Removal of the anti-cancer drug methotrexate from water by advanced oxidation processes: Aerobic biodegradation and toxicity studies after treatment.

Anti-cancer drugs are discussed as high risk substances in regard to human health and considered as problematic for the environment. They are of potential environmental relevance due to their poor biodegradability and toxicological properties. Methotrexate (MTX) is an antimetabolite that was introduced in the pharmaceutical market in the 40's and still today is one of the most consumed cytotoxic compounds around the world. In the present study MTX was only partially biodegraded in the closed bottle test (CBT). Therefore, it was submitted to three different advanced oxidation processes (AOPs): UV/H2O2, UV/Fe(2+)/H2O2 and UV/TiO2. The irradiation was carried out with a Hg medium-pressure lamp during 256min whereas the analytical monitoring was done through LC-UV-MS/MS and DOC analysis. MTX was easily removed in all the irradiation experiments, while the highest mineralization values and rates were achieved by the UV/Fe(2+)/H2O2 treatment. The lowest resulted from the UV/H2O2 reactions. The UV/H2O2 treatment resulted in little biodegradable transformation products (TPs). However, the same treatment resulted in a reduction of the toxicity of MTX by forming less toxic TPs. Analysis by LC-UV-MS/MS revealed the existence of nine TPs formed during the photo-catalytic treatments. The pH of the solutions decreased from 6.4 (t 0min) to 5.15 in the UV/H2O2 and from 6.4 (t 0min) to 5.9 in the UV/TiO2 at the end of the experiments. The initial pH of the UV/Fe(2+)/H2O2 experiments was adjusted to 5 and after the addition of H2O2 the pH decreased to around 3 and remained in this range until the end of the treatments.

Molecular and functional characterization of the gilthead seabream ß-defensin demonstrate its chemotactic and antimicrobial activity.

Antimicrobial peptides (AMPs) are important mediators of the innate immune response against bacteria and viruses. We have found a ß-defensin (BD) gene searching the expressed sequence tags (ESTs) of the teleost fish gilthead seabream (Sparus aurata). The clone contains an open reading frame of 201 bp mRNA that encodes a putative seabream ß-defensin (saBD) propeptide of 66 amino acids containing the six conserved cysteines as the main signature of this AMP. The phylogenetic tree shows that saBD, and its fish orthologues, are closely related to the human BD-4. Transcripts of the saBD gene were mainly detected by real-time PCR in the skin, peritoneal leucocytes and head-kidney but scarcely expressed in the peripheral blood. Interestingly, head-kidney leucocytes incubation with synthetic unmethylated CpG oligodeoxynucleotides and bacterial DNA up-regulated the saBD gene expression. Recombinant protein (saBD-V5-His) was expressed in the HEK293 cell line and its functional activity determined. First, seabream head-kidney leucocytes showed chemotactic activity towards supernatants containing saBD-V5-His whilst failed to do so to human recombinant BD-1 y BD-4. Moreover, both cell lysates and supernatants containing saBD-V5-His showed strong antimicrobial activity against Vibrio anguillarum (a seabream pathogenic bacterium) and Bacillus subtilis whilst little on other fish pathogens such as Vibrio harvey and Photobacterium damselae. Further studies will elucidate the existence of other BD genes and their implications on the seabream defense against bacteria and virus.

Dual function of fish hepcidin: response to experimental iron overload and bacterial infection in sea bass (Dicentrarchus labrax).

The role of hepcidin in iron metabolism regulation and bacterial infection has been the focus of recent attention. However, in spite of the growing number of hepcidin genes known from different organisms, little is known about its putative dual function in fish. The aim of this study was to characterize the sea bass hepcidin gene and to study its role in iron metabolism and infection. The novel sea bass hepcidin gene was found to be organized into two introns and three exons with several copies present in the genome. The transcript showed a constitutive low basal expression being mainly expressed in liver and encoding a putative 85 residues long peptide. Fish were submitted either to iron status modulation or bacterial infection and the hepcidin transcript levels were analysed along with a number of other parameters. Liver hepcidin expression was found to increase in both the iron-overloaded and infected fish, while in the iron-deficient fish no alteration in expression levels was detected. These results point to the evolutionary conservation of hepcidin's dual functions.

Virulence of Photobacterium damselae subsp. piscicida in cultured cobia Rachycentron canadum.

An outbreak of serious mortality among the cultured cobia Rachycentron canadum (weighing 3 kg) characterized by the presence of whitish granulomatous deposits on the kidney, liver and spleen occurred in July of 2000 in Taiwan. A non-motile strain CP1 was isolated from kidney and/or liver on tryptic soy agar and/or brain heart infusion agar plates (both supplemented with 1% NaCl, w/v). This strain was characterized and identified as Photobacterium damselae subsp. piscicida using biochemical characteristics and Bionor mono-Pp tests. The bacterium and its extracellular products (ECP) were lethal to the cobia (weighing 10 g) with LD50 values of 1.03 x 10(4) colony forming units and 1.26 microg protein/g fish body weight, respectively. All the moribund/dead fish exhibited darkness in color with no gross or internal leasions. However, the bacteria could be reisolated from kidney and liver after bacterial challenge. The present results reveal that Ph. damselae subsp. piscicida is the causative agent of fish photobacteriosis in the cobia and the bacterium isolated from sub-adult cobia (chronic form) is virulent to young cobia causing acute form of the disease.

Solute accumulation in the deep-sea bacterium Photobacterium profundum.

The identity and amounts of intracellular solutes in the deep-sea bacterium Photobacterium profundum strain SS9 were studied using nuclear magnetic resonance techniques. P. profundum strain SS9, a moderate piezophile which grows optimally at 20-30 MPa primarily accumulated glutamate and betaine, with lesser amounts of alanine, beta-hydroxybutyrate (beta-HB) and oligomers composed of the beta-HB units when grown at 0.1 MPa to early stationary phase. When grown at the optimal pressure, the cells preferentially increased intracellular concentrations of beta-HB and beta-HB oligomers, while the amino acid pools remained relatively constant. Since the organic solutes increased with increasing external NaCl in the medium, they are functioning as osmolytes. The beta-HB molecules represent a novel class of osmolytes, termed 'piezolytes,' whose cellular levels responded to hydrostatic pressure as well as osmotic pressure. Factors such as cell growth stage and temperature were also examined for their effect on the solute distribution in these cells.

Structure and regulation of the omega-3 polyunsaturated fatty acid synthase genes from the deep-sea bacterium Photobacterium profundum strain SS9.

Omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) have been shown to be of major importance in the promotion of cardiovascular health, proper human development and the prevention of some cancers. A high proportion of bacterial isolates from low-temperature and high-pressure marine environments produce EPA or DHA. This paper presents the sequence of a 33 kbp locus from the deep-sea bacterium Photobacterium profundum strain SS9 which includes four of the five genes required for EPA biosynthesis. As with other bacterial pfa (polyunsaturated fatty acid) genes, the deduced amino acid sequences encoded by the SS9 genes reveal large multidomain proteins that are likely to catalyse EPA biosynthesis by a novel polyketide synthesis mechanism. RNase protection experiments separated the SS9 pfa genes into two transcriptional units, pfaA-C and pfaD. The pfaA transcriptional start site was identified. Cultivation at elevated hydrostatic pressure or reduced temperature did not increase pfa gene expression despite the resulting increase in percentage composition of EPA under these conditions. However, a regulatory mutant was characterized which showed both increased expression of pfaA-D and elevated EPA percentage composition. This result suggests that a regulatory factor exists which coordinates pfaA-D transcription. Additional consideration regarding the activities required for PUFA synthesis is provided together with comparative analyses of bacterial pfa genes and gene products.

[Effect of cAMP on the growth and development of luminescence in Photobacterium belozerskii]. / Vliianie tsAMF na rost i razvitie liuminestsentsii Photobacterium belozerskii.

The effect of cAMP on the parameters characterizing the development of luminescence in Photobacterium belozerskii is discussed. An addition of cAMP to the culture medium shortened the latent time of luminescence development by 3--6 hours. The intensity of bacterial luminescence increased with a varying rate. During luminescence enhancement the rate of luciferase synthesis increased by a factor of 10 to 10(3). The rate of luciferase synthesis and the maxiumum level of bacterial luminescence when cultivated in the glycerol medium containing arginine and proline increased under the influence of cAMP by 100 and 40 times, respectively. After an addition to cAMP into the glucose medium these parameters of luminescence development increased only when the medium contained arginine. After an addition of cAMP into the glycerol medium the rate of bacterial growth increased two-fold. Possible mechanisms regulating luminescence development and cAMP involvement in these processes are discussed.