Activity of salicylic acid on the growth and biochemism of Chlorella vulgaris Beijerinck

This study was conducted to investigate the influence of salicylic acid (SA) on the growth and changes of nucleic acids, protein, photosynthetic pigments, sugar content and photosynthesis levels in the green alga Chlorella vulgaris Beijerinck (Chlorophyceae). The most significant changes in the content of nucleic acids and proteins was observed at the concentration 10⁻⁴ M SA between 8 and 12 day of cultivation. This concentration of SA increased the number of cells (about 40 %) and content of proteins (about 60 %) and its secretion to the medium. The slight stimulation of protein secretion occurred on the 12th day of cultivation at concentration 10⁻⁴ M, while in the range of 10⁻⁵ M to 10⁻⁶ M the protein secretion was inhibited. SA also stimulated the content of nucleic acids, especially RNA by 20–60 %, compared with the control. The most stimulating influence upon the contents of chlorophylls a and b (50–70 %), total carotenoids (25–57 %), sugar (27–41 %) and intensity of net photosynthesis (18–33 %) was found at 10⁻⁴ M of SA. At the concentration of 10⁻⁶ M SA the slight inhibition of growth and biochemical activity of the algae was recorded at the first days of cultivation.     

The effect of oxygen on methanol oxidation by an obligate methanotrophic bacterium studied by in vivo 13C nuclear magnetic resonance spectroscopy

¹³C NMR was used to study the effect of oxygen on methanol oxidation by a type II methanotrophic bacterium isolated from a bioreactor in which methane was used as electron donor for denitrification. Under high (35–25%) oxygen conditions the first step of methanol oxidation to formaldehyde was much faster than the following conversions to formate and carbon dioxide. Due to this the accumulation of formaldehyde led to a poisoning of the cells. A more balanced conversion of ¹³C-labelled methanol to carbon dioxide was observed at low (1–5%) oxygen concentrations. In this case, formaldehyde was slowly converted to formate and carbon dioxide. Formaldehyde did not accumulate to inhibitory levels. The oxygen-dependent formation of formaldehyde and formate from methanol is discussed kinetically and thermodynamically. Journal of Industrial Microbiology & Biotechnology (2001) 26, 9–14. Received 04 March 2000/ Accepted in revised form 07 November 2000     

Influence of glutathione on zinc-mediated cellular toxicity

The effect of zinc on various pulmonary cell lines has been studied by measuring the depletion of total cellular glutathione after exposure to zinc(II) chloride at different concentrations. Total cellular glutathione (cGS) was measured at 31 ± 3 nmol/mg, 3.8 ± 0.6 nmol/mg, and 3.7 ±1.2 nmol/mg protein in A549, L2, and 11Lu cells, respectively. After treatment with buthionine sulfoximine (BSO), the cGS levels decreased by 20% in A549 cells and below 0.2 nmol/mg in L2 and 11Lu cells. Exposure of A549 cells to 25–200 μM ZnCl₂ for 4 h alone decreased the cGS content to 60–80%. There was little additional effect in BSO-pretreated cells. In L2 and 11Lu cells, the decrease of cGS was 70–85% following exposure to 15–150 μM ZnCl₂ for 2 h. If BSO was also used, the decrease in cGS was 85–95% in L2 cells and 75–85% in 11Lu cells. Exposure to 25–250 μM ZnCl₂ for 2 h diminished protein synthesis as determined by radiolabeled methionine incorporation, with half-maximum inhibition (EC₅₀) from 40–160 μM ZnCl₂. To attain similar EC₅₀ values in BSO-pretreated cells, only about half the zinc concentrations were required as compared to cells without pretreatment. The decrease of cGS was accompanied by an increased ratio of oxidized : reduced glutathione that was more pronounced in cells with low glutathione content.     

The influence of differentiation-inducing agents on plasma membrane surface characteristics of THP-1 cells

Undifferentiated THP-1 cells from Cell Culture Collection of the Institute of Cytology, RAS (St. Petersburg), are characterized by weak expression of Toll-like receptor-4 (TLR4) on the cell surface (up to 2%) and by almost undetectable expression of CD14 and CD11b receptors. Differentiation agent phorbol-12-myristate-13-acetate independently of its concentration (2 × 10⁻⁷ M or 10⁻⁸ M) and incubation time (24 or 48 h) did not initiate CD11b surface expression and did not change the parameter S_app (0.605 ± 0.005 at 37°C) reflecting the cell membrane viscosity. Differentiation of THP-1 cells induced by another differentiation agent, 1α,25-dihydroxyvitamin D₃, caused expression of CD14 (up to 70–80%) and CD11b (up to 15–20%) receptors, again without changes in plasma membrane viscosity. The rate constants of the reduction of 5- and 16-doxyl-stearic acids by THP-1 cells were in the range of 6–8 × 10⁻³ s⁻¹ at 37°C. During cell differentiation significant changes in cell electrophoretic mobility (EM, μm s⁻¹ V⁻¹ cm) were observed. Mean value of EM for undifferentiated THP-1 cells was −1.332 ± 0.011, whereas for phorbol-12-myristate-13-acetate- and 1α,25-dihydroxyvitamin D₃-treated cells it was −1.432 ± 0.030 and −1.212 ± 0.016, respectively.     

Induction of human cytotoxic T lymphocytes that preferentially recognise tumour cells bearing a conformational p53 mutant

 The tumour-suppressor gene p53 is pivotal in the regulation of apoptosis, and point mutations within p53 are the commonest genetic alterations in human cancers. Cytotoxic T lymphocytes (CTL) recognise peptide-MHC complexes on the surface of tumour cells and bring about lysis. Therefore, p53-derived peptides are potential candidates for immunisation strategies designed to induce antitumour CTL in patients. Conformational changes in the p53 protein, generated as a result of point mutations, frequently expose the 240 epitope, RHSVV (amino acids 212–217), which may be processed differently from the wild-type protein resulting in an altered MHC-associated peptide repertoire recognised by tumour-specific CTL. In this study 42 peptides (37 overlapping nonameric peptides, from amino acids 193–237 and peptides 186–194, 187–197, 188–197, 263–272, 264–272, possessing binding motifs for HLA-A2) derived from the wild-type p53 protein sequence were assayed for their ability to stabilise HLA-A2 molecules in MHC class I stabilisation assays. Of the peptides tested, 24 stabilised HLA-A2 molecules with high affinity (fluorescence ratio>1.5) at 26 °C, and five (187–197, 193–200, 217–224, 263–272 and 264–272) also stabilised the complexes at 37 °C. Peptides 188–197, 196–203 and 217–225 have not previously been identified as binders of HLA-A2 molecules and, of these, peptide 217–225 stabilised HLA-A2 molecules with the highest fluorescence ratio. Peptide 217–225 was chosen to generate HLA-A2-restricted CTL in vitro; peptide 264–272 was used as a positive control. The two primary CTL thus generated (CTL-217 using peptide 217–225; and CTL-264 using peptide 264–272) were capable of specifically killing peptide-pulsed T2 or JY cells. In order to determine whether these peptides were endogenously processed and to test the hypothesis that mutants expressing different protein conformations would generate an alternative peptide repertoire at the cell surface, a panel of target cells was generated. HLA-A2⁺ SaOs-2 cells were transfected with p53 cDNA containing point mutations at either position 175 (R → H) or 273 (R → H) (SaOs-2/175 and SaOs-2/273). Two HLA-A2-negative cell lines, A431 and SKBr3, naturally expressing p53 mutations at positions 273 and 175 respectively, were transfected with a cDNA encoding HLA-A2. The results showed that primary CTL generated in response to both peptides were capable of killing SaOs-2/175 and SKBr3-A2 cells, which possess the same mutation, but not SaOs-2/273, A431-A2 or SKBr3 cells transfected with control vector. This suggests that these peptides are presented on the surface of SaOs-2/175 and SKBr3-A2 cells in a conformation-dependent manner and represent potentially useful target peptides for immunotherapy. Received: 23 March 2000 / Accepted: 22 June 2000     

Formaldehyde stress

Formaldehyde, one of the most toxic organic compounds, is produced and processed in human cells. The level of human endogenous formaldehyde is maintained at a low concentration (0.01–0.08 mmol L⁻¹ in blood) under physiological conditions, but the concentration increases during ageing (over 65 years old). Clinical trials have shown that urine formaldehyde concentrations are significantly different between elderly Alzheimer’s patients (n=91) and normal elderly volunteers (n=38) (P<0.001). Abnormally high levels of intrinsic formaldehyde lead to dysfunction in cognition such as learning decline and memory loss. Excess extracellular and intracellular formaldehyde could induce metabolic response and abnormal modifications of cellular proteins such as hydroxymethylation and hyperphosphorylation, protein misfolding, nuclear translocation and even cell death. This cellular response called formaldehyde stress is dependent upon the concentration of formaldehyde. Chronic impairments of the brain resulted from formaldehyde stress could be one of the mechanisms involved in the process of senile dementia during ageing.     

The impact of melaphene on the expression of chloroplastic chaperone protein HSP70B and photosynthetic pigments in cells of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The effects of growth regulator of the new generation—melamine salt of bis(oxymethyl)phosphine acid (melaphene)—on culture growth, pigment and protein content, and the induction of protective chloroplastic chaperone HSP70B in Chlamydomonas reinhardtii CW15 cells were studied. Melaphene exhibited 10–30% growth inhibition at 10⁻⁹–10⁻²% concentration. At 10⁻⁹–10⁻⁴% of melaphene electrophoretic concentration, the pattern of cellular proteins was similar to the control. The alterations in protein content of algae cells were detected only at 10⁻²% concentration. The content of chlorophyll and carotenoids in melaphene-treated cells was 17–40% lower than in the control. Melaphene at 10⁻⁹–10⁻²% concentration inhibited HSP70B induction by 39–43% compared to untreated cells. The potential mechanism of melaphene effect might involve its influence on nuclear gene expression.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers, pH 3.5–7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2–4.6 and 7.5–8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 °C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 °C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 × 10³kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors. Received: 8 November 1996 / Received revision: 31 January 1997 / Accepted: 31 January 1997     

Encapsulation technology for short-term preservation and germplasm distribution of the African mahogany <Emphasis Type="Italic">Khaya senegalensis</Emphasis>

A protocol was developed for short-term preservation and distribution of the medicinal and timber plantation tree, Khaya senegalensis, using alginate-encapsulated shoot tips. The study assessed the effects of culture medium, storage temperature, auxin concentration and planting substrate on shoot regrowth or conversion into plantlets of four different clones. Optimal shoot growth was obtained, with high frequencies (92–100%) of shoot emergence, on Murashige and Skoog (MS) culture media containing 4.4 μM benzyladenine (BA). Encapsulated shoot tips survived longer at 25°C than at 4°C, with viability of 73–88% after 8 weeks. Conversion into plantlets was achieved on half-strength MS medium by pre-culture treatment of shoot tips with 49–490 μM indole-3-butyric acid (IBA) before encapsulation. Treatment with 245 μM IBA provided 52–98% conversion, and 90–95% of plantlets survived acclimatisation under nursery conditions. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile substrates. Highest frequencies of plantlet formation from pre-converted synthetic seeds (42–86%) were obtained by transferring synthetic seeds to organic compost, and these plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion is a novel and convenient method for producing synthetic seeds that are suitable for distribution to commercial nurseries.     

A target-specific chimeric toxin composed of epidermal growth factor and Pseudomonas exotoxin A with a deletion in its toxin-binding domain

 We have fused the epidermal growth factor (EGF) to the amino terminus of Pseudomonas exotoxin A (PE) to create a cytotoxic agent, designated EGF-PE, which preferentially kills EGF-receptor-bearing cells. In this study, we analyzed the effect of the Ia domain, the binding domain, of PE on the cytotoxicity of EGF-PE towards EGF-receptor-bearing cells and tried to develop a more potent EGF-receptor-targeting toxin. EGF-PE molecules with sequential deletions at the amino terminus of PE were constructed and expressed in E. coli strain BL21(DE3). The cytotoxicity of these chimeric toxins was then examined. Our results show that the amino-terminal and carboxy-terminal regions of the Ia domain of PE are important for the cytotoxicity of a PE-based targeting toxin. To design a more potent PE-based EGF-receptor-targeting toxin, a chimeric toxin, named EGF-PE(Δ34–220), which had most of the Ia domain deleted but retained amino acid residues 1–33 and 221–252 of this domain, was constructed. EGF-PE(Δ34–220) has EGF-receptor-binding activity but does not show PE-receptor-binding activity and is mildly cytotoxic to EGF-receptor-deficient NR6 cells. As expected, EGF-PE(Δ34–220) is a more potent cytotoxic agent towards EGF-receptor-bearing cells than EGF-PE(Δ1–252), where the entire Ia domain of PE was deleted. In addition, EGF-PE(Δ34–220) was shown to be extremely cytotoxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, and KB-3-1 cells. We also found that EGF-PE(Δ34–220) was highly expressed in BL21(DE3) and could be easily purified by urea extraction. Thus, EGF-PE(Δ34–220) can be a useful cytotoxic agent towards EGF-receptor-bearing cells. Received : 20 May 1994 / Received last revision : 9 September 1994 / Accepted : 28 September 1994     

Environmental and physiological factors affecting the uptake of phosphate by Chlorobium limicola

The uptake of soluble phosphate by the green sulfur bacterium Chlorobium limicola UdG6040 was studied in batch culture and in continuous cultures operating at dilution rates of 0.042 or 0.064 h^–1. At higher dilution rates, washout occurred at phosphate concentrations below 7.1 μM. This concentration was reduced to 5.1 μM when lower dilution rates were used. The saturation constant for growth on phosphate (K _μ) was between 2.8 and 3.7 μM. The specific rates of phosphate uptake in continuous culture were fitted to a hyperbolic saturation model and yielded a maximum rate (Va _max) of 66 nmol P (mg protein)^–1 h^–1 and a saturation constant for transport (K _t) of 1.6 μM. In batch cultures specific rates of phosphate uptake up to 144 nmol P (mg protein)^–1 h^–1 were measured. This indicates a difference between the potential transport of cells and the utilization of soluble phosphate for growth, which results in a significant change in the specific phosphorus content. The phosphorus accumulated within the cells ranged from 0.4 to 1.1 μmol P (mg protein)^–1 depending on the growth conditions and the availability of external phosphate. Transport rates of phosphate increased in response to sudden increases in soluble phosphate, even in exponentially growing cultures. This is interpreted as an advantage that enables Chl. limicola to thrive in changing environments. Received: 9 February 1998 / Accepted: June 1998     

Redox chemistry of tungsten and iron–sulfur prosthetic groups in Pyrococcus furiosus formaldehyde ferredoxin oxidoreductase

Formaldehyde oxidoreductase (FOR) is one of the tungstopterin iron–sulfur enzymes of the five-membered family of aldehyde oxidoreductases in the hyperthermophilic archaeon Pyrococcus furiosus. In dye-mediated equilibrium redox titrations, the tungsten in active P. furiosus FOR is a two-electron acceptor, W(VI/IV). The intermediate, paramagnetic W(V) state can be trapped only by reduction with substrate, with consecutive one-electron intraprotein electron transfer to the single [4Fe–4S]^(2+;+) cluster and partial comproportionation of the tungsten over W(IV, V, VI); this is a stable state in the absence of an external electron acceptor. Electron paramagnetic resonance (EPR) spectroscopy reveals a single “low-potential” W(V) spectrum with g _xyz values 1.847, 1.898, and 1.972, and a [4Fe–4S]⁺ cubane in a spin mixture of S = 1/2 (10%) and S = 3/2 (90%) of intermediate rhombicity (E/D = 0.21, g _real = 1.91). The development of this intermediate in vitro is slow even at elevated temperature and with a nominal 50:1 excess of substrate over enzyme presumably owing to the very unfavorable hydration equilibrium of the formaldehyde/methylene glycol couple with K _D ≈ 10³. Rapid intermediate formation of enzyme at concentrations suitable for EPR spectroscopy (200 μM) is only obtained with extremely high nominal substrate concentration (1 M formaldehyde) and is followed by a slower phase of denaturation. The premise that the free formaldehyde, and not the methylene glycol, is the enzyme’s substrate implies that K _M for formaldehyde is 3 orders of magnitude less that the previously reported value.     

Oxidation of methanol,formaldehyde and formic acid by methanol-utilizing yeast

Methanol-utilizing yeast,Candida boidini 11 Bh, characterized by high tolerance to methanol during growth, displays even higher tolerance when the oxidation rate by intact cells is tested. Low respiration activity is found even at 22% v/v of methanol. The half-saturation constant was 17–18mM. The half-saturation constants for the two oxidation intermediates, formaldehyde and formic acid were 3.6–4.0 and 30–33mM, respectively. When applied together with standard concentration of methanol, very low concentrations of both intermediates stimulated the oxidation rate. These results are discussed in connection with the relationship between growth and oxidation, the tolerance to high concentrations of inhibitory products and the mechanism of inhibition.     

Micropropagation of Hemidesmus indicus for cultivation and production of 2-hydroxy 4-methoxy benzaldehyde

Caulogenic responses of various explant types from 12-month-old plants of Hemidesmus indicus were tested. Second and third visible nodes (0.5 cm) from the apex and root segments (0.5 cm) were the most and least regenerative respectively, with the formation of 9.37 and 2.6 shoots in 4 weeks on half strength MS medium supplemented with 2.22 μM BA and 1.07 μM NAA and 4.44 μM BA and 2.69 μM NAA respectively. Caulogenic ability of the nodes decreased with increasing maturity. Shoot buds initiated upon the young nodes on day 10 developed into 7.2 cm long shoots within 4 weeks thereby making a shoot elongation phase unnecessary. Nodal explants of the in vitro raised shoots subcultured in the same medium produced 9.32 shoots of 7.1 cm length in 3–4 weeks, similar to those of the mature plant-derived nodes. Multiplication through subculture of the nodes up to 25 passages of 4 weeks each was achieved without decline. Shoot cultures were rooted in quarter salt strength MS medium containing 9.8 μM IBA and the rooted plants were hardened for establishment in pots at 96% rate. Four months after establishment, the micropropagated plants were stable and showed uniform morphological and growth characteristic. After 12 months of cultivation in the field, on an average micropropagated plant consisted of 4–5 shoots, 5–8 branches per shoot and increased root biomass (13.5 g) compared to the poor growth (single shoot and 2–3 branches) and root production (4.6 g) values obtained with plants raised from conventional rooted stem cuttings. The concentration of the root specific compound, 2-hydroxy 4-methoxy benzaldehyde per plant was 2–3 fold higher in micropropagated plants though on unit dry root biomass (0.12% per g dry wt) basis it remained the same between two sources of plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Media effects on black walnut (<Emphasis Type="Italic">Juglans nigra</Emphasis> L.) shoot culture growth <Emphasis Type="Italic">in vitro</Emphasis>: evaluation of multiple nutrient formulations and cytokinin types

The growth of black walnut shoot cultures was compared on media differing in nutrient formulation (MS, DKW, WPM, and 1/2X DKW), cytokinin type (ZEA, BA, and TDZ), and cytokinin concentration. On WPM and 1/2X DKW media, hyperhydricity was observed at frequencies of 60–100% compared with frequencies of 10–40% on the high-salt media (DKW and MS). All three cytokinins facilitated shoot regeneration from nodal cuttings, but recurrent elongation was only observed for BA (5–12.5 μM) and ZEA (5–25 μM) with mean shoot heights of 70–80 mm being possible after two culture periods (6–8 wk) for the fastest elongating lines. ZEA was effective across all six shoot lines with mean shoot heights of at least 35 mm over two culture periods, but two of the shoot lines were ‘nonresponsive’ to BA with mean shoot heights of <15 mm. In contrast, when shoot tip explants were used for culture multiplication, ZEA was the least effective cytokinin with proliferation frequencies of only 30–40%. The proliferation frequencies were twice as great (75–87%) for TDZ (0.05–0.1 μM), but most of the shoots regenerated were swollen or fasciated in morphology. High rates of proliferation (61–88%) were also possible using BA (12.5–25 μM), but axillary shoots did not elongate well, growing to heights of only 5–10 mm, on average, after 4–5 wk. Since the cytokinin types and concentrations required for high-frequency (>50%) axillary proliferation had adverse effects on the morphology and growth potential of the shoots, multiplication strategies based on the use of nodal cuttings are recommended.     

Phylogenetic analysis and characterization of lipolytic activity of halophilic archaeal isolates

Five isolates designated as B45, D83A, A206A, A85 and E49 found to possess lipolytic activities were taxonomically classified on the basis of their phylogenetic, phenotypic and chemotaxonomic characteristics. The isolates were determined to be gram-negative, catalase and oxidase positive, hydrolyzing Tween 80 and 60 but not starch, need 3.5–4 M NaCl for optimal growth and lack of anaerobic growth with arginine or DMSO. All isolates had the highest lipolytic activity at pH 8.5. Lipase and esterase activities increased with salt concentration up to 3–4.5 M NaCl, and decreased at 5 M NaCl. Esterase and lipase showed their maximal activities at 50–55°C and 60–65°C, respectively. The phylogenetic tree constructed by the neighbor-joining method indicated that the strain B45 and A85 were closely related to the members of genera Halovivax and Natrinema, respectively. The closest relative of the strain A206A and D83A were found to be Haloterrigena saccharevitans. The strain E49 displayed a more distant relationship to known strains.     

Metabolism of dynorphin A1–13 in human CSF

In-vitro incubation of human cerebrospinal fluid (CSF) obtained from patients ranging from 22–78 years with 10 μM of dynorphin A1–13 (Dyn A1–13) resulted in several cleavage products. Dyn A1–12 and A2–13 were identified as the major CSF metabolites by matrix-assisted laser desorption mass spectrometry (LD-MS). Further metabolites were Dyn A1–6, A2–12 and A4–12. LD-MS further suggested the formation of Dyn A1–8, A1–7, A1–10, A7–10, A3–12, A7–12, A3–13, A7–13 and A8–13. The metabolic half-life of Dyn A1–13 at 37°C was approximately 2.5 h (range 1.75–8.5 h), compared to less than one minute in plasma. The half-life of Dyn A1–13 decreased markedly with age or age-associated processes (n=20, r²=0.498). Noncompartmental kinetic analysis in the absence or presence of enzyme inhibitors (leucinethiol 10 μM, captopril 100 μM and GEMSA 20 μM) suggested that Dyn A1–13 is mainly metabolized by carboxypeptidase to A1–12 (51%) and by aminopeptidases to A2–13 (35%). The generation of A1–6 (13%) was only detected under enzyme inhibition. The extent of conversion into the main metabolites did not follow an age-associated trend, thus over-all enzyme levels but no specific enzymatic systems are elevated with age.     

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Purification,characterization and cloning of aldehyde dehydrogenase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> UPV-1

The enzyme responsible for formaldehyde removal in industrial wastewaters by cells of Rhodococcus erythropolis UPV-1 was identified as a broad-specific aldehyde dehydrogenase (EC 1.2.1.3). The enzyme was purified to electrophoretic homogeneity from ethanol-grown cells with a specific activity of 19.5 U mg⁻¹ protein and an activity recovery of 56%. The enzyme showed an isoelectric point (pI) of 5.3 and was a trimer of 162 kDa consisting of three identical 54-kDa subunits. It was specific for NAD⁺ and showed hyperbolic kinetics for this coenzyme (K _m=90 μM), but sigmoidal kinetics for the aliphatic aldehydes used as substrates. The enzyme affinity for aldehydes increased with their hydrocarbon chain length, ranging from 333 μM for formaldehyde to 85 nM for n-octanal. The corresponding calculated Hill coefficients were in the 1.55–2.77 range. With n-propanal as substrate, the optimum pH and temperature for activity were 9.5–10.0 and 47.5°C, respectively, with an E _a for catalysis of 28.6 kJ mol⁻¹. NAD⁺ protected the enzyme against thermal inactivation, but aldehydes were ineffective. The activity was severely inhibited by p-hydroxymercuribenzoate, indicating that a thiol was essential for catalysis. The 1,524-bp aldhR gene encoding a 507-amino-acid protein was expressed in cells of Escherichia coli M15 as a hexahistidine-tagged protein.