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.     

Nitrogen utilization by the raphidophyte Heterosigma akashiwo: Growth and uptake kinetics in laboratory cultures

The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m⁻² s⁻¹), the growth rate of H. akashiwo was slightly greater for cells grown on NH₄⁺ (0.89 d⁻¹) compared to cells grown on NO₃⁻ or urea, which had identical growth rates (0.82 d⁻¹). At sub-saturating PPFD (40 μE m⁻² s⁻¹), both urea- and NH₄⁺-grown cells grew faster than NO₃⁻-grown cells (0.61, 0.57 and 0.46 d⁻¹, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the ¹⁵N-tracer technique. Maximum specific uptake rates (V_max) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m⁻² s⁻¹) were 28.0, 18.0 and 2.89 × 10⁻³ h⁻¹ for NH₄⁺, NO₃⁻ and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (K_s) were similar for NH₄⁺ and NO₃⁻ (1.44 and 1.47 μg-at N L⁻¹), but substantially lower for urea (0.42 μg-at N L⁻¹). Whereas the α parameter (α = V_max/K_s), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<K_s), were 19.4, 12.2 and 6.88 × 10⁻³ h⁻¹/(μg-at N L⁻¹) for NH₄⁺, NO₃⁻ and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH₄⁺ > NO₃⁻ > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.     

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.     

Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f)azepine as chromogenic probe

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H₂O₂) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H₂O₂ by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H₂O₂ is found between 5 × 10⁻⁶ and 45 × 10⁻⁶ mol L⁻¹ at pH 4.0 and a temperature of 25 °C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415 × 10⁶ M⁻¹ min⁻¹ and 9.81 × 10⁻⁴ min⁻¹, respectively. The catalytic constant (k_cat) and specificity constant (k_cat/K_m) at saturated concentration of the cosubstrates were 163.2 min⁻¹ and 4.156 × 10⁶ L mol⁻¹ min⁻¹, respectively. This method can be incorporated into biochemical analysis where H₂O₂ undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.     

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.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

Molecular mass and chain conformations of Rhizoma Panacis Japonici polysaccharides

The molecular mass and size of five water-soluble polysaccharides isolated from Rhizoma Panacis Japonici (RPJ) were determined with laser light scattering (LLS), size-exclusion chromatography (SEC) combined with LLS (SEC–LLS), dynamic light scattering (DLS), as well as transmission electron microscope (TEM). Their weight-average molecular masses (M_w) were 3.5 × 10⁴, 1.47 × 10⁵, 1.24 × 10⁶, 9.26 × 10⁵ and 1.36 × 10⁶, radii of gyration (<s²>_z^1/2) were 14.7, 31.7, 50.8, 41.8 and 40.4 nm, and hydrodynamic radii (R_h) were 19.9, 37.5, 66.2, 52.1, and 55.2 nm, respectively. The results showed that molecular masses and sizes of the polysaccharides were influenced by the pH and temperature of the extraction mediums. The conformation parameters were calculated from the above data according to polymer solution theory. The values of ρ (= <s²>_z^1/2/R_h) were from 0.7 to 0.8, exponents (ν) of <s²>_z^1/2 = k M_w^ν were from 0.31 to 0.43, and fractal dimension (d_f) were from 2.3 to 3.2, respectively. The results revealed that all of the polysaccharides existed as spheres in 0.15 M NaCl aqueous solution. TEM and atomic force microscope (AFM) further confirmed the spherical morphologies of these molecules. The spherical conformations of the polysaccharides were a result of their highly branched structures.     

Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules

Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 μM and 10 μM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 μM concentrations. Comparing control and REN concentration of 1 μM, JHCO₃⁻, nmol cm^− 2 s^− 1 − 1,76 ± 0,11_control × 1,29 ± 0,08_{REN 10 μM}; P < 0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 μM (JHCO₃⁻, nmol cm^− 2 s⁻ 1 − 0.80 ± 0.07_control × 0.60 ± 0.06_{REN 1 μM}; P < 0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na⁺/H⁺exchanger and H⁺-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.     

Glycan-binding profile of a D-galactose binding lectin purified from the annelid, Perinereis nuntia ver. vallata

A lectin recognizing D-galactose was purified from the pacific annelid Perinereis nuntia ver. vallata (Polychaeta) by affinity chromatography. Hemagglutinating activity, with a very low titer suggesting the presence of lectin appeared in the supernatant from the homogenization of body with Tris-buffered saline. However, dialyzed supernatant from the precipitate homogenized by galactose in the buffer revealed strong hemagglutinating activity against human erythrocytes. The crude supernatant was applied onto lactosyl–agarose column, and only the supernatant eluted from precipitate with galactose was obtained a galactose-binding lectin with 32 kDa polypeptide was obtained from the supernatant of the precipitate, extracted in presence of galactose. It suggests that the lectin tightly binds with glycoconjugate as endogenous ligand(s) in the tissue. Hemagglutinating activity against trypsinized and glutaraldehyde-fixed human erythrocytes was specifically inhibited by D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and asialofetuin. Glycan-binding profile of the lectin analyzed by frontal affinity chromatography shows that the lectin recognizes branched complex type N-linked oligosaccharides and both type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) lactosamine. The surface plasmon resonance study of the lectin against asialofetuin showed the k_ass and k_diss values are 5.14 × 10⁴ M^− 1 s^− 1 and 2.9 × 10⁻³ s^− 1, respectively. The partial primary structure of the lectin reveals 182 amino acids with novel sequence.     

Red tide blooms of Cochlodinium polykrikoides in a coastal cove

Successive blooms of the dinoflagellate Cochlodinium polykrikoides occurred in Pettaquamscutt Cove, RI, persisting from September through December 1980 and again from April through October 1981. Cell densities varied from <100 cells L⁻¹ at the onset of the bloom and reached a maximum density exceeding 3.4 × 10⁶ cells L⁻¹ during the summer of 1981. The bloom was mainly restricted to the mid to inner region of this shallow cove with greatest concentrations localized in surface waters of the southwestern region during summer/fall periods of both years. Highly motile cells consisting of single, double and multiple cell zooids were found as chains of 4 and 8 cells restricted to the late August/September periods. The highest cell densities occurred during periods when annual temperatures were between 19 and 28 °C and salinities between 25 and 30. A major nutrient source for the cove was Crying Brook, located at the innermost region at the head of the cove. Inorganic nitrogen (NH₃ and NO₂ + NO₃) from the brook was continually detectable throughout the study with maximum values of 57.5 and 82.5 μmol L⁻¹, respectively. Phosphate (PO₄-P) was always present in the source waters and rarely <0.5 μmol L⁻¹; silicate always exceeded 30 μmol L⁻¹ with maximum concentrations reaching 226 μmol L⁻¹. Chlorophyll a and ATP concentrations during the blooms varied directly with cell densities. Maximum Chl a levels were 218 mg m⁻³ and ATP-carbon was >20 g C m⁻³. Primary production by the dinoflagellate-dominated community during the bloom varied between 4.3 and 0.07 g C m⁻³ d⁻¹. Percent carbon turnover calculated from primary production values and ATP-carbon varied from 6 to 129% d⁻¹. The dinoflagellates dominated the entire summer period; other flagellates and diatoms were present in lesser amounts. A combination of low washout rate due to the cove dynamics, active growth, and life cycles involving cysts allowed C. polykrikoides to maintain recurrent bloom populations in this area.     

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.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Synthesis, characterization and applications of graft copolymer (Chitosan-g-N,N-dimethylacrylamide)

An unreported graft copolymer of N,N-dimethylacrylamide (DMA) with chitosan has been synthesized under nitrogen atmosphere using peroxymonosulphate/mandelic acid redox pair. The effect of reaction conditions on grafting parameters i.e. grafting ratio, efficiency, conversion, add on and homopolymer has been studied. Experimental results show that maximum grafting has been obtained at 1.0 g dm⁻³ concentration of chitosan, 30 × 10⁻² mol dm⁻³ concentration of N,N-dimethylacrylamide and 7.0 × 10⁻³ mol dm⁻³ concentration of hydrogen ion. It has also been observed that grafting ratio, add on, conversion and efficiency increase upto 3.2 × 10⁻³ mol dm⁻³ of mandelic acid, 12.0 × 10⁻³ mol dm⁻³ of potassium peroxymonosulphate, 150 min of time and 40 °C of temperature. Grafted polymer has been characterized by FTIR spectroscopy and thermogravimetric analysis. Water swelling capacity of chitosan-g-N,N-dimethylacrylamide has been determined. It has been observed that the graft copolymer is thermally more stable than parent backbone.     

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.     

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K_a) and binding and dissociation rate constants (k_on and k_off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K_a = (4 ± 1) × 10⁶ M⁻¹, k_on = (4 ± 1) × 10⁴ M⁻¹ s⁻¹, and k_off = (12 ± 2) × 10^–3 s⁻¹. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K_a = (14 ± 2) × 10⁶ M⁻¹, k_on = (14 ± 2) × 10⁴ M⁻¹ s⁻¹, and k_off = (7 ± 2) × 10^–3 s⁻¹. Thus, K_a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k_on) and a three times smaller dissociation rate constant (k_off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.     

Allelochemical interactions and short-term effects of the dinoflagellate Alexandrium on selected photoautotrophic and heterotrophic protists

The marine dinoflagellate genus Alexandrium (Halim) Balech contains members that produce highly potent phycotoxins (PSP toxins or spirolides) as well as lytic substances and other allelochemicals of unknown structure and ecological significance. One isolate each of six Alexandrium species (A. tamarense, A. ostenfeldii, A. lusitanicum, A. minutum, A. catenella, A. taylori), of the closely related gonyaulacoid dinoflagellate Fragilidium subglobosum, and of the peridinioid Scrippsiella trochoidea were tested in 24 h co-incubation experiments for their short-term deleterious effects on a diversity of marine protists. Both autotrophs (Rhodomonas salina, Dunaliella salina, Thalassiosira weissflogii) and heterotrophs (Oxyrrhis marina, Amphidinium crassum, Rimostrombidium caudatum) were included as target species. All donor isolates except S. trochoidea exhibited lytic effects on at least some target species. Lytic effects were observed with all Alexandrium species, for both whole cell samples and culture filtrate (<10 μm and <0.2 μm). Antibiotic treated cultures with drastically reduced bacterial numbers did not show any general reduction in lytic capacity, therefore direct involvement of extracellular bacteria in allelochemical production is unlikely. Values of EC₅₀, defined as the Alexandrium cell concentration causing lysis of 50% of target cells, differed by two orders of magnitude depending on the donor/target combination, from 3.1 × 10³ cells ml⁻¹ (A. minutum/O. marina) down to 0.02 × 10³ cells ml⁻¹ (A. catenella/D. salina). Within the array of nine donor Alexandrium/target combinations, variable ratios in EC₅₀ values between donor/target combination cannot be explained by quantitative differences in allelochemical production, but rather indicate qualitative differences in the composition of compounds produced by different Alexandrium strains. In conclusion, our study confirms the widespread lytic capacity within the genus Alexandrium, although allelochemical effects are not restricted to this genus. Allelochemical interactions mediated by such lytic substances may be significant in explaining the formation and maintenance of Alexandrium blooms through direct destructive effects on competing algae or unicellular grazers.     

Characterization of a root-specific β-thioglucoside glucohydrolase gene in Carica papaya and its recombinant protein expressed in Pichia pastoris

Plant β-thioglucoside glucohydrolases (TGGs or myrosinases) are a young class of enzymes in the glycosyl hydrolase family 1 and have a narrow distribution. TGG genes have mainly been cloned from crucifers, while TGGs in other species have received little attention. The TGG gene CpTGG2 and its recombinant protein from papaya were characterized in this paper. This is the first plant TGG gene without unusual intron splicing borders, as present in all other available TGG genes. Phylogenetic analysis indicated that plant myrosinases are divided into two major lineages. CpTGG2 is located in the lineage constituted by AtTGG4–6 from Arabidopsis thaliana, while the rest of myrosinases (including MA, MB and MC subfamilies) are grouped into another lineage. RT-PCR analysis indicated that CpTGG2 was specifically expressed in the root. The recombinant CpTGG2 expressed in yeast had a subunit mass of 70 kDa, and had low basal TGG activity without addition of ascorbate. Low concentrations of ascorbate stimulated CpTGG2 activity, while high concentrations were inhibitory. CpTGG2 was active in broad pH and temperature ranges, similar to AtTGG4 and AtTGG5. The apparent K_m and V_max were 2.24 mM and 24.3 μmol min⁻¹ mg⁻¹ when sinigrin was the substrate. The calculated k_cat/K_m value was 1.3 × 10⁴ S⁻¹ M⁻¹_. Our results reshaped and expanded the myrosinase family structure and provided clues to the evolution of myrosinase genes.     

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.     

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.     

Ascaris suum NADH-methemo(myo)globin reductase systems recovering differential functions of hemoglobin and myoglobin, adapting to environmental hypoxia

We reported previously that Ascaris suum cytochrome b₅, specifically expressed in this nematode at the adult stage and dually localized in extracellular perienteric fluid and hypodermis, is involved in both perienteric NADH-methemoglobin and cytosolic NADH-metmyoglobin reduction, where cytochrome b₅ functions as an electron carrier between NADH-mediated cytochrome b₅ reductase and substrates, methemo(myo)globins to reduce the nonfunctional globins back to functional ferrous hemo(myo)globins. To further characterize NADH-methemo(myo)globin reductase systems, the midpoint potentials of A. suum perienteric hemoglobin and body wall myoglobin, as well as the affinities of Ascaris methemoglobin and metmyoglobin toward cytochrome b₅, were evaluated using potentiometric titration and surface plasmon resonance techniques, respectively. Midpoint potentials of + 7.2 mV and + 19.5 mV were obtained for Ascaris perienteric hemoglobin and body wall myoglobin, respectively. The affinities of Ascaris perienteric methemoglobin and body wall metmyoglobin toward the nematode cytochrome b₅ were comparable to that for mammalian hemoglobin and cytochrome b₅; association constants were 0.585 × 10³ M^− 1 and 2.32 × 10³ M^− 1, respectively, with rapid equilibration kinetics. These observations highlight the physiological importance of A. suum perienteric NADH-methemoglobin and cytosolic metmyoglobin reductase systems. Differential roles of A. suum perienteric hemoglobin and body wall myoglobin are also discussed from the viewpoint of oxygen homeostasis under hypoxic conditions.