Fluoxetine modulates the transcription of genes involved in serotonin, dopamine and adrenergic signalling in zebrafish embryos.

Neurotransmitters pathways in fish and mammals are phylogenetically conserved. Therefore, the environmental presence of psychopharmaceuticals, such as fluoxetine (FLU), are likely to interact with fish serotonergic, dopaminergic and adrenergic systems, affecting their response and associated biological functions. Hence, the present work aimed at evaluating the effects of FLU in the transcription of genes involved in serotonin, dopamine and adrenergic transporters and receptors signalling in early stages of Danio rerio development. Embryos (1 hpf) were exposed for 80 h to different concentrations of FLU (0.0015, 0.05, 0.1, 0.5 and 0.8 µM) and mRNA levels of sert, 5-ht1a, 5-ht2c, dat, drd1b, drd2b, net, adra2a, adra2b, adra2c, vmat and mao were evaluated. A sensorimotor reflex assay was also performed demonstrating a significant decrease in tail reflex at 0.1 and 0.5 µM. The transcription levels of serotonergic and dopaminergic transporters (sert and dat) and vmat were down-regulated at environmentally relevant concentration (0.0015 µM). Receptors 5-ht2c, drd2b adra2b and adra2c mRNA levels also displayed a down regulation pattern after FLU exposure. In conclusion, this study demonstrated the interaction of FLU with the neurotransmission system at environmentally relevant concentrations by changing transcription patterns. Therefore, given the importance of these signalling pathways it is possible that their disruption can ultimately disturb the escape behaviour and biological functions in fish. Hence, evaluating the presence of this psychopharmaceutical in the aquatic environment should be implemented in future monitoring programmes.

Simvastatin modulates gene expression of key receptors in zebrafish embryos.

Nuclear receptors (NR) are involved in the regulation of several metabolic processes and it is well known that these constituents may be modulated by different chemicals classes, including pharmaceuticals that may activate or antagonize NR. In mammals, some pharmaceuticals modulate the transcription of pregnane X receptor, Pxr, peroxisome proliferator activated receptor, Ppars, and aryl hydrocarbon receptor, Ahr, affecting mRNA expression of genes belonging to various regulatory pathways, including lipid metabolism and detoxification mechanisms. The aim of this study was to determine the effects of simvastatin (SIM), an anticholesterolemic drug, on selected NR and AhR mRNA transcription levels during zebrafish early development. Embryos were collected at different development stages (0, 2, 6, 14, 24, 48, and 72 hr post fertilization (hpf)) and mRNA of all target NR was detected at all time points. Embryos (1 and 24 hpf) were exposed to different concentrations of SIM (5 or 50 µg/L) in two differing assays with varying exposure times (2 or 80 hr). The transcription levels of ahr2, raraa, rarab, rarga, pparαa, pparß1, pparγ, pxr, rxraa, rxrab, rxrbb, rxrga, rxrgb, as well as levels of cholesterol (Chol) were measured after exposure. SIM exerted no marked effect on Chol levels, and depending upon exposure duration mRNA levels of NR and AhR either increased or decreased. After 2 hr SIM treatment in 24 hpf embryos, transcription of ppars, pxr, and ahr was up-regulated, while after 80 hr mRNA levels of pxr and ahr were decreased with no marked changes in ppars. Data demonstrate that SIM produced alterations in gene expression of NR which are involved in varying physiological functions and that may disturb regulation of different physiological processes which might impair fish survival and ecosystems regeneration.

Effects of pharmaceuticals and personal care products (PPCPs) on multixenobiotic resistance (MXR) related efflux transporter activity in zebrafish (Danio rerio) embryos.

Certain ATP binding cassette (ABC) transporter proteins, such as zebrafish Abcb4, are efflux pumps acting as a cellular defence against a wide range of different, potentially toxic chemical compounds thus mediating so called multixenobiotic resistance (MXR). Certain chemicals target MXR proteins and, as so called chemosensitisers, inhibit the activity of these proteins thus increasing the toxicity of other chemicals that would normally be effluxed. In this study 14 pharmaceuticals and personal care products (PPCPs) that are being increasingly detected in aquatic systems, were assessed for interference with the MXR system of zebrafish (Danio rerio). Concentration dependent effects of test compounds were recorded with the dye accumulation assay using zebrafish embryos and in ATPase assays with recombinant zebrafish Abcb4. In the dye accumulation assay embryos at 24h post fertilisation (hpf) were exposed to 8µm rhodamine 123 along with test compounds for 2h. The rhodamine 123 tissue levels upon the exposure served as a measure for MXR transporter efflux activity of the embryo (low rhodamine levels - high activity; high levels - low activity). The known ABC protein inhibitors MK571, vinblastine and verapamil served as positive controls. All tested PPCPs affected rhodamine 123 accumulation in embryos. For seven compounds rhodamine tissue levels were either both decreased and increased depending on the compound concentration indicating both stimulation and inhibition of rhodamine 123 efflux by those compounds, only increased (inhibition, six compounds) or only decreased (stimulation, one compound). Recombinant zebrafish Abcb4 was obtained with the baculovirus expression system and PPCPs were tested for stimulation/inhibition of basal transporter ATPase activity and for inhibition of the transporter ATPase activity stimulated with verapamil. Eight of the tested PPCPs showed effects on Abcb4 ATPase activity indicating that their effects in the dye accumulation assay may have indeed resulted from interference with Abcb4-mediated rhodamine 123 efflux. Slight stimulatory effects were found for musk xylene, nerol, isoeugenol, α-amylcinnamaldehyde, α-hexylcinnamaldehyde and simvastatin indicating Abcb4 substrate/competitive inhibitor properties of those compounds. Likewise, decreases of the verapamil-stimulated Abcb4 ATPase activity by diclofenac and fluoxetine may indicate competitive transporter inhibition. Sertraline inhibited the basal and verapamil-stimulated Abcb4 ATPase activities suggesting its property as non-competitive Abcb4 inhibitor. Taken together, our finding that chemically diverse PPCPs interfere with MXR efflux activity of zebrafish indicates that (1) efflux transporters may influence bioaccumulation of many PPCPs in fish and that (2) many PPCPs may act as chemosensitisers. Furthermore, it appears that interference of PPCPs with efflux activity in zebrafish embryos is not only from effects on Abcb4 but also on other efflux transporter subtypes.

Danio rerio embryos on Prozac - Effects on the detoxification mechanism and embryo development.

In the past decade the presence of psychopharmaceuticals, including fluoxetine (FLU), in the aquatic environment has been associated with the increasing trend in human consumption of these substances. Aquatic organisms are usually exposed to chronic low doses and, therefore, risk assessments should evaluate the effects of these compounds in non-target organisms. Teleost fish possess an array of active defence mechanisms to cope with the deleterious effects of xenobiotics. These include ABC transporters, phase I and II of cellular detoxification and oxidative stress enzymes. Hence, the present study aimed at characterising the effect of FLU on embryo development of the model teleost zebrafish (Danio rerio) concomitantly with changes in the detoxification mechanisms during early developmental phases. Embryos were exposed to different concentrations of FLU (0.0015, 0.05, 0.1, 0.5 and 0.8µM) for 80hours post fertilization. Development was screened and the impact in the transcription of key genes, i.e., abcb4, abcc1, abcc2, abcg2, cyp1a, cyp3a65, gst, sod, cat, ahr, pxr, pparα, pparß, pparγ, rxraa, rxrab, rxrbb, rxrga, rxrgb, raraa, rarab, rarga evaluated. In addition, accumulation assays were performed to measure the activity of ABC proteins and antioxidant enzymes (CAT and Cu/ZnSOD) after exposure to FLU. Embryo development was disrupted at the lowest FLU concentration tested (0.0015µM), which is in the range of concentrations found in WWTP effluents. Embryos exposed to higher concentrations of FLU decreased Cu/Zn SOD, and increased CAT (0.0015 and 0.5µM) enzymatic activity. Exposure to higher concentrations of FLU decreased the expression of most genes belonging to the detoxification system and upregulated cat at 0.0015µM of FLU. Most of the tested concentrations downregulated pparα, pparß, pparγ, and raraa, rxraa, rxrab, rxrbb rxrgb and ahr gene expression while pxr was significantly up regulated at all tested concentrations. In conclusion, this study shows that FLU can impact zebrafish embryo development, at concentrations found in effluents of WWTPs, concomitantly with changes in antioxidant enzymes, and the transcription of key genes involved in detoxification and development. These finding raises additional concerns supporting the need to monitor the presence of this compound in aquatic reservoirs.

Simvastatin effects on detoxification mechanisms in Danio rerio embryos.

The transcription and protein activity of defence mechanisms such as ABC transporters, phase I and II of cellular detoxification and antioxidant enzymes can be altered in the presence of emerging contaminants such as pharmaceuticals impacting the overall detoxification mechanism. The present work aimed to characterise the effects of simvastatin on the detoxification mechanisms of embryonic stages of Danio rerio. In a first approach, constitutive transcription of key genes involved in detoxification was determined. Embryos were collected at different developmental stages, and transcription patterns of genes coding for ABC transporters, phase I and II and oxidative stress were analysed. With exception of abcc2, all genes seem to be from maternal transfer (0-2 hpf). Embryos were then exposed to different concentrations of simvastatin (5 and 50 µg/L), verapamil and MK571 (10 µM; ABC protein inhibitors) and a combination of simvastatin and ABC inhibitors. mRNA expression levels of abcb4, abcc1, abcc2, abcg2, cyp1a, cyp3a65, gst, sod, cat was evaluated. Accumulation assays to measure ABC proteins activity and activity of EROD, GST, CAT and Cu/ZnSOD, were also undertaken. Simvastatin acted as a weak inhibitor of ABC proteins and increased EROD and GST activity, whereas Cu/ZnSOD and CAT activity were decreased. Simvastatin up-regulated abcb4 and cyp3a65 transcription (both concentrations), as well as abcc1 and abcc2 at 50 µg/L, and down-regulated gst, sod, cat at 5 µg/L. In conclusion, our data revealed the interaction of simvastatin with detoxification mechanisms highlighting the importance of monitoring the presence of this emerging contaminant in aquatic environments.

Osteoblast adhesion and morphology on TiO2 depends on the competitive preadsorption of albumin and fibronectin.

This study aims at assessing the influence of the competitive preadsorption of human serum albumin (HSA) and human plasma fibronectin (FN) from binary solutions and 10% plasma on MC3T3-E1 osteoblast adhesion and morphology on two types of TiO2 substrates. One was commercially pure titanium with a titanium oxide layer formed in an H2O2 solution and the other TiO2 sputtered on Si (Sousa et al., Langmuir 2004; 20:9745-9754.). The strategy applied in the present investigation was to compare osteoblast adhesion to surfaces preadsorbed with HSA, FN, HSA/FN = 1, HSA/FN = 200, and 10% plasma. The adsorption of proteins was evaluated measuring the amount and the effectiveness of binding with radiolabeled proteins, 125 I-FN and 125 I-HSA. Our results indicated that MC3T3-E1 osteoblast adhesion correlates well with the amounts of FN and HSA adsorbed on TiO2 surfaces. Also, we found that fewer osteoblasts adhered to both substrates preadsorbed with HSA, HSA/FN = 200, and 10% plasma, after 4 and 24 h, than to the surfaces preadsorbed with FN and HSA/FN = 1. For the latter, FN was able to compensate the inhibitory effect of HSA on osteoblast adhesion. Therefore, the presence of lower amounts of coadsorbed albumin may improve presentation of FN in a more integrin-recognized conformation, suggesting that some degree of molecular packing prevents loss of integrin-binding activity. FN reversibility does not seem to be dependent on the HSA/FN adsorption mass ratio in solution, suggesting that FN competitively adsorbs to TiO2 in a favorable conformation and does not suffers subsequent conformational changes allowing exchange with other FN molecules in solution.

Dynamics of fibronectin adsorption on TiO2 surfaces.

In the present work we analyze the dynamics of fibronectin (FN) adsorption on two different stable titanium oxides, with varied surface roughness, and chemically similar to those used in clinical practice. The two types of titanium oxide surfaces used were TiO2 sputtered on Si (TiO2 sp) and TiO2 formed on commercially pure titanium after immersion in H2O2 (TiO2 cp). Surface characterization was previously carried out using different techniques (Sousa, S. R.; Moradas-Ferreira, P.; Melo, L. V.; Saramago, B.; Barbosa, M. A. Langmuir 2004, 20 (22), 9745-9754). Imaging and roughness analysis before and after FN adsorption used atomic force microscopy (AFM) in tapping mode, in air, and in magnetic alternating current mode, in liquid (water). FN adsorption as a function of time was followed by X-ray photoelectron spectroscopy (XPS), by radiolabeling of FN with 125I (125I-FN), and by ellipsometry. Exchangeability studies were performed using FN and HSA. AFM roughness analysis revealed that, before FN adsorption, both TiO2 surfaces exhibited a lower root-mean-square (Rq) and maximum peak with the depth of the maximum valley (Rmax) roughness in air than in water, due to TiO2 hydration. After protein adsorption, the same behavior was observed for the TiO2 sp substrate, while Rq and Rmax roughness values in air and in water were similar in the case of the TiO2 cp substrate, for the higher FN concentration used. Surface roughness was always significantly higher on the TiO2 cp surfaces. AFM led to direct visualization of adsorbed FN on both surfaces tested, indicating that after 10 min of FN incubation the TiO2 sp surface was partially covered by FN. The adsorbed protein seems to form globular aggregates or ellipsoids, and FN aggregates coalesce, forming clusters as the time of adsorption and the concentration increase. Radiolabeling of FN revealed that a rapid adsorption occurs on both surfaces and the amount adsorbed increased with time, reaching a maximum after 60 min of incubation. Time dependence is also observed for the evolution of the atomic (%) of N determined by XPS and by the increase of the thickness by ellipsometry. TiO2 cp adsorbs more FN than the TiO2 sp surfaces, after 60 min of adsorption, as shown by the radiolabeling data. FN molecules are also more strongly attached to the former surface as indicated by the exchangeability studies. The overall results provide novel evidence that FN spontaneously adsorbs as a self-assembly at TiO2 surfaces as a function of time. The aggregate structure is an intermediate feature shared by some protein fibrillar assemblies at interfaces, which is believed to promote cell adhesion and cytoskeleton organization (Pellenc, D.; Berry, H.; Gallet, O. J. Colloid Interface Sci. 2006, 298 (1), 132-144. Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. J. Cell Sci. 2000, 113 (10), 1677-1686).

A novel approach for the identification of bacterial taxa-specific molecular markers.

AIMS: To develop and establish a methodology for an oriented and fast identification of species taxa-specific molecular markers useful for the identification of micro-organisms. METHODS AND RESULTS: From the complete microbial genomes available in Pfam database, taxa-specific protein domains were identified which lead to the selection of taxa-specific loci. This strategy was used to identify six genetic markers: four specific for Pseudomonas syringae pv. tomato, one specific for P. syringae pv. syringae and one specific for P. putida. The discriminatory potential of these loci was evaluated by Southern hybridization using several pseudomonad species and pathovars, by dot-blot hybridization and by multiplex PCR optimized for the simultaneous detection of P. putida, P. syringae pv. syringae and P. syringae pv. tomato. Sensitivity assays indicated a detection limit of approximately 10 pg of chromosomal DNA template needed for each bacterium. CONCLUSIONS: The proposed methodology was efficient on the selection of six Pseudomonas-specific markers able to discriminate Pseudomonas at the species and pathovar level. SIGNIFICANCE AND IMPACT OF THE STUDY: The oriented search of taxa-specific molecular probes described in this work, which can be easily extended to other groups of bacteria, will improve the accuracy and expedite the identification of micro-organisms by DNA-based molecular methods.

The effect of long-term depuration on levels of oxidative stress biomarkers in mullets (Mugil cephalus) chronically exposed to contaminants.

The present study aimed to obtain additional data on the effect of long-term depuration on the levels of oxidative stress biomarkers, and to clarify the role of mullets for monitoring pollution in River Douro estuary. Mullets chronically exposed to a mixture of contaminants in Douro estuary were captured in Spring of 2001, 2002 and 2003. The activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX); and oxidative damages in lipids (lipid peroxidation) and in proteins (protein carbonyl content) were assessed at capture day and after transfer to unpolluted seawater for 1, 4 and 8 months. An overall decrease in the activities of the antioxidant enzymes was detected, except for the GPX after 4 months depuration. CAT activity exhibited the more significant decrease at the end of the long-term depuration. The decrease in SOD activity after 1 month of depuration was then maintained during the remaining depuration period. Regarding oxidative damages, a decrease in lipid peroxidation as well as the content of oxidised proteins was observed during depuration. Indeed, at capture the activities of antioxidant defences were higher as a result of the formation of reactive oxygen species (ROS) from the metabolism of pollutants. The oxidative damaged molecules were repaired or degraded during the depuration period, supporting the use of such damages as indicators of exposure to pro-oxidant pollutants.

The effect of long-term depuration on phase I and phase II biotransformation in mullets (Mugil cephalus) chronically exposed to pollutants in River Douro Estuary, Portugal.

Pollutants such polycyclic aromatic hydrocarbons (PAHs) are released into the environment by urban communities and industries and the enzymes that catalyse the biotransformation of pollutants play a key role regarding the accumulation of these compounds in fish species inhabiting these areas. In this study the relationship between phase I (EROD activity) and phase II (GST activity) and PAH metabolites was measured in grey mullet (Mugil cephalus) after capture in the Douro estuary, and after long-term depuration in an unpolluted laboratory environment. The results showed a significant decrease in EROD activity after 1 month and in bile metabolites after 4 months in captivity, with both maintaining reduced levels at 4 and 8 months depuration. Liver GST activity did not showed significant changes. This study provides evidence that Douro estuary waters contain bioavailable PAHs that can be associated with the induction of cytochrome P450, and that mullets have the ability to metabolise and eliminate PAHs.

TiO2 type influences fibronectin adsorption.

Human fibronectin (FN) plays a key role in the biointegration of implants as the success depends on adsorption of proteins like FN [1]. Indeed FN can be an intermediary between the biomaterial surface and cells. The adsorption of human fibronectin (FN) on commercially pure titanium with a titanium oxide layer formed in a H2O2 solution (TiO2 cp) and TiO2 sputtered on Si (TiO2 sp) was studied. Adsorption isotherms and the work of adhesion were assessed by wettability studies, X-ray photoelectron spectroscopy (XPS), and by radiolabelling of FN with 125I, (125)I-FN. Exchangeability of bound FN by free FN, was also evaluated by the radiolabelling technique. Contact angle determinations have shown that FN displays higher affinity for the TiO2 cp surface than for the TiO2 sp. As expected from the surface free energy values, the work of adhesion of FN is higher for the TiO2 cp substrate, the more hydrophilic one, and lower for the TiO2 sp substrate, the more hydrophobic one. The adsorption isotherms were evaluated by two different techniques: radiolabelling of FN (125I-FN) and XPS. TiO2 cp adsorbs more FN than the TiO2 sp surfaces as shown by the radiolabelling data. FN molecules are also more strongly attached to the former surface as indicated by the work of adhesion and by the exchangeability studies. Results using 125I-FN also suggests that FN adsorbs as a multilayer for FN concentrations in solution higher than 100 microg/mL.

Oxidative stress biomarkers in two resident species, mullet (Mugil cephalus) and flounder (Platichthys flesus), from a polluted site in River Douro Estuary, Portugal.

Exposure of marine animals to certain pollutants can enhance reactive oxygen species (ROS) production with subsequent damage to macromolecules and alterations in oxidant defences levels. Aimed at correlating the tissue concentration of certain contaminants (PCBs, DDT) with antioxidant defence levels and oxidative damages, two fish species with different life strategies (mullet, Mugil cephalus, and flounder, Platichthys flesus) were collected in the Douro Estuary (NW Portugal). After capture, the fish were left to depurate for 1 month in clean seawater. The levels of the two antioxidant enzyme activities revealed that they are species-dependent with mullet's livers showing higher superoxide dismutase (SOD) (13.2+/-0.5 U/mg protein) and catalase (CAT) (15.5+/-1.0 mmol/min/mg protein) activities than flounder (SOD: 7.9+/-0.9 U/mg protein; CAT: 11.1+/-0.8 mmol/min/mg protein). After 1 month in captivity the antioxidant enzymes activities in liver decreased in mullets, while for flounders the responses were not consistent because during the experimental period flounders did not ate and responses of antioxidant enzymes and oxidative damages were dependent on the fasting condition. The liver oxidative damages were evaluated by estimating oxidised lipids and proteins. Both species showed similar levels for these two parameters. The hepatic lipid peroxidation in flounder increased after 1 month in captivity, while in mullet an increase was observed only in summer and autumn. The oxidised protein content increased for both species after the depuration period. This study reveals differences between species under oxidative stress when exposed to pollutants. In a clean environment, the mullet's primary antioxidant defences decreased indicating that the animals living in Douro estuary were facing an oxidative stress. The data indicate that, namely in mullet, the presence of pollutants induce oxidative stress responses.

Human serum albumin adsorption on TiO2 from single protein solutions and from plasma.

In the present work, the adsorption of human serum albumin (HSA) on commercially pure titanium with a titanium oxide layer formed in a H(2)O(2) solution (TiO(2) cp) and on TiO(2) sputtered on Si (TiO(2) sp) was analyzed. Adsorption isotherms, kinetic studies, and work of adhesion determinations were carried out. HSA exchangeability was also evaluated. Surface characterization was performed by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and wettability studies. The two TiO(2) surfaces have very distinct roughnesses, the TiO(2) sp having a mean R(a) value 14 times smaller than the one of TiO(2) cp. XPS analysis revealed consistent peaks representative of TiO(2) on sputtered samples as well as on Ti cp substrate after 48 h of H(2)O(2) immersion. Nitrogen was observed as soon as protein was present, while sulfur, present in disulfide bonds in HSA, was observed for concentrations of protein higher than 0.30 mg/mL. The work of adhesion was determined from contact angle measurements. As expected from the surface free energy values, the work of adhesion of HSA solution is higher for the TiO(2) cp substrate, the more hydrophilic one, and lower for the TiO(2) sp substrate, the more hydrophobic one. The work of adhesion between plasma and the substrates assumed even higher values for the TiO(2) cp surface, indicating a greater interaction between the surface and the complex protein solutions. Adsorption studies by radiolabeling of albumin ((125)I-HSA) suggest that rapid HSA adsorption takes place on both surfaces, reaching a maximum value after approximately 60 min of incubation. For the higher HSA concentrations in solution, a multilayer coverage was observed on both substrates. After the adsorption step from single HSA solutions, the exchangeability of adsorbed HSA molecules by HSA in solution was evaluated. The HSA molecules adsorbed on TiO(2) sp seem to be more easily exchanged by HSA itself than those adsorbed on TiO(2) cp after 24 h. In contrast, after 72 h, nearly all the adsorbed albumin molecules effectively exchange with other albumin molecules.

Oxidative stress and signal transduction in Saccharomyces cerevisiae: insights into ageing, apoptosis and diseases.

In yeast, as in higher eukaryotes, reactive oxygen species are produced as normal by-products of cellular metabolism. Under physiological conditions, the cell defence mechanisms are able to avoid molecular damages. This balance is disturbed when yeast cells are exposed to diverse environmental stress conditions, such as the presence of oxidants, heat shock, ethanol and metal ions. The increased production of reactive oxygen species is sensed by the cell, leading to the induction of defence mechanisms - the oxidative stress response. The present review discusses the mechanisms by which reactive oxygen species are sensed and the signalling pathways that are coupled with changes in genomic expression programs. Yeast has been used as an eukaryotic cell system to characterise the molecular mechanisms underlying the oxidative stress response. Furthermore, yeast has been utilised to elucidate the role of oxidative stress in ageing, apoptosis, and diseases, such as familial amyotrophic lateral sclerosis and Friedreich's ataxia.

Isolation and characterization of two new methanesulfonic acid-degrading bacterial isolates from a Portuguese soil sample.

Two novel bacterial strains that can utilize methanesulfonic acid as a source of carbon and energy were isolated from a soil sample collected in northern Portugal. Morphological, physiological, biochemical and molecular biological characterization of the two isolates indicate that strain P1 is a pink-pigmented facultative methylotroph belonging to the genus Methylobacterium, while strain P2 is a restricted methylotroph belonging to the genus Hyphomicrobium. Both strains are strictly aerobic, degrade methanesulfonate, and release small quantities of sulfite into the medium. Growth on methanesulfonate induces a specific polypeptide profile in each strain. This, together with the positive hybridization to a DNA probe that carries the msm genes of Methylosulfonomonas methylovora strain M2, strongly endorses the contention that a methanesulfonic acid monooxygenase related to that found in the previously known methanesulfonate-utilizing bacteria is present in strains P1 and P2. The isolation of bacteria containing conserved msm genes from diverse environments and geographical locations supports the hypothesis that a common enzyme may be globally responsible for the oxidation of methanesulfonate by natural methylotrophic communities.

Flocculation of Saccharomyces cerevisiae is induced by transformation with the GAP1 gene from Kluyveromyces marxianus.

A non-flocculent strain of Saccharomyces cerevisiae was transformed with the GAP1 gene which encodes p37, a GAPDH-like protein present in the cell wall of Kluyveromyces marxianus flocculent cells. The transformed cells were characterized with respect to flocculation behaviour, morphology, growth, cell wall integrity and GAPDH activity. A flocculent phenotype was acquired by the transformed cells, showing a behaviour in respect to flocculation/deflocculation very similar to that of K. marxianus. The presence of p37 in the cell wall was assessed by immunoprecipitation of biotinylated cell wall proteins and an accumulation of p37 was evident in the cell wall of transformed cells. This result was confirmed by studies using a chimeric protein resulting from fusing the p37 with a yeast-enhanced green fluorescent protein, yEGFP. The recombinant protein was localized mainly in the cell wall of the transformed strain, although the presence of p37 in the cytosol was indicated by an increase in GAPDH activity. Calcofluor white sensitivity tests indicated that the cell wall structure is affected by the accumulation of p37. These results provided further evidence of p37 function regarding flocculation and that although lacking a N-terminal signal peptide p37 is targeted to the cell wall.

Adaptive response of the yeast Saccharomyces cerevisiae to reactive oxygen species: defences, damage and death.

The yeast Saccharomyces cerevisiae has been extensively utilised to address the mechanisms underlying the oxidative stress response. The antioxidant defences can be induced either by respiratory growth or in the presence of pro-oxidants. The cell response involves the transcriptional control of genes by protein regulators that have been recently identified and post-translational activation of pre-existing defences. The current state of the art regarding the induction of antioxidant defences during respiratory growth and by exposure to hydrogen peroxide is reviewed.

The influence of Cu concentration on ethanolic fermentation by Saccharomyces cerevisiae.

In the present work, the influence of Cu concentration on alcoholic fermentation by Saccharomyces cerevisiae was studied in white grape musts and in YNB medium containing glucose. In the YNB medium, the yield of ethanol, relative to the control, doubled in the presence of 0.50 and 1.0 mM Cu. As for production of ethanol from musts, only minor effects were observed at different Cu concentrations, which indicates that Cu levels do not effect changes in fermentation, and, therefore, are below any toxic level regarding the yeast performance.

Molecular analysis of a novel methanesulfonic acid monooxygenase from the methylotroph Methylosulfonomonas methylovora.

Methylosulfonomonas methylovora M2 is an unusual gram-negative methylotrophic bacterium that can grow on methanesulfonic acid (MSA) as the sole source of carbon and energy. Oxidation of MSA by this bacterium is carried out by a multicomponent MSA monooxygenase (MSAMO). Cloning and sequencing of a 7.5-kbp SphI fragment of chromosomal DNA revealed four tightly linked genes encoding this novel monooxygenase. Analysis of the deduced MSAMO polypeptide sequences indicated that the enzyme contains a two-component hydroxylase of the mononuclear-iron-center type. The large subunit of the hydroxylase, MsmA (48 kDa), contains a typical Rieske-type [2Fe-2S] center with an unusual iron-binding motif and, together with the small subunit of the hydroxylase, MsmB (20 kDa), showed a high degree of identity with a number of dioxygenase enzymes. However, the other components of the MSAMO, MsmC, the ferredoxin component, and MsmD, the reductase, more closely resemble those found in other classes of oxygenases. MsmC has a high degree of identity to ferredoxins from toluene and methane monooxygenases, which are enzymes characterized by possessing hydroxylases containing mu-oxo bridge binuclear iron centers. MsmD is a reductase of 38 kDa with a typical chloroplast-like [2Fe-2S] center and conserved flavin adenine dinucleotide- and NAD-binding motifs and is similar to a number of mono- and dioxygenase reductase components. Preliminary analysis of the genes encoding MSAMO from a marine MSA-degrading bacterium, Marinosulfonomonas methylotropha, revealed the presence of msm genes highly related to those found in Methylosulfonomonas, suggesting that MSAMO is a novel type of oxygenase that may be conserved in all MSA-utilizing bacteria.