Ligninolytic activity of tropical rainforest basidiomycetes

A total of 116 strains of Brazilian tropical rainforest basidiomycetes were evaluated in terms of their ability to oxidize the dye rhemazol brilliant blue R (RBBR) and guaiacol. Laccase and peroxidase activities were detected by the drop test using solutions of α-naphthol and pyrogallol, respectively. RBBR and guaiacol oxidation occurred in 96.6 and 87.1% of the strains tested, respectively. One hundred strains oxidized both substrates. In the drop test, most strains presented laccase (96.6%) and peroxidase (92.2%) activity. The quick screening method used here can be useful to identify ligninolytic fungal strains to be used in various biotechnological applications. This revised version was published online in November 2006 with corrections to the Cover Date.     

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

Summary The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to -galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229 Ile) and Val274 (Asp274 Val) substitutions over the N-terminal His4l (Arg4l His) substitution, and the intraallelic dominance of Thr45 (Arg45 Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88 Leu) and Arg256 (Pro256 Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Va1288 (Asp288 Val), and the double mutant was susceptible to activation by benzoates. The results suggest that intramolecular interactions between the C- and N-terminal regions of XylS are critical for activation of the regulator by the effector.     

The relative impact of lichen symbiotic partners to repeated copper uptake

The relative impact of lichen photobiont and mycobiont was evaluated by submitting nine lichen species with: (i) different photobiont types; (ii) different lichen growth forms; and (iii) different nutrients, pH, humidity preferences; to a range of Cu concentrations (μM) supplied in repeated cycles to simulate the natural process of uptake under field conditions. The physiological performance of the photosystem II photochemical reactions was measured using F_v/F_m and the metabolic activity of the mycobiont was evaluated using ergosterol and intracellular K-loss as indicators. Lichens with higher cation exchange capacity showed higher intracellular Cu uptake and their ecology seemed to be associated with low-nutrient environments. Thus the wall and external matrix, mainly characteristic of the mycobiont partner, cannot be ignored as the first site of interaction of metals with lichens. No common intracellular Cu concentration threshold was found for the physiological impacts observed in the different species. Most physiological effects of Cu uptake in sensitive lichens occurred for intracellular Cu below 200 μg/g dw whereas more tolerant species were able to cope with intracellular Cu at least 3 times higher. Cyanobacterial lichens showed to be more sensitive to Cu uptake than green-algal lichens. Within the Trebouxia lichens, different species showed different sensitivities to Cu uptake, suggesting that the mycobiont may change the microenvironment close to the photobiont partner providing different degrees of protection. Despite the fact that the photobiont is the productive partner, the metabolic activity of the mycobiont of lichen species adapted to environments rich in nutrients, showed to be more sensitive to Cu uptake than the photochemical performance of the photobiont.     

Advantages and limitations in the use of hairy root cultures for the production of tropane alkaloids: Use of anti-auxins in the maintenance of normal root morphology

Summary Brugmansia candida hairy roots, obtained by infection withAgrobacterium rhizogenes LBA 9402, exhibit, after subculturing in liquid media, a tendency towards dedifferentiation. It has been found that the following strategies can be applied to inhibit this dedifferentiation and preserve normal root morphology: (a) lowering both the mineral and sucrose concentration in the media employed so as to diminish osmotic stress (a condition to which these roots appear to be particularly susceptible); (b) employing antiauxins in appropriate concentrations; and (c) maintaining the hairy roots on solid media prior to use in production processes in liquid media. The first strategy suggested does not favor alkaloid productivity, but in this case a two-step method could be attempted: biomass with normal root morphology could be obtained in a first stage using low sucrose concentrations, and in a second stage, sucrose could be increased in order to achieve higher productivity. In all the clones ofB. candida obtained, alkaloid production was biased towards scopolamine.     

Association between IGF2 and CYP21 gene polymorphisms and characteristics of economic interest in Nellore cattle

We analyzed two single nucleotide polymorphisms (SNPs) of the IGF2 and CYP21 genes in Nellore cattle participating in the Brazilian Animal Breeding Program. The SNPs were found in exon 6 of the IGF2 (insulin-like growth factor 2) gene (RFLP/MboII) as well as in the promoter region of the CYP21 (steroid 21-hydroxylase) gene (RFLP/HpaII) of these animals. The TC heterozygotes were significantly more frequent than CC and TT homozygotes in the RFLP/MboII polymorphism. The T allele was significantly more frequent than the C allele in RFLP/HpaII polymorphism. This population was found to be in Hardy-Weinberg equilibrium for these SNPs. Association of these polymorphisms with expected progeny differences of reproductive and productive traits was investigated, but proved to be significant only for DP550 (expected progeny differenced for weight at 365 days - IGF2 - RFLP/MboII) and DP450 (expected progeny differenced for weight at 450 days - CYP21 - RFLP/HpaII). This is the first study on the occurrence of these two polymorphisms in this Zebu breed of cattle. A total of 147 Nellore animals participating in the Breeding Program of the Nellore Breed (PMGRN) under the management of the National Association of Breeders and Researchers (ANCP) in the city of Ribeir?o Preto were analyzed.     

A new methodology combining PCR, cloning, and sequencing of clones discriminated by RFLP for the study of microbial populations: application to an UASB reactor sample

This work describes a methodology combining DNA extraction, polymerase chain reaction amplification with primers targeting 16S ribosomal RNA genes, cloning, and sequencing of clones previously analyzed by restriction fragment length polymorphism (RFLP), which can be applied to study the microbial diversity in a given habitat. The methodology allows the minimization of the sequencing effort, which is particularly relevant when analyzing large numbers of clones. The methodology does not require particularly skilled personnel and can easily be adaptable to the molecular characterization of virtually any particular microbial population, provided that both adequate primers and suitable restriction enzymes for RFLP analysis of the clone library have been chosen. An example of application is presented, in which a sample taken from a continuously operating upflow anaerobic sludge blanket reactor was analyzed. RFLP analysis of the initial 162 clones with HaeIII allowed the identification of only 28 distinct profiles. As expected, identical RFLP profiles corresponded to identical nucleotide sequences.     

Interpretability of bi-level variable selection methods

Variable selection is usually performed to increase interpretability, as sparser models are easier to understand than full models. However, a focus on sparsity is not always suitable, for example, when features are related due to contextual similarities or high correlations. Here, it may be more appropriate to identify groups and their predictive members, a task that can be accomplished with bi-level selection procedures. To investigate whether such techniques lead to increased interpretability, group exponential LASSO (GEL), sparse group LASSO (SGL), composite minimax concave penalty (cMCP), and least absolute shrinkage, and selection operator (LASSO) as reference methods were used to select predictors in time-to-event, regression, and classification tasks in bootstrap samples from a cohort of 1001 patients. Different groupings based on prior knowledge, correlation structure, and random assignment were compared in terms of selection relevance, group consistency, and collinearity tolerance. The results show that bi-level selection methods are superior to LASSO in all criteria. The cMCP demonstrated superiority in selection relevance, while SGL was convincing in group consistency. An all-round capacity was achieved by GEL: the approach jointly selected correlated and content-related predictors while maintaining high selection relevance. This method seems recommendable when variables are grouped, and interpretation is of primary interest.