Exploiting the versatility of human cytochrome P450 enzymes: The promise of blue roses from biotechnology

The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries.

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY 1002/3A4. which express respective human P450 enzymes and NADPH-cytochrome P350 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA 1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double me promoter and the other, pOA 102, carrying O-AT and umuClacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 135 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 1-Amino-1,4-dimethyl-5H-pyrido[4.3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B-1 exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta -Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrom P450 enzyme involved in bioactivation of HCAs. (C) 2001 Elsevier Science B.V. All rights reserved.

Pea rms6 mutants exhibit increased basal branching

Our studies on two branching mutants of pea (Pisum sativum L.) have identified a further Ramosus locus, Rms6, with two recessive or partially recessive mutant alleles: rms6-1 (type line S2-271) and rms6-2 (type line K586). Mutants rms6-1 and rms6-2 were derived from dwarf and tall cultivars, Solara and Torsdag, respectively. The rms6 mutants are characterized by increased branching from basal nodes. In contrast, mutants rms1 through rms5 have increased branching from both basal and aerial (upper stem) nodes. Buds at the cotyledonary node of wild-type (WT) plants remain dormant but in rms6 plants these buds were usually released from dormancy. Their growth was either subsequently inhibited, sometimes even prior to emergence above ground, or they grew into secondary stems. The mutant phenotype was strongest for rms6-1 on the dwarf background. Although rms6-2 had a weak single-mutant phenotype, the rms3-1 rms6-2 double mutant showed clear transgression and an additive branching phenotype, with a total lateral length almost 2-fold greater than rms3-1 and nearly 5-fold greater than rms6-2 . Grafting studies between WT and rms6-1 plants demonstrated the primary action of Rms6 may be confined to the shoot. Young WT and rms6-1 shoots had similar auxin levels, and decapitated plants had a similar magnitude of response to applied auxin. Abscisic acid levels were elevated 2-fold at node 2 of young rms6-1 plants. The Rms6 locus mapped to the R to Gp segment of linkage group V (chromosome 3). The rms6 mutants will be useful for basic research and also have possible agronomical value.

The influence of light spectra, UV-A, and growth regulators on the in vitro seed germination of Senecio cineraria DC.

This study was carried out to investigate the effects of light spectra, additional UV-A, and different growth regulators on the in vitro germination of Senecio cineraria DC. Seeds were surface-sterilized and inoculated in MS medium to evaluate the following light spectra: white, white plus UV-A, blue, green, red or darkness. The maximum germinability was obtained using MS0 medium under white light (30%) and MS + 0.3 mg L-1 GA3 in the absence of light (30.5%). S. cineraria seeds were indifferent to light. Blue and green lights inhibited germination. Different concentrations of gibberellic acid (GA3) (0.1; 0.4; 0.6; 0.8; 1.0 and 2.0 mg L-1) and indole-3-acetic acid IAA (0.1; 0.3 and 1.0 mg L-1) were evaluated under white light and darkness. No concentration of GA3 enhanced seed germination percentage under white light. However, when the seeds were maintained in darkness, GA3 improved germination responses in all tested concentrations, except at 1.0 mg L-1. Under white light, these concentrations also increased the germination time and reduced germination rate. Germination rate, under light or darkness, was lower using IAA compared with GA3.

Efeitos da ingestão de simbiótico e indol-3-carbinol sobre o processo de carcinogênese química de cólon em ratos Wistar alimentados com dieta contendo heme

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Insights into the mechanims underlyng the antiproliferative potential of a Co(II) coordination compound bearing 1,10-Phenanthroline-5,6-Dione: DNA and protein interaction studies

The very high antiproliferative activity of [Co(Cl)(H2O)(phendione)(2)][BF4] (phendione is 1,10-phenanthroline-5,6-dione) against three human tumor cell lines (half-maximal inhibitory concentration below 1 mu M) and its slight selectivity for the colorectal tumor cell line compared with healthy human fibroblasts led us to explore the mechanisms of action underlying this promising antitumor potential. As previously shown by our group, this complex induces cell cycle arrest in S phase and subsequent cell death by apoptosis and it also reduces the expression of proteins typically upregulated in tumors. In the present work, we demonstrate that [Co(Cl)(phendione)(2)(H2O)][BF4] (1) does not reduce the viability of nontumorigenic breast epithelial cells by more than 85 % at 1 mu M, (2) promotes the upregulation of proapoptotic Bax and cell-cycle-related p21, and (3) induces release of lactate dehydrogenase, which is partially reversed by ursodeoxycholic acid. DNA interaction studies were performed to uncover the genotoxicity of the complex and demonstrate that even though it displays K (b) (+/- A standard error of the mean) of (3.48 +/- A 0.03) x 10(5) M-1 and is able to produce double-strand breaks in a concentration-dependent manner, it does not exert any clastogenic effect ex vivo, ruling out DNA as a major cellular target for the complex. Steady-state and time-resolved fluorescence spectroscopy studies are indicative of a strong and specific interaction of the complex with human serum albumin, involving one binding site, at a distance of approximately 1.5 nm for the Trp214 indole side chain with log K (b) similar to 4.7, thus suggesting that this complex can be efficiently transported by albumin in the blood plasma.

Inoculation with Metal-Mobilizing Plant-GrowthPromoting Rhizobacterium Bacillus sp. SC2b and Its Role in Rhizoremediation

A plant growth-promoting bacterial (PGPB) strain SC2b was isolated from the rhizosphere of Sedum plumbizincicola grown in lead (Pb)/zinc (Zn) mine soils and characterized as Bacillus sp. based on (1) morphological and biochemical characteristics and (2) partial 16S ribosomal DNA sequencing analysis. Strain SC2b exhibited high levels of resistance to cadmium (Cd) (300 mg/L), Zn (730 mg/L), and Pb (1400 mg/L). This strain also showed various plant growth-promoting (PGP) features such as utilization of 1-aminocyclopropane-1-carboxylate, solubilization of phosphate, and production of indole-3-acetic acid and siderophore. The strain mobilized high concentration of heavy metals from soils and exhibited different biosorption capacity toward the tested metal ions. Strain SC2b was further assessed for PGP activity by phytagar assay with a model plant Brassica napus. Inoculation of SC2b increased the biomass and vigor index of B. napus. Considering such potential, a pot experiment was conducted to assess the effects of inoculating the metal-resistant PGPB SC2b on growth and uptake of Cd, Zn and Pb by S. plumbizincicola in metal-contaminated agricultural soils. Inoculation with SC2b elevated the shoot and root biomass and leaf chlorophyll content of S. plumbizincicola. Similarly, plants inoculated with SC2b demonstrated markedly higher Cd and Zn accumulation in the root and shoot system, indicating that SC2b enhanced Cd and Zn uptake by S. plumbizincicola through metal mobilization or plant-microbial mediated changes in chemical or biological soil properties. Data demonstrated that the PGPB Bacillus sp. SC2b might serve as a future biofertilizer and an effective metal mobilizing bioinoculant for rhizoremediation of metal polluted soils.

The hyperaccumulator Sedum plumbizincicola harbors metal-resistant endophytic bacteria that improve its phytoextraction capacity in multi-metal contaminated soil

Endophyte-assisted phytoremediation has recently been suggested as a successful approach for ecological restoration of metal contaminated soils, however little information is available on the influence of endophytic bacteria on the phytoextraction capacity of metal hyperaccumulating plants in multi-metal polluted soils. The aims of our study were to isolate and characterize metal-resistant and 1-aminocyclopropane-1-carboxylate (ACC) utilizing endophytic bacteria from tissues of the newly discovered Zn/Cd hyperaccumulator Sedum plumbizincicola and to examine if these endophytic bacterial strains could improve the efficiency of phytoextraction of multi-metal contaminated soils. Among a collection of 42 metal resistant bacterial strains isolated from the tissues of S. plumbizincicola grown on Pb/Zn mine tailings, five plant growth promoting endophytic bacterial strains (PGPE) were selected due to their ability to promote plant growth and to utilize ACC as the sole nitrogen source. The five isolates were identified as Bacillus pumilus E2S2, Bacillus sp. E1S2, Bacillus sp. E4S1, Achromobacter sp. E4L5 and Stenotrophomonas sp. E1L and subsequent testing revealed that they all exhibited traits associated with plant growth promotion, such as production of indole-3-acetic acid and siderophores and solubilization of phosphorus. These five strains showed high resistance to heavy metals (Cd, Zn and Pb) and various antibiotics. Further, inoculation of these ACC utilizing strains significantly increased the concentrations of water extractable Cd and Zn in soil. Moreover, a pot experiment was conducted to elucidate the effects of inoculating metal-resistant ACC utilizing strains on the growth of S. plumbizincicola and its uptake of Cd, Zn and Pb in multi-metal contaminated soils. Out of the five strains, B. pumilus E2S2 significantly increased root (146%) and shoot (17%) length, fresh (37%) and dry biomass (32%) of S. plumbizincicola as well as plant Cd uptake (43%), whereas Bacillus sp. E1S2 significantly enhanced the accumulation of Zn (18%) in plants compared with non-inoculated controls. The inoculated strains also showed high levels of colonization in rhizosphere and plant tissues. Results demonstrate the potential to improve phytoextraction of soils contaminated with multiple heavy metals by inoculating metal hyperaccumulating plants with their own selected functional endophytic bacterial strains.

Synthesis, characterization and applications of new schiff base fluorescent chemosensors for metal and DNA interactions: conventional and "green" approaches

Dissertação apresentada para a obtenção do Grau de Doutor em Química Sustentável, especialidade de Química-Física Inorgânica, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Curare - Botany, Chemistry, and Pharmacology

Some aspects of curare research carried out over the last 25 years are discussed. Accepting a pharmacological rather than purely ethnological definition means, that curares are not limited to South America but that they are also known from Central Africa and South-EastAsia. Among the criteria that have been suggested for classifying South American curares: type of container, geographical origin, botanical sourcesof the active, constituent!, and chemical composition. A combination of botanical and geographical criteria leads to much the same regional ;groupings a combination of criteria involving the type of container and the chemical composition. The active principles in curares may derive from members of thr Loganiaceae (Strychnos) and/or Menispermaceae mainly Chondrodendron and Curarea, but also Abuta,Anomospermum, Cissampelos, Sciadotenia, and Telitoxicum). Certain of the Strychnos dimeric indole alkaloids can undergo a variety of cleavages, oxidations, and isomerizations; hence., some of the compounds obtained by normal isolation procedures one almost certainly artefacts. The different genera of, Menispermaceae a wide range of bisbenzyl and other types of isoquinoline alkaloids. Many of the plant additives also contain a variety of isoquinoline bases, and this has to be taken into account in assessing the contribution these ingredients may make to the ovzJuxll activity of, curare. Loganiaceae-bated curares with toxiferinzas major alkaloid tend to be the most toxic. In the case of Menispermaceae-based products, there-is evidence that the process by which they are made may lead to a considerable increase in the toxicity of the finished poisons as compared with the original plant materials. The mechanism of action of the alkaloids it, outlined, and the role of curare alkaloids in the development of, present-day muscle-relaxant drugs used in surgery is indicated. Attention lb drawn to reported medicinal uses of some of the alkaloid-bearing plants incorporated into curares, suggesting that further evaluation of these plants may be of interest.

Otimização com restrições de complementaridade: algoritmos e aplicações

Tese de Doutoramento em Engenharia Industrial e de Sistemas (PDEIS)

Biotechnological approaches for yield improvement of anticancer alkaloids in the plant Catharanthus roseus

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Diversity and capacity to promote maize growth of bacteria isolated from the Amazon region

ABSTRACT Maize plants can establish beneficial associations with plant growth-promoting bacteria. However, few studies have been conducted on the characterization and inoculation of these bacteria in the Amazon region. This study aimed to characterize endophytic bacteria isolated from maize in the Amazon region and to assess their capacity to promote plant growth. Fifty-five bacterial isolates were obtained from maize grown in two types of ecosystems, i.e., a cerrado (savanna) and a forest area. The isolates were characterized by the presence of the nifH gene, their ability to synthesize indole-3-acetic acid (IAA) and solubilize calcium phosphate (CaHPO4), and 16S rRNA partial gene sequencing. Twenty-four bacteria contained the nifH gene, of which seven were isolated from maize plants cultivated in a cerrado area and seventeen from a forest area. Fourteen samples showed the capacity to synthesize IAA and only four solubilized calcium phosphate. The following genera were found among these isolates: Pseudomonas; Acinetobacter; Enterobacter; Pantoea; Burkholderia and Bacillus. In addition, eight isolates with plant growth-promoting capacity were selected for a glasshouse experiment involving the inoculation of two maize genotypes (a hybrid and a variety) grown in pots containing soil. Inoculation promoted the development of the maize plants but no significant interaction between maize cultivar and bacterial inoculation was found. A high diversity of endophytic bacteria is present in the Amazon region and these bacteria have potential to promote the development of maize plants.

Peroxisomal beta-oxidation--a metabolic pathway with multiple functions.

Fatty acid degradation in most organisms occurs primarily via the beta-oxidation cycle. In mammals, beta-oxidation occurs in both mitochondria and peroxisomes, whereas plants and most fungi harbor the beta-oxidation cycle only in the peroxisomes. Although several of the enzymes participating in this pathway in both organelles are similar, some distinct physiological roles have been uncovered. Recent advances in the structural elucidation of numerous mammalian and yeast enzymes involved in beta-oxidation have shed light on the basis of the substrate specificity for several of them. Of particular interest is the structural organization and function of the type 1 and 2 multifunctional enzyme (MFE-1 and MFE-2), two enzymes evolutionarily distant yet catalyzing the same overall enzymatic reactions but via opposite stereochemistry. New data on the physiological roles of the various enzymes participating in beta-oxidation have been gathered through the analysis of knockout mutants in plants, yeast and animals, as well as by the use of polyhydroxyalkanoate synthesis from beta-oxidation intermediates as a tool to study carbon flux through the pathway. In plants, both forward and reverse genetics performed on the model plant Arabidopsis thaliana have revealed novel roles for beta-oxidation in the germination process that is independent of the generation of carbohydrates for growth, as well as in embryo and flower development, and the generation of the phytohormone indole-3-acetic acid and the signal molecule jasmonic acid.

Gas emission during laparoscopic colorectal surgery using a bipolar vessel sealing device: A pilot study on four patients.

BACKGROUND: Dissection during laparoscopic surgery produces smoke containing potentially toxic substances. The aim of the present study was to analyze smoke samples produced during laparoscopic colon surgery using a bipolar vessel sealing device (LigaSuretrade mark). METHODS: Four consecutive patients undergoing left-sided colectomy were enrolled in this pilot study. Smoke was produced by the use of LigaSuretrade mark. Samples (5,5l) were evacuated from the pneumoperitoneum in a closed system into a reservoir. Analysis was performed with CO2-laser-based photoacoustic spectroscopy and confirmed by a Fourier-transform infrared spectrum. The detected spectra were compared to the available spectra of known toxins. RESULTS: Samples from four laparoscopic sigmoid resections were analyzed. No relevant differences were noted regarding patient and operation characteristics. The gas samples were stable over time proven by congruent control measurements as late as 24 h after sampling. The absorption spectra differed considerably between the patients. One broad absorption line at 100 ppm indicating H2O and several unknown molecules were detected. With a sensitivity of alpha min ca 10-5 cm-1 no known toxic substances like phenol or indole were identified. CONCLUSION: The use of a vessel sealing device during laparoscopic surgery does not produce known toxic substances in relevant quantity. Further studies are needed to identify unknown molecules and to analyze gas emission under various conditions.