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.     

Production, characterization and evaluation of methylcellulose from sugarcane bagasse for applications as viscosity enhancing admixture for cement based material

In previous works, methylcellulose (MC) was prepared from sugarcane bagasse cellulose in heterogeneous medium using dimethyl sulfate (DMS) as etherification agent. MC was produced in a range of degrees of substitution (DS) from 0.70 to 1.40 and the materials showed low water solubility. In this work methylcellulose was prepared with 5 h (MC5h) of reaction with reagent substitution at each hour. MC5h showed a DS of 1.89 ± 0.04. An aqueous viscous suspension was produced with MC5h for application as viscosity enhancing admixture of cimentitious adhesive mortars. It was observed a 40.37% increase on the consistency index (CI) and a 27.70% increase on the Potential Tensile Adhesion Strength. Such characteristics show the potential of this material for the utilization in situations that require good workability, improve viscosity and adhesive properties such as for tile setting in civil engineering.     

Molecular mass and antitumor activities of sulfated derivatives of α-glucan from Poria cocos mycelia

Two kinds of water-insoluble (1 → 3)-α-d-glucan samples, ab-PCM3-I and ac-PCM3-I, isolated from different Poria cocos mycelia were sulfated, to produce two series of water-soluble derivatives ab-PCM3-I-S1–S5 and ac-PCM3-I-S1–S5, respectively. The derivatives having different weight-average molecular mass (M_w) were produced by changing reaction temperature and time as well as molar ratios between chlorosulfonic acid and number of hydroxyl groups in the glucan. The degrees of substitution (DS) of the sulfated derivatives were analyzed by elemental analysis (EA) to be 0.39–0.67 for ab-PCM3-I-S and 0.73–0.96 for ac-PCM3-I-S, respectively. The M_w and the intrinsic viscosity ([η]) of the samples ab-PCM3-I-S and the ac-PCM3-I-S were measured by size exclusion chromatography combined with laser light scattering (SEC–LLS) and viscometry in phosphate buffer solution (PBS) at 37 °C. The results indicated that their M_w ranged from 2.0 to 11.3 × 10⁴ for the samples ab-PCM3-I-S, and 4.7 to 40.0 × 10⁴ for the samples ac-PCM3-I-S. Moreover, the antitumor activities of the sulfated derivatives ab-PCM3-I-S and ac-PCM3-I-S against Sarcoma 180 tumor cell tested both in vitro and in vivo are significantly higher than those of the native α-d-glucans. Therefore, a moderate range of molecular mass from 2.0 × 10⁴ to 40.0 × 10⁴, relatively high chain stiffness and good water solubility of the sulfated derivatives are beneficial to the enhancement of their antitumor activities.     

Structure and chain conformation of five water-soluble derivatives of a beta-D-glucan isolated from Ganoderma lucidum

A linear water-insoluble (1-->3)-beta-D-glucan, coded as GL-IV-I, was isolated from the fruit body of Ganoderma lucidum by extracting with NaOH solution. Its derivatives were prepared by using sulfation, carboxymethylation, hydroxyethylation, hydroxypropylation, and methylation, respectively, and these were labeled as S-GL, CM-GL, HE-GL, HP-GL and M-GL. Five derivatives exhibited good water solubility. Their structures and chain conformations were investigated with infrared spectroscopy, elemental analysis (EA), one- and two-dimensional NMR spectroscopy, laser light scattering (LLS), and size-exclusion chromatography combined with LLS (SEC-LLS). The reactivity of the hydroxyl group of GL-IV-I was ordered as C-6>C-4>C-2 for the five derivatives. The degree of substitution (DS) of the derivatives was calculated from EA and NMR spectroscopy to be from 0.32 to 1.18. The weight-average molecular mass (M(w)) of GL-IV-I, S-GL, CM-GL, HE-GL, HP-GL, and M-GL was 13.3 x 10(4), 10.1 x 10(4), 6.3 x 10(4), 7.2 x 10(4), 5.1 x 10(4), and 14.1 x 10(4), respectively. The conformation analysis studies revealed that GL-IV-I exists as a compact coil in dimethyl sulfoxide, whereas the five derivatives are slightly expanded flexible chains in 0.9% aqueous NaCl solution.     

AFM imaging of surface adsorbed polymeric 3-alkylpyridinium salts from the marine sponge Reniera sarai

Bioactive 3-alkylpyridinium polymers (poly-APS) have recently been isolated from the marine sponge Reniera sarai. Previous results have shown that these molecules in aqueous solutions form supramolecular aggregates with an average hydrodynamic radius of 23±2 nm. To obtain additional evidences about the shape and the dimensions of poly-APS aggregates, we used atomic force microscopy (AFM) operating in tapping mode. The images clearly showed adsorbed aggregates with a lateral dimension of ≈40 nm and a thickness of the order of ≈1 nm. The distribution of volumes of the adsorbed aggregates is very similar to the distribution of hydrodynamic radii as obtained from the dynamic light scattering experiments. The volume distribution of these aggregates shows a maximum at 1750 nm³, which corresponds to a sphere with a radius of 7.5 nm.     

Molecular dynamics studies of the conformation of sorbitol

Molecular dynamics simulations of a 3 molal aqueous solution of d-sorbitol (also called d-glucitol) have been performed at 300 K, as well as at two elevated temperatures to promote conformational transitions. In principle, sorbitol is more flexible than glucose since it does not contain a constraining ring. However, a conformational analysis revealed that the sorbitol chain remains extended in solution, in contrast to the bent conformation found experimentally in the crystalline form. While there are 243 staggered conformations of the backbone possible for this open-chain polyol, only a very limited number were found to be stable in the simulations. Although many conformers were briefly sampled, only eight were significantly populated in the simulation. The carbon backbones of all but two of these eight conformers were completely extended, unlike the bent crystal conformation. These extended conformers were stabilized by a quite persistent intramolecular hydrogen bond between the hydroxyl groups of carbon C-2 and C-4. The conformational populations were found to be in good agreement with the limited available NMR data except for the C-2–C-3 torsion (spanned by the O-2–O-4 hydrogen bond), where the NMR data support a more bent structure.     

Structure and chain conformation of five water-soluble derivatives of a β-d-glucan isolated from Ganoderma lucidum

A linear water-insoluble (1→3)-β-d-glucan, coded as GL-IV-I, was isolated from the fruit body of Ganoderma lucidum by extracting with NaOH solution. Its derivatives were prepared by using sulfation, carboxymethylation, hydroxyethylation, hydroxypropylation, and methylation, respectively, and these were labeled as S-GL, CM-GL, HE-GL, HP-GL and M-GL. Five derivatives exhibited good water solubility. Their structures and chain conformations were investigated with infrared spectroscopy, elemental analysis (EA), one- and two-dimensional NMR spectroscopy, laser light scattering (LLS), and size-exclusion chromatography combined with LLS (SEC-LLS). The reactivity of the hydroxyl group of GL-IV-I was ordered as C-6 > C-4 > C-2 for the five derivatives. The degree of substitution (DS) of the derivatives was calculated from EA and NMR spectroscopy to be from 0.32 to 1.18. The weight-average molecular mass (M_w) of GL-IV-I, S-GL, CM-GL, HE-GL, HP-GL, and M-GL was 13.3 × 10⁴, 10.1 × 10⁴, 6.3 × 10⁴, 7.2 × 10⁴, 5.1 × 10⁴, and 14.1 × 10⁴, respectively. The conformation analysis studies revealed that GL-IV-I exists as a compact coil in dimethyl sulfoxide, whereas the five derivatives are slightly expanded flexible chains in 0.9% aqueous NaCl solution.     

A dynamic laser light-scattering study of chitosan in aqueous solution

The solution behavior of four chitosans (91% deacetylated chitin) with different molecular weights in 0.2M CH₃COOH/0.1M CH₃COONa aqueous solution was investigated at 25°C by dynamic laser light scattering (LLS). The Laplace inversion of the precisely measured intensity-intensity lime correlation function leads us to an estimate of the line-width distribution G(Γ), which could be further reduced too translational diffusion coefficient distributions G(D). By using a combination of static and dynamic LLS results, i.e., M_w and G (D), we were able to establish a calibration of D = k_DM with k_D = (3.14 ± 0.20) × 10⁻⁴ and α_D = 0.655 ± 0.015. By using this calibration, we successfully converted G(D) into a molecular weight distribution f_w(M). The larger α_D value confirms that the chitosan chain is slightly extended in aqueous solution even in the presence of salts. This is mainly due to its backbone and polyelectrolytes nature. As a very sensitive technique, our dynamic LLS results also revealed that even in dilute solution chitosan still forms a small amount of larger sized aggregates that have been overlooked in previous studies. The calibration obtained in this study will provide another way to characterize the molecular weight distribution of chitosan in aqueous solution at room temperature. © 1995 John Wiley & Sons, Inc.     

A study of the distribution of chitosan onto and within a paper sheet using a fluorescent chitosan derivative

Acidic glutaraldehyde (Gh) crosslinked chitosan (ChGhH) when deprotonated the biopolymer (ChGh) presents high content of free amino groups. These modified biopolymers are comparable to epichlorohydrin (Ep) crosslinked (ChEp). C/N molar ratio of 6.1 for chitosan increases to 7.3, 7.5 and 7.1 for ChGhH, ChGh and ChEp. The effectiveness of the carbon-6 hydroxyl group in interconnecting chitosan units was supported by IR and ¹³CNMR, where Ep promotes increase in crystallinity. Copper uptake gave the order Ch > ChGh > ChGhH > ChEp, as: 1.35 ± 0.06, 1.30 ± 0.05, 1.05 ± 0.07 and 0.96 ± 0.22 mmol g⁻¹, reflecting the availability of nitrogen basic centers in adsorbing. The favorable thermodynamic data of adsorption through calorimetric titration gave exothermic enthalpic values: −28.98 ± 0.05, −6.68 ± 0.04, −6.13 ± 0.07 and −0.65 ± 0.23 kJ mol⁻¹ for Ch, ChGh, ChGhH and ChEp. Free Gibbs energy reflected spontaneity of interactions and, with the exception of chitosan, the entropic values are positive.     

The Planktonic Index of Biotic Integrity (P-IBI): An approach for assessing lake ecosystem health

Using historical (1970) and more recent (1996) Lake Erie plankton and trophic status data, we developed a Planktonic Index of Biotic Integrity (P-IBI) to measure changes in lake ecosystem health. We used discriminant analysis to determine phytoplankton and zooplankton community characteristics (metrics) that distinguished among levels of impairment. Traditional measures of lake trophic status classes (i.e., oligotrophic, mesotrophic, eutrophic), such as chlorophyll a and total phosphorus concentrations, were used to classify sites on a gradient of impairment. We then judged the ability of plankton metrics to distinguish among trophic status classes. Because of the temporal variability found in plankton communities, we conducted analyses on a monthly basis (May–September). For June, July and August we found five unique metrics that could distinguish among trophic status classes. The P-IBI showed an increase in water quality in Lake Erie between 1970 (<3 = eutrophic) and the mid-1990s (1996 and 1997) (3–4 = mesotrophic) (which reflected mean (±standard error) total phosphorus concentrations (μg/L) 1970 > 1996; western basin (41.53 ± 2.68 > 29.75 ± 1.39), eastern basin (14.84 ± 0.82 > 7.74 ± 0.28) and mean (±standard error) chlorophyll a concentrations (μg/L) uncorrected for pheophytin 1970 > 1996; western basin (12.58 ± 1.82 > 5.40 ± 0.22), central basin (5.90 ± 0.36 > 3.17 ± 0.54), and eastern basin (5.17 ± 0.38 > 1.67 ± 0.18)), with declining water quality in the late 1990s (1998 and 1999) (3) and 2002 (<3). We recommend that the techniques used in creating the P-IBI be investigated for determining ecosystem health of other lakes.     

Global conformation analysis of irradiated xyloglucans

Xyloglucan isolated and purified from tamarind seed was subjected to various degrees of γ-irradiation treatments, from 10 to 70 kGy, monitored for radiation damage and then studied using a new combined hydrodynamic approach with regards to conformation and flexibility. Radiation products were analysed with regard to molecular weight (weight average) M_w from size exclusion chromatography coupled to multi-angle laser light scattering (SEC–MALLs), intrinsic viscosity [η] and sedimentation coefficient s^o_20,w. Sedimentation coefficient distributions and elution profiles from SEC–MALLs confirmed the unimodal nature of the molecular weight distribution for each sample in solution. The chain flexibility was then investigated in terms of the persistence length, L_p of the equivalent worm-like chain model. The traditional Bushin–Bohdanecky (intrinsic viscosity) and Yamakawa–Fujii (sedimentation coefficient) relations were used separately then combined together by minimisation of a target function according to a recently published procedure [Ortega, A., & García de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464–2475 [see also Ortega, A. Metodologías computacionales para propiedades en disolución de macromoléculas rígidas y flexibles. Ph.D. Dissertation, Universidad de Murcia, 2005]] and yielded an estimate for L_p in the range 4–9 nm using floated and fixed mass per unit length analysis protocols and “point” global analysis: irradiated xyloglucans behave as flexible structures in common with pressure/heat treated materials.     

Homogeneous modification of cellulose with succinic anhydride in ionic liquid using 4-dimethylaminopyridine as a catalyst

Cellulose, extracted from sugarcane bagasse, was successfully succinylated in ionic liquid 1-buty-3-methylimidazolium (BMIMCl) using 4-dimethylaminopyridine (DMAP) as a catalyst. Parameters investigated included the mass ratio of DMAP/succinic anhydride in a range from 0% to 15%, reaction time (from 30 to 120 min), reaction temperature (from 60 to 110 °C). The succinylated cellulosic derivatives had a degree of substitution (DS) ranging from 0.24 to 2.34. It was found that the DS of succinylated cellulosic derivatives using DMAP as a catalyst was higher than that without any catalyst under the same reaction conditions. The products were characterized by FT-IR, solid-state CP/MAS ¹³C NMR, and thermal analysis. FT-IR and solid-state CP/MAS ¹³C NMR spectra showed that succinoylation occurred at C-6, C-2 and C-3 positions. The thermal stability of the succinylated cellulose decreased upon chemical modification.     

Ethylenesulfide as a useful agent for incorporation into the biopolymer chitosan in a solvent-free reaction for use in cation removal

Chitosan (Ch) was chemically modified with ethylenesulfide (Es) under solvent-free conditions to give (ChEs), displaying a high content of thiol groups due to opening of the three member cyclic reagent. Elemental analysis showed a decrease in nitrogen content. This result indicated the incorporation of two ethylenesulfide molecules for each unit of the polymeric structure of the precursor biopolymer. Infrared spectroscopy, thermogravimetry, and ¹³C NMR in the solid state demonstrated the effectiveness of the reaction, with signals at 30 ppm for ChEs due to the change in the methylene group environment. Divalent metal uptake by chemically modified biopolymer gave the order Cu > Ni > Co > Zn, reflecting the corresponding acidity of these cations in bonding to the sulfur and the basic nitrogen atoms available on the pendant chains. The equilibrium data were fitted to Freundlich, Temkin, and Langmuir models. The maximum monolayer adsorption capacity for the cations was found to be 1.54 ± 0.02, 1.25 ± 0.03, 1.13 ± 0.01, and 0.83 ± 0.03 mmol g⁻¹, respectively. The Langmuir model best explained the cation–sulfur bond interactions at the solid–liquid interface. The thermodynamics for these interactions gave exothermic enthalpic values of −43.02 ± 0.03, −28.72 ± 0.02, −26.27 ± 0.04, and −17.32 ± 0.02 kJ mol⁻¹, respectively. The spontaneity of the systems is given by negative Gibbs free energies of −31.2 ± 0.1, −32.7 ± 0.1, −31.7 ± 0.1, and −32.2 ± 0.1 kJ mol⁻¹, respectively, in spite of the unfavorable negative entropic values of −39 ± 1, −13 ± 1, −18 ± 1, and −49 ± 1 J K⁻¹ mol⁻¹ due to solvent ordering in the course of complexation. This newly synthesized biopolymer is presented as a chemically useful material for cation removal from aqueous solution.     

Acetylation of α-chitin in ionic liquids

Acetylation of α-chitin using acetic anhydride in an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), was performed. First, a mixture of chitin and AMIMBr (2% w/w) was heated at 100 °C for 24 h for dissolution. Then, acetic anhydride (5–20 equiv) was added to the solution and the mixture was heated with stirring at desired temperatures for 24 h. The product was precipitated by the addition of the reaction mixture into methanol. The IR spectrum of the product indicated the progress of acetylation. The degrees of substitution (DS), which were determined from the IR spectra, increased with increasing the amounts of acetic anhydride used for the reaction. The highest DS was 1.86, which was obtained by the reaction using 20 equiv of acetic anhydride at 100 °C. The product with this DS value was soluble in DMSO, and thus the structure of the product was further confirmed by ¹H NMR spectroscopy in DMSO-d₆. The DS value estimated by the integrated ratio of signals due to acetyl protons to a signal due to anomeric protons was in good agreement with that determined from the IR spectrum.     

A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein–protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 °C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 °C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein–protein interactions at 37 °C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein–protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein–protein interactions in living cells and offer the potential to visualize protein–protein interactions in living animals.     

Kinetic and thermodynamic characterization of the cold activity acquired upon single amino-acid substitution near the active site of a thermostable α-glucosidase

The α-glucosidase of Bacillus sp. SAM1606, a thermophilic bacterium, is a thermostable enzyme that has maximal activity at an apparent optimal temperature between 65 and 70 °C and only very low activity at low temperatures (0–25 °C). In this study, we identified Thr272, which is located adjacent to Glu271 (a catalytic residue) and Gly273 (a determinant of specificity), as a determinant of the optimal temperature, as substitution of Thr272 with other residues significantly altered the temperature–activity profile of the enzyme. Substitution of Thr272 with other amino acids, in particular bulky hydrophobic residues such as valine, methionine and phenylalanine, resulted in a significant downward shift (by 30 °C) of the apparent optimal temperature with an increase in catalytic activity at low temperatures. The observed downward shift of the apparent optimal temperature was not due to instability of the mutants at 40–65 °C, as the mutants were stable at temperatures up to 65 °C. Among the mutants examined, T272V displayed the highest k_cat values at 10–25 °C, which was at least 11-fold greater than the k_cat value observed for the wild-type enzyme. The thermodynamic characteristics of reactions catalyzed by T272V, T272M, T272F, and wild type at 25 °C were examined in greater detail. The T272V, T272M and T272F mutants displayed large K_s (or K_m) values and reduced and values at 25 °C, consistent with the general features of cold adaptation. The observed cold activities of T272V, T272M and T272F most likely arose from local flexibility of the active site at low temperatures due to loss of a Thr272-mediated hydrogen bond. However, this hydrogen-bond loss likely permits reversible conformational changes of the active site to less active forms at elevated temperatures (e.g., 60 °C). This may explain why catalytic activities for T272V, T272M and T272F at high temperatures (e.g., 60 °C) were lower than those at low temperature (e.g., 25 °C), even though the mutant enzymes appeared stable at 60 °C.     

Effect of storage regime on the stability of DNA used as a calibration standard for real-time polymerase chain reaction

This article looks at storage factors influencing the stability of potential DNA calibration standards for use in quantitative polymerase chain reaction (PCR). Target sequences from the bacteria Campylobacter jejuni were cloned into a plasmid vector. Samples of these potential calibration standards were stored at +4, −20, and −80 °C as aqueous and lyophilized samples and were prepared as both single-use aliquots and multiple-use preparations. Results showed that the samples stored as single-use aqueous solutions at +4 °C and lyophilized samples stored at +4 and −20 °C were the most stable. Samples stored as frozen aqueous solutions at −20 °C were the least stable.     

High-performance liquid chromatographic assay of hydroxyl free radical using salicylic acid hydroxylation during in vitro experiments involving thiols

A HPLC method was developed to monitor the production of hydroxyl free radical (°OH) produced during in vitro experiments: (i) a chemical reaction involving EDTA chelated ferric ion and various exogenous and endogenous thiols [glutathione (GSH) and its metabolites], and (ii) an enzymatic reaction corresponding to the breakdown of GSH catalyzed by γ-glutamyltransferase (GGT). The method relies upon the use of a selective trapping reagent of °OH: salicylic acid (SA). The three resulting dihydroxylated products, i.e., 2,3-dihydroxybenzoic acid (DHB), 2,5-DHB and catechol, were measured in an ion-pairing reversed-phase HPLC system coupled with amperometric detection; the sum of the three concentrations was used to quantify the production of °OH during in vitro experiments. Resulting data demonstrate that °OH is produced during Fenton-like reactions involving thiols and GSH catabolism via GGT.     

Temperature induced denaturation of collagen in acidic solution

The denaturation of collagen solution in acetic acid has been investigated by using ultra-sensitive differential scanning calorimetry (US-DSC), circular dichroism (CD), and laser light scattering (LLS). US-DSC measurements reveal that the collagen exhibits a bimodal transition, i.e., there exists a shoulder transition before the major transition. Such a shoulder transition can recover from a cooling when the collagen is heated to a temperature below 35 degrees C. However, when the heating temperature is above 37 degrees C, both the shoulder and major transitions are irreversible. CD measurements demonstrate the content of triple helix slowly decreases with temperature at a temperature below 35 degrees C, but it drastically decreases at a higher temperature. Our experiments suggest that the shoulder transition and major transition arise from the defibrillation and denaturation of collagen, respectively. LLS measurements show the average hydrodynamic radius R(h), radius of gyration R(g)of the collagen gradually decrease before a sharp decrease at a higher temperature. Meanwhile, the ratio R(g)/R(h) gradually increases at a temperature below approximately 34 degrees C and drastically increases in the range 34-40 degrees C, further indicating the defibrillation of collagen before the denaturation.     

Response of cylindrospermopsin production and release in Aphanizomenon flos-aquae (Cyanobacteria) to varying light and temperature conditions

The influence of light and temperature on the cylindrospermopsin (CYN) production of two Aphanizomenon flos-aquae strains, isolated from North-eastern German lakes, was investigated with semi-continuously growing cultures. A light gradient from 10 to 60 μE m⁻² s⁻¹ in combination with temperatures of 16, 20, and 25 °C was tested.CYN concentrations varied by a maximum factor of 2.7 in strain 10E9 with a significant decrease with increasing temperature. Strain 22D11 showed less pronounced changes, i.e. by a factor of 1.6, and without clear relationship to temperature.Reaction patterns of CYN production to changing light intensities are different at different temperatures. In both strains CYN concentrations increase significantly at 20 °C between 10 and 60 μE m⁻² s⁻¹, whereas they decrease significantly at 25 °C in the same light gradient. The amount of synthesised CYN is not reflected by growth rates of the strains in a uniform manner. Nonetheless several temperature–light combinations which constitute physiological stress seem to trigger CYN production and particularly CYN release from cells. The lowest growth rate observed at 16 °C and 60 μE m⁻² s⁻¹ of strain 22D11 may reflect photoinhibition due to the lower temperature and related limited CO₂-fixation. Under these conditions, extracellular CYN concentrations increased to 58% of total CYN, while the share of extracellular CYN of all other light and temperature regimes was 11–26%. From the results and the experimental design we conclude an active release of the toxin into medium to be more likely than mere leakage from cells.