Molecular and structural modeling of the Phanerochaete flavido-alba extracellular laccase reveals its ferroxidase structure

The fungus Phanerochaete flavido-alba is highly efficient in the oxidation of olive oil wastewater-derived polyphenols. This capability is largely due to the action of a multicopper-oxidase (MCO), encoded by the pfaL gene. We describe the sequence and organization of pfaL gene and the biochemical characterization and predicted 3D structural model of the encoded protein. pfaL gene organization and peptide sequence are highly similar to those of P. chrysosporium MCOs. However, PfaL is the first MCO in the Phanerochaete genus to show evident laccase activity. Phylogenetic analysis places PfaL in a differentiated sub-branch of ferroxidases. Protein structure analysis reveals close similarity of PfaL and ferroxidases and provides clues about the differences of activity between both types of enzymes. To summarize, P. flavido-alba laccase is the first enzyme in the novel and biochemically poorly defined group of “ferroxidases/laccases” that shows efficacious oxidation of laccase substrates, biotechnologically exploitable in bioremediation approaches.     

Adherence of Pseudomonas aeruginosa and Candida albicans to urinary catheters

In this study, the in vitro adherence capabilities of Pseudomonas aeruginosa and Candida albicans clinical isolates to urinary catheters were investigated. Quantitative analysis was performed by colony-forming unit counts and scanning electron microscopy. Results demonstrated that the adherence of P. aeruginosa to urinary catheters was enhanced in the presence of C. albicans, while C. albicans adherence was not significantly affected. Further investigations are warranted to fully understand the pathogenic potential of their interaction in order to aid in the design of novel strategies for the prevention and treatment of catheter-related UTIs.     

Quercetin in Lyotropic Liquid Crystalline Formulations: Physical, Chemical and Functional Stability

The purpose of this study was to develop a lyotropic liquid crystalline formulation using the emulsifier vitamin E TPGS and evaluate its behavior after incorporation of a flavonoid, quercetin. The physical (macro and microscopic), chemical (determination of quercetin content by the HPLC method) and functional (determination of quercetin antioxidant activity by DPPH^• assay) stability of the lamellar liquid crystalline formulation containing flavonoid was evaluated when stored at 4 ± 2 °C; 30 ± 2 °C/70 ± 5% RH (relative humidity) and 40 ± 2 °C/70 ± 5% RH during 12 months. The lamellar liquid crystalline structure of the formulation was maintained during the experiment, however chemical and functional stability results showed a great influence of the storage period in all conditions tested. A significant decrease in quercetin content (approximately 40%) was detected during the first month of storage and a similar significant loss in antioxidant activity was detected after 6 months. The remaining flavonoid content was unchanged during the final 6 months of the experimental period. The results suggest possible interactions between quercetin and the liquid crystalline formulation, which could inhibit or reduce the quercetin activity incorporated in the system. In conclusion, the present study demonstrated that incorporation of quercetin (1%) did not affect the liquid crystalline structure composed of vitamin E TPGS/IPM/PG–H₂O (1:1) at 63.75/21.25/15 (w/w/w). Nevertheless, of the total quercetin incorporated in the system only 60% was free to act as an antioxidant.     

Monarch butterfly oviposition on swallow-worts (Vincetoxicum spp.)

As black swallow-wort, Vincetoxicum nigrum L. Moench, and pale swallow-wort, V. rossicum (Kleopow) Barbar., spread throughout the northeastern United States and southern Canada, there is concern about the impact of these invasive plants on populations of the native North American monarch butterfly Danaus plexippus L. Recent laboratory studies in New York and Ontario, Canada, have found little or no oviposition by monarch butterflies on the two Vincetoxicum species. In Rhode Island, we found 10.5-21.7% oviposition on Vincetoxicum species relative to common milkweed Asclepias syriaca L. in choice tests and 11.9-20.3% in no-choice tests in 2 yr of laboratory testing. These results were supported by field cage trials where monarchs given a choice between V. nigrum and A. syriaca laid 24.5% of their eggs on V. nigrum. In surveys of three pasture fields in Rhode Island where relative coverage of A. syriaca exceeded that of V. nigrum by a 0.77:0.23 ratio, 15.4% of monarch eggs were found on V. nigrum plants. In V. nigrum stands with very little A. syriaca (6.25 stems/ha), monarch egg density on V. nigrum was found to be over five times greater than in the three mixed pasture fields. In none of our laboratory or field evaluations was there any survival of monarch larvae on Vincetoxicum species. It seems that in Rhode Island, Vincetoxicum species serve as an oviposition sink for monarch butterflies. These findings suggest that East Coast butterflies may differ in host selection from those in central New York and southern Ontario, Canada.     

A consensus list of microsatellite markers for olive genotyping

Cultivar identification is a primary concern for olive growers, breeders, and scientists. This study was aimed at examining the SSR markers retrieved from the literature and currently used in olive study, in order to select those most effective in characterizing the olive accessions and to make possible the comparison of data obtained by different laboratories. Olive microsatellite profiles were assessed by four independent laboratories, which analyzed 37 pre-selected SSR loci on a set of 21 cultivars. These SSR markers were initially tested for their reproducibility, power of discrimination and number of amplified loci/alleles. Independent segregation was tested for each pair of SSRs in a controlled cross and the allelic error rate was quantified. Some of them were finally selected as the most informative and reliable. Most of the alleles were sequenced and their sizes were determined. Profiles of the reference cultivars and a list of alleles with their sizes obtained by sequencing are reported. Several genetic parameters have been analysed on a larger set of cultivars allowing for a deeper characterization of the selected loci. Results of this study provide a list of recommended markers and protocols for olive genotyping as well as the allelic profile of a set of reference cultivars that would be useful for the establishment of a universal database of olive accessions.     

Naringenin Inhibits Superoxide Anion-Induced Inflammatory Pain: Role of Oxidative Stress,Cytokines, Nrf-2 and the NO?cGMP?PKG?KATPChannel Signaling Pathway

In the present study, the effect and mechanism of action of the flavonoid naringenin were evaluated in superoxide anion donor (KO₂)-induced inflammatory pain in mice. Naringenin reduced KO₂-induced overt-pain like behavior, mechanical hyperalgesia, and thermal hyperalgesia. The analgesic effect of naringenin depended on the activation of the NO−cGMP−PKG−ATP-sensitive potassium channel (K_ATP) signaling pathway. Naringenin also reduced KO₂-induced neutrophil recruitment (myeloperoxidase activity), tissue oxidative stress, and cytokine production. Furthermore, naringenin downregulated KO₂-induced mRNA expression of gp91phox, cyclooxygenase (COX)-2, and preproendothelin-1. Besides, naringenin upregulated KO₂-reduced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) mRNA expression coupled with enhanced heme oxygenase (HO-1) mRNA expression. In conclusion, the present study demonstrates that the use of naringenin represents a potential therapeutic approach reducing superoxide anion-driven inflammatory pain. The antinociceptive, anti-inflammatory and antioxidant effects are mediated via activation of the NO−cGMP−PKG−K_ATP channel signaling involving the induction of Nrf2/HO-1 pathway.     

Allele mining for stress tolerance genes in Oryza species and related germplasm

Allele mining exploits the deoxyribonucleic acid (DNA) sequence of one genotype to isolate useful alleles from related genotypes. The international project to sequence the genome of Oryza sativa L cv. Nipponbare will make allele mining possible for all genes of rice and possibly related cereals. We used a rice calmodulin gene, a rice gene encoding a late embryogenesis-associated protein, and salt-inducible rice gene to optimize the polymerase chain reaction (PCR) for allele mining of stress tolerance genes on identified accessions of rice and related germplasm. Two sets of PCR primers were designed for each gene. Primers based on the 5′ and 3′ untranslated region of genes were found to be sufficiently conserved so as to be effective over the entire range of germplasm in rice for which the concept of allelism is applicable. However, the primers based on the adjacent amino (N) and carboxy (C) termini amplify additional loci.     

Inter-population variation of carotenoids in Galápagos land iguanas (Conolophus subcristatus)

Carotenoids have received much attention from biologists because of their ecological and evolutionary implications in vertebrate biology. We sampled Galápagos land iguanas (Conolophus subcristatus) to investigate the types and levels of blood carotenoids and the possible factors affecting inter-population variation. Blood samples were collected from populations from three islands within the species natural range (Santa Cruz, Isabela, and Fernandina) and one translocated population (Venecia). Lutein and zeaxanthin were the predominant carotenoids found in the serum. In addition, two metabolically modified carotenoids (anhydrolutein and 3'-dehydrolutein) were also identified. Differences in the carotenoid types were not related to sex or locality. Instead, carotenoid concentration varied across the localities, it was higher in females, and it was positively correlated to an index of body condition. Our results suggest a possible sex-related physiological role of xanthophylls in land iguanas. The variation in the overall carotenoid concentration between populations seems to be related to the differences in local abundance and type of food within and between islands.     

Identification of a DNA fragment that increases mitotic stability of episomal linear DNAs in Leishmania major

The centromere is a specialized region of eukaryotic chromosomes, the site of kinetochore formation, spindle attachment and regulation of chromosome segregation during mitotic and meiotic cell divisions. To identify sequences which increase mitotic stability and/or act as potential centromeres in Leishmania major, we first generated libraries of Leishmania linear artificial chromosomes (LACs) bearing 30 kb inserts of randomly selected genomic DNAs. These were introduced into parasites, and then their stability was assessed following a period of 10 passages of growth in the absence of selective pressure. Approximately 80% of the 108 transfectants tested lost their LACs promptly and only 20% of the recombinants were retained; of these six showed strong but partial stability (maintained in 30-46% of cells). Mapping and sequencing of one clone (cSC10), which confers the highest degree of maintenance, revealed the presence of a sequence that was found within another stable episome, and which is dispersed in the genome of L. major. The implications of these data to the possible mechanisms of chromosomal maintenance are discussed.