Laccase-catalyzed oxidation of phenolic compounds in organic media

Rhus vernificera laccase-catalyzed oxidation of phenolic compounds, i.e., (+)-catechin, (−)-epicatechin and catechol, was carried out in selected organic solvents to search for the favorable reaction medium. The investigation on reaction parameters showed that optimal laccase activity was obtained in hexane at 30 °C, pH 7.75 for the oxidation of (+)-catechin as well as for (−)-epicatechin, and in toluene at 35 °C, pH 7.25 for the oxidation of catechol. E_a and Q₁₀ values of the biocatalysis in the reaction media of the larger log p solvents like isooctane and hexane were relatively higher than those in the reaction media of lower log p solvents like toluene and dichloromethane. Maximum laccase activity in the organic media was found with 6.5% of buffer as co-solvent. A wider range of 0–28 μg protein/ml in hexane than that of 0–16.7 μg protein/ml in aqueous medium was observed for the linear increasing conversion of (+)-catechin. The kinetic studies revealed that in the presence of isooctane, hexane, toluene and dichloromethane, the K_m values were 0.77, 0.97, 0.53 and 2.9 mmol/L for the substrate of (+)-catechin; 0.43, 0.34, 0.14 and 3.4 mmol/L for (−)-epicatechin; 2.9, 1.8, 0.61 and 1.1 mmol/L for catechol, respectively, while the corresponding V_max values were 2.1 × 10⁻², 2.3 × 10⁻², 0.65 × 10⁻² and 0.71 × 10⁻² δA/μg protein min); 1.8 × 10⁻², 0.88 × 10⁻², 0.19 × 10⁻² and 1.0 × 10⁻² δA/μg protein min); 0.48 × 10⁻², 0.59 × 10⁻², 0.67 × 10⁻² and 0.54 × 10⁻² δA/μg protein min), respectively. FT-IR indicated the formation of probable dimer from (+)-catechin in organic solvent. These results suggest that this laccase has higher catalytic oxidation capacity of phenolic compounds in suitable organic media and favorite oligomers could be obtained.     

Microbiological degradation of the herbicide dicamba

Summary Pseudomonas paucimobilis was isolated from a consortium which was capable of degrading dicamba (3,6-dichloro-2-methoxybenzoic acid) as the sole source of carbon. The degradation of dicamba byP. paucimobilis and the consortium was examined over a range of substrate concentration, temperature, and pH. In the concentration range of 100–2000 mg dicamba L^–1 (0.5–9.0 mM), the degradation was accompanied by a stoichiometric release of 2 mol of Cl^– per mol of dicamba degraded. The cultures had an optimum pH 6.5–7.0 for dicamba degradation. Growth studies at 10°C, 20°C, and 30°C yielded activation energy values in the range of 19–36 kcal mol^–1 and an average Q₁₀ value of 4.0. Compared with the pure cultureP. paucimobilis, the consortium was more active at the lower temperature.     

Graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose and study of its swelling, metal ion sorption and flocculation behaviour

The present paper reports the graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose by free radical polymerization using potassium peroxymonosulphate/thiourea redox system in an inert atmosphere. The reaction conditions for maximum grafting have been optimized by varying the reaction variables, including the concentration of N-vinylformamide (12.0 × 10⁻²–28.0 × 10⁻² mol dm⁻³), potassium peroxymonosulphate (4.0 × 10⁻³–12.0 × 10⁻³ mol dm⁻³), thiourea (1.2 × 10⁻³–4.4 × 10⁻³ mol dm⁻³), sulphuric acid (2.0 × 10⁻³–10.0 × 10⁻³ mol dm⁻³), sodium carboxymethylcellulose (0.2–1.8 g dm⁻³) along with time duration (60–180 min) and temperature (25–45° C). Water swelling capacity, metal ion sorption and flocculation studies of synthesized graft copolymer have been performed with respect to the parent polymer. The graft copolymer has been characterized by FTIR spectroscopy and thermogravimetric analysis.     

Strong small-scale temporal bacterial changes associated with the migrations of bloom-forming dinoflagellates

Bacterial abundances in nearshore Mediterranean planktonic environments tend to change seasonally by 10-fold. Strong daily changes in bacterial abundance, at least as large as seasonal range, occurred in the presence of large dinoflagellate populations performing daily vertical migrations. The daily variability of heterotrophic bacteria was associated with the daily migrations of a bloom of Alexandrium taylori in La Fosca Bay, and Gymnodinium impudicum in Barcelona harbor. Bacterial abundance in surface waters can change daily as much as from 1 × 10⁶ to 5 × 10⁶ with apparent net change rates of 0.24 h⁻¹. We suggest that the migrating dinoflagellates create microstructures exploited by the bacteria, and that the large algal populations (>10⁶ cells l⁻¹) make this microstructure visible with conventional sampling protocols. We also show evidence of the link between dinoflagellate abundance and relative bacterial activity in these waters, as measured by the percentage of bacteria with high nucleic acid content.     

In vitro cytogenetic effects of Andrographis paniculata (kalmegh) on arsenic

In vitro effects of arsenic in human peripheral lymphocytes (HPL) at three different doses – 3.6×10⁻⁴, 1.4×10⁻³ and 0.72×10⁻³ μM for 24 h before harvesting on sister chromatid exchanges (SCE), Cell cycle proliferative index/replicative index (CCPI/RI), %M₁, %M₂ and %M₃, population doubling time (PDT) and average generation time (AGT) were examined. Andrographis paniculata (commonly referred to as ‘kalmegh’) has been used for centuries in traditional Indian and Chinese herbal medicine as a safe, natural folk remedy for assorted health concerns. In the present study, kalmegh (0.01 μg/7 ml culture media) was used along with the highest dose of arsenic; the results showed that arsenic induced increase in these genotoxic endpoints were fairly diminished by kalmegh. In addition, mutagenic in vitro effect of ethyl methanesulphonate (EMS) was used as a positive control in this study. It is thus concluded from this study that Andrographis has a protective role in arsenic toxicity.     

Molecular identification of intestinal microflora in Takifugu niphobles

We investigated the intestinal microflora of coastal fish including Takifugu niphobles using both culture techniques and library cloning. As a result, the numbers of bacteria appeared on agar media were 1.0 × 10⁴ to 1.4 × 10⁹ CFU/g (colony forming units/gram), whereas those of total bacteria stained with 4′,6-diamidino-2-phenylindole were 4.7 × 10¹⁰ to 1.9 × 10¹¹ cells/gram, irrespective of different fish species. In addition, the culture technique showed that the intestinal microflora in all specimens was mainly composed of the genus Vibrio. In contrast, the direct count method showed that spirochaetes with length of 2.5-4.5 μm were present in the intestinal contents of T. niphobles at high densities, whereas such bacteria could not be detected in those of other fish species. Library cloning yielded the sequences of 16S rRNA genes that were divided into seven taxonomic categories of bacteria including Actinobacteria, Bacilli, Clostridia, Gammaproteobacteria, Mollicutes, Spirochaetes and an unclassified bacterial group. These results demonstrate that the molecular diversity of the intestinal bacteria in T. niphobles based on the clone library method reflects the direct observation by fluorescence microscopy to some extent.     

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III under laboratory culture

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III, two species of causative bloom dinoflagellates in China, were investigated using bi-algal cultures under controlled laboratory conditions. The growth of P. donghaiense and S. trochoidea were significantly suppressed when the initial cell densities were set at 1.9 × 10⁴ cells mL⁻¹ or 1.9 × 10⁵ cells mL⁻¹ for P. donghaiense and 1.0 × 10⁴ cells mL⁻¹ for S. trochoidea when the initial size/density ratio was 1:1 or 10:1, respectively, but no out-competement was observed in either bi-algal culture by the end. The simultaneous assay on the culture filtrate showed that P. donghaiense filtrate prepared at a lower initial density (1.9 × 10⁴ cells mL⁻¹) stimulated the co-cultured S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹, but filtrate at a higher density (1.9 × 10⁵ cells mL⁻¹) depressed its growth. Differently, the filtrate of S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹ significantly suppressed the growth of P. donghaiense at a density of 1.9 × 10⁴ cells mL⁻¹, but had little stimulatory effect on P. donghaiense at a density of 1.9 × 10⁵ cells mL⁻¹compared to the control (P > 0.05). It is likely that these two species of microalgae interact with each other mainly by releasing allelochemical substance(s) into the culture medium, and a direct cell-to-cell contact was not necessary for their mutual interaction. We then quantify their interactions in the bi-algal culture by using a mathematical model. The estimated parameters from the model showed that the inhibition exerted by S. trochoidea on P. donghaiense was about 43 and 24 times stronger than the inhibitory effect that P. donghaiense exerted on S. trochoidea when the initial size/density were 1:1 and 10:1, respectively. S. trochoidea seemed to have a survival strategy that was superior to P. donghaiense in the bi-algal culture under controlled laboratory conditions. We also observed a closely positive relationship between the initial cell density and its effect on the co-cultured microalga by measuring the fluorenscence: filtrate prepared from higher initial cell density had stronger interference on the co-cultured microalga. Moreover, pre-treated under different temperature conditions (30 °C, 60 °C and 100 °C) would significantly changed the effect of culture filtrate on the co-cultured microalga. Result inferred that P. donghaiense or S. trochoidea would release allelochemicals into the bi-algal culture medium and the allelochemicals might be a mixture with temperature-sensitive components in it.     

Chromate reduction by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> TKW3 isolated from marine sediments

Summary Bacillus megaterium strain TKW3 was isolated from multiple-metal-contaminated marine sediments of Tokwawan, Hong Kong SAR. This facultative aerobe utilized arabinose, mannitol, N-acetylglucosamine, maltose, caprate, citrate, butyrate or lactate as the sole source of carbon and energy for growth.B. megaterium TKW3 reduced highly toxic and soluble Cr⁶⁺ (as CrO₄²⁻) into almost non-toxic and insoluble Cr³⁺ under aerobic conditions. Complete reduction of 0.20 mM Cr⁶⁺ by B. megaterium TKW3 was achieved within 360 h. Initial Cr⁶⁺ concentration below 0.90 mM or inoculum less than 10⁷ cells ml⁻¹ did not have significant effect on ⁶⁺ reduction, while the residue Cr⁶⁺ concentration was the lowest at 10⁷ cells ml⁻¹. Cr⁶⁺ reduction by this strain was inhibited by high levels of NaCl (55%). B. megaterium TKW3 was also resistant to other oxyanions including 0.34 mM Cr₂O₇²⁻ 0.32 mM AsO₄³⁻, 0.58 mM SeO₃²⁻ and 0.53 mM SeO₄²⁻, and reduced soluble Se⁴⁺ (as SeO₃²⁻) to insoluble red amorphous Se⁰. B. megaterium TKW3 might have potential application in bioremediation of Cr-laden sediments associated with other oxyanions.     

Application of response-surface methodology to evaluate the optimum medium components for the enhanced production of lichenysin by Bacillus licheniformis R2

Biosurfactants have gained attention because they exhibit some advantages such as biodegradability, low toxicity, ecological acceptability and ability to be produced from renewable and cheaper substrates. They are widely used for environmental applications for bioremediation and also in biomedical field. However, the high cost of production is the limiting factor for widespread industrial applications. Thus, optimization of the growth medium for biosurfactant-lichenysin production by Bacillus licheniformis R2 was carried out using response-surface methodology. A preliminary screening phase based on a two-level fractional factorial design led to the identification of NH₄NO₃, glucose, Na₂HPO₄ and MnSO₄·4H₂O concentrations as the most significant variables affecting the fermentation process. The 2⁴ full-factorial central composite design was then applied to further optimize the biosurfactant production. The optimal levels of the aforementioned variables were (g/l): NH₄NO₃, 1.0; glucose, 34.0; KH₂PO₄, 6.0; Na₂HPO₄, 2.7; MgSO₄·7H₂O, 0.1; CaCl₂, 1.2 × 10⁻³; FeSO₄·7H₂O, 1.65 × 10⁻³; MnSO₄·4H₂O, 1.5 × 10⁻³ and Na–EDTA, 2.2 × 10⁻³. With the optimization procedure, the relative lichenysin yield expressed as the critical micelle dilution (CMD) was fourfold higher than that obtained in the non-optimized reference medium.     

Bottlenecks to water transport in Quercus robur L.: the abscission zone and its physiological consequences

Cladoptosis, the abscission of twigs, is the main mechanism of changes in crown structure in senescing pedunculate oak (Quercus robur L.). We tested the hypotheses that abscission zones in nodes of old pedunculate oak trees reduce leaf-specific hydraulic conductance of shoots and thereby limit the stomatal conductance and assimilation.Hydraulic conductance and leaf-specific hydraulic conductance, measured with a high pressure flowmeter in 0.5–1.5 m long shoots, were significantly lower in shoots of low vigour compared to vigorous growing shoots in a 165-years-old stand in the southeast of Germany. Two types of bottlenecks to water transport could be identified in shoots of old oak trees, namely nodes and abscission zones. In young twigs, vessel diameter and vessel density in nodes with abscission zones were significantly reduced compared with internodes. In nodes without abscission zones, vessel density was significantly reduced. The reduction of hydraulic conductance was especially severe in the smallest and youngest shoots with diameters less than 2 mm. Internodes of 1–5 mm sapwood diameter had an average hydraulic conductance of 7.13×10⁻⁶±0.2×10⁻⁶ kg s⁻¹ m⁻¹ MPa⁻¹, compared to 4.54×10⁻⁶±0.3×10⁻⁶ kg s⁻¹ m⁻¹ MPa⁻¹ in those with nodes.Maximum stomatal conductance and maximum net assimilation rate increased significantly with hydraulic conductance and leaf-specific hydraulic conductance. Maximum rate of net photosynthesis A_max of the most vigorous shoots (VC0) (7.34±0.55 μmol m⁻² s⁻¹) was significantly higher (P<0.001) than in shoots of other vigour classes (5.97±0.28 μmol m⁻² s⁻¹). Our data support the hypothesis that the changes in shoot and consequently crown architecture that are observed in ageing and declining trees can limit photosynthesis by reducing shoot hydraulic conductance. Abscission zones increase the hydraulic disadvantage of less vigorous compared to vigorously growing twigs. Cladoptosis might serve as a mechanism of selection between twigs of different efficiency.     

Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.     

o-Phthalaldehyde–N-acetylcysteine polyamine derivatives: formation and stability in solution and in C18 supports

A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10⁻² min⁻¹ in solution versus 7.7±1.1×10⁻⁴ min⁻¹ on the (C₁₈) solid support. The results obtained showed that forming the derivative on the packing support (C₁₈) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples.     

Prostaglandin I2 as a potentiator of acute inflammation in rats

Prostaglandin I₂ potentiated the paw swelling induced by carrageenin in rats. Prostaglandin I₂ (0.1 μg) showed similar activity to PGE₁ (0.01 μg). This potentiating property disappeared in 60 minutes and was completely abolished by diphenhydramine (25 mg kg⁻¹, i.p.). In vascular permeability tests, PGI₂ itself (2.5 × 10⁻¹⁰ mol, 88 ng) caused no dye leakage reaction, but PGE₁ (2.5 × 10⁻¹⁰ mol, 88.5 ng) caused a significant dye leakage. This effect of PGE₁ was statistically significant compared with vehicle- or PGI₂-treated group (p<0.05). Prostaglandin I₂ potentiated the increased vascular permeability induced by 5-hydroxytriptamine (2.5 × 10⁻¹⁰ mol), bradykinin (5 × 10⁻¹⁰ mol) and histamine (2 × 10⁻¹⁰ to 2 × 10⁻⁸ mol). The potentiation was the most evidence in the case of histamine.     

Physico-chemical and Catalytic Properties of Clostridium perfringens Hyaluronidase: An Update

Hyaluronidase (E.C. 4.2.2.1 hyaluronate lyase) or Mu toxin is one of the main components ofClostridium perfringens toxin complex. Although this enzyme has been studied for many years, data on its physico-chemical and catalytic characteristics are still quite contradictory and lack lucidity and completeness. In order to update knowledge of enzymatic properties of clostridial hyaluronidase, a chromatographically purified preparation from C. perfringens type A BP6K free of side phospholipase C (alpha toxin), neuraminidase (sialidase) and collagenase (kappa toxin) activities was obtained and characterized. The purification procedure included the following steps: processing the culture liquid with calcium phosphate gel, precipitation of the enzyme with acetone, ultrafiltration, and chromatography on Sephadex G-100 column. The purified hyaluronidase was homogenous as judged by rechromatography, SDS-PAGE and isoelectric focusing. Being a glycoprotein, the enzyme was most active at pH 5.7–6.2 (depending on the nature of the buffer used), at temperatures 37–45°C and at a relatively high ionic strength (0.15 and higher). The hyaluronidase was unstable when at pH values below 5.0 and above 9.0 as well as at temperatures below 30°C and above 50°C. The enzyme was most sensitive to Cu²⁺, Pb²⁺and Al³⁺ions, while the inhibitory effect of EDTA was moderate. Molecular mass of hyaluronidase was 96kDa as estimated by gel filtration and 48kDa when estimated by SDS-PAGE, suggesting that enzyme is composed of two subunits. The isoelectric point of the enzyme was 4.4. Substrate specificity of the enzyme was narrow (appart from hyaluronate, it slightly split chondroitin, but did not split heparin and various chondroitinsulphates). Moreover, unsplit glycosaminoglycans appeared to be competitive inhibitors with K_ivalues 5.3×10⁻², 4.9×10⁻², 4.5×10⁻²and 4.2×10⁻²mg/mL for heparin, chondroitinsulphates A, B and C, respectively. The Michaelis constant in regard to potassium hyaluronate was calculated to be (15.4±2.6)×10⁻²mg/mL.     

Production and recovery of recombinant protease inhibitor α1-antitrypsin

Human α₁-antitrypsin (AAT) was produced in the recombinant yeast Saccharomyces cerevisiae ATCC 20699 grown in batch and fed-batch culture. The final biomass concentration and antitrypsin concentration attained were 55 g·L⁻¹ and 1.23 g·L⁻¹, respectively, in the fed-batch. The maximum productivities of biomass and antitrypsin were 1.6 and > 0.04 g L⁻¹h⁻¹, respectively, or substantially greater than the highest productivity values reported in the past. For recovering the antitrypsin, the cell slurry was concentrated 4-fold (231 g·L⁻¹ biomass, 122 min of processing) by cross-flow microfiltration and the cells were disrupted by bead milling (3 passes of 3 min total retention time). The cell homogenate was treated with aluminum chloride or PBS (pH 7) to aid separation of the cell debris by flocculation and sedimentation. The clarified cell homogenate was subjected to ammonium sulfate fractionation to precipitate the recombinant antitrypsin. The AAT precipitated at 45–75% saturation of ammonium sulfate, depending on the age of the homogenate. The crude AAT in the homogenate degraded at room temperature (25°C), with a zero order deactivation rate of 1.815 × 10⁻³ ± 3.43 × 10⁻⁴ g AAT L⁻¹h⁻¹.     

Occurrence and toxicity of Amphidinium carterae Hulburt in the North Arabian Sea

The occurrence and toxicity of Amphidinium carterae Hulburt is hereby reported for the first time from the North Arabian Sea on the coast of Pakistan. The concentrations of 1.2 × 10⁴ cells ml⁻¹ were found in intertidal pools that were also inhabited by the brown macroalga Sargassum wightii. Both wild and cultured A. carterae cells were tested for ciguatera toxicity through exposure to brine shrimp nauplii (Artemia salina) and albino mice. Although the brine shrimp did not appear to be affected mortalities in mice ranged between 13 and 16% at doses of 7.2 × 10⁴ and 2.5 × 10⁵ cells ml⁻¹, respectively. When mice were affected pharmacological effects such as muscle contraction in lower back area, increased respiration, immobility and paralysis in hind limbs were observed for 2 h. These effects appeared to be reversible and gradually disappeared within 24 h.     

Entropy decrease associated to solute compartmentalization in the cell

We have deduced equations to quantify the entropy associated to the compartmentalization of components in eukaryotic cells as a function of cell and compartment volumes, and of the concentration of solutes. On the basis of known and plausible values of volume and solute concentrations and the deduced equations, we estimate that the contribution of compartmentalization to the decrease of entropy is approximately −14.4 × 10⁻¹⁴ J K⁻¹ cell⁻¹ (−0.7 J K⁻¹ L⁻¹) in the case of Saccharomyces cerevisiae, a typical eukaryotic cell, and approximately −49.6 × 10⁻¹⁴ J K⁻¹ cell⁻¹ (−1.0 J K⁻¹ L⁻¹) in the more complex Chlamydomonas reinhardtii. When compared with other potential contributing factors, such as the informational entropy of DNA and the conformational entropy of proteins, compartmentalization appears as an essential development that significantly decreased the entropy of living cells during biological evolution.     

Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode

Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10⁻⁶ to 1.3 × 10⁻² M (r = 0.9982) with a detection limit of 4.5 × 10⁻⁷ M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (K_m) and the maximum current density (I_m) were estimated to be 23.9 mM and 378 μA/cm², respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.     

The development and multiple uses of a standardised bioassay method to select hypocrealean fungi for biological control of aphids

A technically standardised bioassay method was designed, evaluated and used to assess virulence and host range of hypocrealean fungi against aphids. A track mounted sprayer was used to apply conidia because hand held versions of the same sprayer can be used for field applications, thereby allowing the outcome from laboratory experiments to predict activity in the field accurately. Eighteen fungal isolates were assessed in single concentration bioassays against the black bean aphid Aphis fabae Scopoli. Isolates comprised commercially available mycoinsecticides (based on Beauveria bassiana and Lecanicillium longisporum) and isolates of B. bassiana, Lecanicillium spp., Paecilomyces fumosoroseus and Metarhizium anisopliae. Aphid mortality was in excess of 80% for 15 isolates, and HRI 1.72 (L. longipsorum), Z11 (P. fumosoroseus), Mycotech strain GHA (B. bassiana) and ARSEF 2879 (B. bassiana) were studied further. Multiple concentration bioassays identified HRI 1.72 as the most virulent isolate against A. fabae with significantly smaller LC₅₀ and LT₅₀ values compared to other isolates. A precise LC₅₀ value (2.95 × 10² conidia ml⁻¹) was calculated for HRI 1.72 using a second multiple concentration assay with smaller concentrations of conidia. The four isolates were applied at a single concentration (1 × 10⁸ conidia ml⁻¹) against Myzus persicae, A. fabae, Acyrthosiphon pisum, Metopolophium dirhodum, Sitobion avenae and Rhopalosiphum padi. A ranking of aphid susceptibility was obtained, such that S. avenae > M. persicae, A. pisum, A. fabae > R. padi. Results indicate the importance of standardising bioassay methods to reduce bioassay variability without compromising the ability to use the bioassay to investigate fungus–host interactions under varying abiotic and biotic conditions.     

Effects of arsenic exposure on the frequency of HPRT-mutant lymphocytes in a population of copper roasters in Antofagasta, Chile: a pilot study

A pilot biomarker study was conducted to investigate the feasibility of using the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in peripheral blood lymphocytes as a biomarker for detecting genetic effects of arsenic exposure. Blood and urine samples were obtained from workers highly exposed to arsenic in a copper roasting plant in Antofagasta, Chile. Individuals were classified according to their job titles into three potential exposure groups: high, medium, and low. To confirm exposure, arsenic concentration was determined in urine samples. The HPRT mutant frequencies were measured in lymphocytes from 15 individuals ranging in age from 24 to 66 years. The mean mutant frequencies for the three exposure groups were: low (9×10⁻⁶), medium (11×10⁻⁶), and high (24×10⁻⁶). An increased mutant frequency was observed in the highly exposed group, but the response was so slight that it is not likely that this assay will be capable of providing dose–response information across a range of lower, more typical environmental arsenic levels.