Cloning sequencing and expression of the gene for cytochrome P450meg,the steroid-15β-monooxygenase from Bacillus megaterium ATCC 13368

A 4.3 kb EcoRI fragment carrying the gene for cytochrome P450meg, the steroid-15β-monooxygenase from Bacillus megaterium ATCC 13368, was cloned and completely sequenced. The gene codes for a protein of 410 amino acids and was expressed in Escherichia coli and B. subtilis. Protein extracts from the recombinant E. coli strains were able to hydroxylate corticosteroids in the 15β position when supplemented with an extract from a P450- mutant of B. megaterium ATCC 13368 as a source of megaredoxin and megaredoxin reductase. In contrast, 15β-hydroxylation was obtained in vitro and in vivo without the addition of external electron transfer proteins, when cytochrome P450meg was produced in B. subtilis 168. Protein extracts from nonrecombinant B. subtilis 168 could also support the in vitro hydroxylation by cytochrome P450meg produced in E. coli.     

LEVELS OF NOREPINEPHRINE AND DOPAMINE IN MOUSE BRAIN REGIONS FOLLOWING MICROWAVE INACTIVATION-RAPID POST-MORTEM DEGRADATION OF STRIATAL DOPAMINE IN DECAPITATED ANIMALS

—The effects of 2 methods of killing on norepinephrine and dopamine in mouse brain regions were examined. One method utilized decapitation, while the other method utilized heating with microwave irradiation concentrated on the head. The norepinephrine and dopamine contents of the cerebellum, medulla-pons, midbrain, diencephalon, hippocampus, corpus striatum, and cerebral cortex were determined by methods using liquid chromatography with electrochemical detection. Dopamine content in striatum was also quantitated by the method of gas chromatography with mass fragmentography. A significantly lower value for decapitated animals, as compared to the microwave heated group, was found only for dopamine exclusively in the striatum. Activities of the enzymes tyrosine hydroxylase, DO PA decarboxylase, monoamine oxidase, and catechol-o-methyltransferase in the striatum were also examined. These enzymes were totally inactivated by the microwave heating, except catechol-o-methyltransferase which was decreased approx 80%. These results support either (1) the existence of a substantial pool of dopamine in the striatum with a very rapid turnover rate or (2) a decapitation-related release and destruction of striatal dopamine. Measurements of 3-methoxytyramine in the striatum exhibit post-mortem increases corresponding to the decreases of dopamine. Use of the rapid tissue enzyme inactivation technique suggests that in vivo levels of this O-methylated dopamine metabolite are an order of magnitude lower than the results normally obtained after killing by decapitation.     

Optimizing seeding density of fast‐growing native trees for restoring the Brazilian Atlantic Forest

Direct seeding is a promising method for reducing restoration costs, but methodological adjustments are still needed to reduce the uncertainties to achieve a desired seedling density in the field. Here, we investigated the technical approaches and outcomes of direct seeding of fast‐growing native trees for cost‐effective restoration of the Brazilian Atlantic Forest. Sixteen tree species were manually sown at three seeding densities in planting lines prepared with a subsoiler, in two experimental areas, which were weeded with hoes and had leaf‐cutter ants controlled with insecticide baits. Seedling density was monitored for 30, 90, and 180 days after sowing. No substantial change in tree density was observed 30 days after sowing, thus allowing fast corrective actions to adjust tree density. Only a minor proportion of the sown viable seeds resulted in established seedlings at 180 days (4–12% for the community; approximately 25% for the species with the best performance). However, tree density was high (6,000 on average; approximately 1,400–13,000 trees/ha) and allowed an effective canopy development. Overall, seedling density was linearly and positively associated with seeding density, was highly influenced by the species used, and was higher in the soil with higher sum of bases. Buying seeds would be, for most species, less costly than buying nursery‐grown seedlings for achieving the expected tree densities in the field. These results evidence the potential of direct seeding for reducing restoration costs, as well as the need to select species with better performance and adjust seeding densities to optimize the use of this method.     

Predictive Factors for Sustained Virological Response after Treatment with Pegylated Interferon α-2a and Ribavirin in Patients Infected with HCV Genotypes 2 and 3

Background

Previous trials have often defined genotype 2 and 3 patients as an “easy to treat” group and guidelines recommend similar management.

Aims

The present study looks for differences between the two genotypes and analyzes predictive factors for SVR.

Methods

Prospective, community-based cohort study involving 421 physicians throughout Germany. The analysis includes 2,347 patients with untreated chronic HCV genotype 2 (n = 391) and 3 (n = 1,956) infection treated with PEG-IFN α-2a plus ribavirin between August 2007 and July 2012.

Results

When compared with genotype 2 patients, those with genotype 3 were younger, had a shorter duration of infection, lower values of total cholesterol, LDL cholesterol and BMI, a higher frequency of drug use as infection mode and male gender (p<0.0001, respectively), and a higher APRI score (p<0.005). SVR was higher in genotype 2 when compared with genotype 3 (64.7% vs. 56.9%, p = 0.004). By multivariate analysis of genotype 2 patients, low baseline γ -GT and RVR predicted SVR. In genotype 3 age ≤45 years, cholesterol>130 mg/dl, a low APRI score, and a γ-GT ≥3-times ULN, RVR, and RBV starting dose were associated with SVR by multivariate analysis.

Conclusions

The present study corroborates that liver fibrosis is more pronounced in genotype 3 vs. 2. SVR is higher in genotype 2 versus genotype 3 partly because of follow-up problems in genotype 3 patients, in particular in those infected by drug use. Thus, subgroups of genotype 3 patients have adherence problems and need special attention also because they often have significant liver fibrosis.

Trial Registration

Verband Forschender Arzneimittelhersteller e.V., Berlin, Germany ML21645 ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02106156","term_id":"NCT02106156"}}NCT02106156     

The rise of the Brazilian Network for Ecological Restoration (REBRE): what Brazilian restorationists have learned from networking

As ecological restoration is gaining importance worldwide, researchers, practitioners, and interested people are beginning to self‐organize, in order to exchange knowledge and experiences in this growing area. Latin America has recently shown some examples, and the Brazilian Network for Ecological Restoration (REBRE) is one of them. Working on a nonhierarchical basis, its members, some of whom are important actors in the proposing and implementing of public policies, are able to freely express their doubts and share experiences, thereby contributing to legislative improvements, developing relevant restoration initiatives, and adopting novel approaches and paradigms. Through its three main communication platforms (website, Google Groups, and Facebook), REBRE has reached more than 2,300 members, and although a more equitable distribution through Brazilian regions should still be reached, it is gradually becoming stronger and more representative. In this scenario, REBRE will act with its branch organization, SOBRE (Brazilian Society for Ecological Restoration), to host the next Society for Ecological Restoration Conference, to be held in Brazil, in 2017, exchanging relevant experiences and welcoming restorationists from all over the world.     

Production of human adrenocorticotropin by cleavage of alkaline-phosphatase-derived fusion proteins containing repetitive recognition sequences for collagenases

Recombinant plasmids coding for fusion proteins which consist of human adrenocorticotropin joined to N-terminal sequences of Escherichia coli alkaline phosphatase via collagenase-sensitive linkers were constructed and used for the production of these proteins by transformed E. coli cells. It was shown that repetitive linkers of the form -Gly-(Pro-Xaa-Gly)n-Pro- with n greater than or equal to 2 were cleaved by clostridiopeptidase A (Clostridium histolyticum) by orders of magnitude faster than corresponding nonrepetitive sequences (n = 1). The C-terminal cleavage product was Gly-Pro-adrenocorticotropin which could be converted to the authentic hormone by dipeptidyl peptidase IV. On the basis of these enzymatic reactions a procedure for the preparation of pure adrenocorticotropin was developed. Derivatives of alkaline phosphatase containing similar repetitive linker sequences were cleaved by clostridiopeptidase A as efficiently as the adrenocorticotropin fusion proteins.