins (GTX1-GTX4). This profile combined with evidence of a low toxin cell quota (1.5 fmol GTX cell⁻¹) supports a close association of this taxon with other members of the A. minutum species complex, particularly from Europe. Toxin analysis from black mussels collected during this bloom indicated that the accumulated PSP toxins originated from A. minutum and not from Alexandrium catenella as is most often the case along the South African coast.     

Mutational spectrum of the lacI gene in Escherichia coli K12 induced by low-energy ion beam

The mutational spectrum of the genomic lacI gene induced by low-energy nitrogen ion irradiation in wild type Escherichia coli strain W3110 were compared with the spontaneous and the vacuum controls. The mutant frequency of irradiated group was dose-dependent and reached 26.3 × 10⁻⁶ at dose of 31.2 × 10¹⁴ ions/cm², which was about 18-fold over the background (1.5 × 10⁻⁶) and 10-fold over the vacuum controls (2.6 × 10⁻⁶). This result indicated that the low-energy ion irradiation was one of many effective mutagens, though the vacuum condition of low-energy ions contributed some low-level gene mutations. It was found that the difference between the spontaneous and the vacuum control was the increases of base-pair substitutions in the vacuum control group. The spectra of irradiated group were quite similar to that of oxygen free-radical induced in the same strain, suggesting free-radicals and other adducts generated by low-energy ions might play an important role in the mutagenesis in vivo. When the spontaneous and the vacuum control group were compared, base-pair substitutions, deletions and additions of the irradiated group were significantly increased, and the +TGGC or −TGGC at hot spot was decreased from 82 to 48%. But the remarkable increase in absolute MF of the +TGGC or −TGGC at hot spot in the irradiated group suggested that low-energy ions did induce the mutations of this type. The spectra of our irradiated group had relative low-level base-pair substitutions, high-level ±TGGC and high proportion additions than those of γ-radiation induced, implying there were some different effects or processes between them.     

Kinetics and mechanism of the stoichiometric oxygenation of [Cu(fla)(idpa)]ClO4 [fla=flavonolate, IDPA=3,3′-imino-bis(N,N-dimethylpropylamine)] and the [Cu(fla)(idpa)]ClO4-catalysed oxygenation of flavonol

Oxygenation of [Cu^II(fla)(idpa)]ClO₄ (fla=flavonolate; IDPA=3,3′-iminobis(N,N-dimethylpropylamine)) in dimethylformamide gives [Cu^II(idpa)(O-bs)]ClO₄ (O-bs=O-benzoylsalicylate) and CO. The oxygenolysis of [Cu^II(fla)(idpa)]ClO₄ in DMF was followed by electronic spectroscopy and the rate law −d[{Cu^II(fla)(idpa)}ClO₄]/dt=k_obs[{Cu^II(fla)(idpa)}ClO₄][O₂] was obtained. The rate constant, activation enthalpy and entropy at 373 K are k_obs=6.13±0.16×10⁻³ M⁻¹ s⁻¹, ΔH^‡=64±5 kJ mol⁻¹, ΔS^‡=−120±13 J mol⁻¹ K⁻¹, respectively. The reaction fits a Hammett linear free energy relationship and a higher electron density on copper gives faster oxygenation rates. The complex [Cu^II(fla)(idpa)]ClO₄ has also been found to be a selective catalyst for the oxygenation of flavonol to the corresponding O-benzoylsalicylic acid and CO. The kinetics of the oxygenolysis in DMF was followed by electronic spectroscopy and the following rate law was obtained: −d[flaH]/dt=k_obs[{Cu^II(fla)(idpa)}ClO₄][O₂]. The rate constant, activation enthalpy and entropy at 403 K are k_obs=4.22±0.15×10⁻² M⁻¹ s⁻¹, ΔH^‡=71±6 kJ mol⁻¹, ΔS^‡=−97±15 J mol⁻¹ K⁻¹, respectively.