Involvement of a cytochrome P450 monooxygenase in thaxtomin A biosynthesis by Streptomyces acidiscabies

The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as (1)H- and (13)C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.     

Three sets of weight training superior to 1 set with equal intensity for eliciting strength

The purpose of this study was to compare single and multiple sets of weight training for strength gains in recreationally trained individuals. Sixteen men (age = 21 +/- 2.0) were randomly assigned to 1 set (S-1; n = 8) or 3 set (S-3; n = 8) groups and trained 3 days per week for 12 weeks. One repetition maximum (1RM) was recorded for bench press and leg press at pre-, mid-, and posttest. Subjects trained according to daily undulating periodization (DUP), involving the bench press and leg press exercises between 4RM and 8RM. Training intensity was equated for both groups. Analysis of variance with repeated measures revealed statistically significant differences favoring S-3 in the leg press (p < 0.05, effect size [ES] = 6.5) and differences approaching significance in the bench press (p = 0.07, ES = 2.3). The results demonstrate that for recreationally trained individuals using DUP training, 3 sets of training are superior to 1 set for eliciting maximal strength gains.     

Improved immunogenicity of an immunodominant epitope of the HER-2/neu protooncogene by alterations of MHC contact residues

The HER-2/neu (HER-2) oncogene is expressed in normal epithelial surfaces at low levels and overexpressed in several types of tumors. The low immunogenicity against this self tumor Ag can be improved by developing epitopes with amino acid replacements in their sequences. In this study, three HER-2/neu.369 (HER-2.369) analogue peptides, produced by modifying both anchor positions by introducing L, V, or T at position 2 and V at the C terminus, were analyzed for their capacity to induce CTLs in vitro from human PBMC and in vivo in HLA-A2.1/Kb transgenic mice. One of the analogues (HER-2.369 V2V9) sensitized target cells for HER-2-specific recognition by human CTLs and induced specific CTLs in vitro at 100-fold lower concentrations than the HER-2.369 wild-type epitope. These CTLs were also able to recognize the wild-type epitope and HER-2-expressing tumors in an MHC-restricted manner. Furthermore, a 100-fold lower amount of the HER-2.369 V2V9 analogue compared with the wild-type epitope was required to induce CTLs in HLA-A2.1/Kb transgenic mice. However, the V2V9 analogue demonstrated only marginally better binding to the MHC class I A2 allele compared with wild type. To establish thermodynamic parameters, we developed radiolabeled F3*Y analogues from both the HER-2.369 epitope and the V2V9 analogue. Our results indicate that the high biological activity of the HER-2.369 V2V9 epitope is associated with a slower dissociation kinetic profile, resulting in an epitope with greater HLA-A2 stability.     

Atrial natriuretic peptide influence on nitric oxide system in kidney and heart

Atrial natriuretic peptide (ANP) and nitric oxide (NO) induce diuresis, natriuresis and diminish vascular tone. Our previous studies showed NO system is involved in ANP hypotensive effect. The aim was to investigate ANP effects on renal and cardiac NO-synthase (NOS) activity. Rats were divided into two groups: group I, infused with saline (1 h, 0.05 ml/min); group II, received ANP bolus (5 microg/kg)+ANP infusion (1 h, 0.2 microg/kg x min). NADPH-diaphorase activity (NADPH-d) was determined in kidney and heart. NOS catalytic activity was determined in renal medulla and cortex and cardiac atria and ventricle by measuring the conversion of l-[U(14)C]-arginine to l-[U(14)C]-citrulline. In group I, NOS activity was determined in basal conditions and plus 1 microM ANP and in group II, NOS activity was determined in basal conditions. NADPH-d was higher in group II than in group I in glomeruli, proximal tubule, cortical and medullar collecting duct, right atria and left ventricle. NOS activity was increased by in vitro ANP addition and, in vivo, ANP infusion in all the studied tissues. ANP treatment increases renal and cardiac NO synthesis. This effect would be independent on the hemodynamic changes induced by ANP. The activation of NO pathway would be one of the mechanisms involved in diuretic, natriuretic and hypotensive effects of ANP.     

Physical fitness and job performance of firefighters

Accurate correlations between a wide range of physical fitness measures and occupational demands are needed in order to identify specific fitness tests and training needs for firefighters. Twenty professional firefighters performed numerous fitness and job-related performance tests. Pearson product moment correlations were performed to identify the relationship between fitness components and job performance. Significant correlations (p <0.05) with job performance were identified for total fitness (r = -0.62), bench press strength (r = -0.66), hand grip strength (r = -0.71), bent-over row endurance (r = -0.61), bench press endurance (r = -0.73), shoulder press endurance (r = -0.71), bicep endurance (r = -0.69), squat endurance (r = -0.47), and 400-m sprint time (r = 0.79). It is apparent that firefighting taxes virtually all aspects of physical fitness. These data can help the exercise specialist choose appropriate tests and prescribe specific fitness programs for firefighters. Traditional firefighter exercise programs focusing mainly on cardiovascular fitness should be replaced with physical conditioning programs that address all components of fitness.     

Characterisation of COPD heterogeneity in the ECLIPSE cohort

Background

Chronic obstructive pulmonary disease (COPD) is a complex condition with pulmonary and extra-pulmonary manifestations. This study describes the heterogeneity of COPD in a large and well characterised and controlled COPD cohort (ECLIPSE).

Methods

We studied 2164 clinically stable COPD patients, 337 smokers with normal lung function and 245 never smokers. In these individuals, we measured clinical parameters, nutritional status, spirometry, exercise tolerance, and amount of emphysema by computed tomography.

Results

COPD patients were slightly older than controls and had more pack years of smoking than smokers with normal lung function. Co-morbidities were more prevalent in COPD patients than in controls, and occurred to the same extent irrespective of the GOLD stage. The severity of airflow limitation in COPD patients was poorly related to the degree of breathlessness, health status, presence of co-morbidity, exercise capacity and number of exacerbations reported in the year before the study. The distribution of these variables within each GOLD stage was wide. Even in subjects with severe airflow obstruction, a substantial proportion did not report symptoms, exacerbations or exercise limitation. The amount of emphysema increased with GOLD severity. The prevalence of bronchiectasis was low (4%) but also increased with GOLD stage. Some gender differences were also identified.

Conclusions

The clinical manifestations of COPD are highly variable and the degree of airflow limitation does not capture the heterogeneity of the disease.     

Enzyme dynamics along the reaction coordinate: critical role of a conserved residue

Conformational flexibility of the enzyme architecture is essential for biological function. These structural transitions often encompass significant portions of the enzyme molecule. Here, we present a detailed study of functionally relevant RNase A dynamics in the wild type and a D121A mutant form by NMR spin-relaxation techniques. In the wild-type enzyme, the dynamic properties are largely conserved in the apo, enzyme-substrate, and enzyme-product complexes. In comparison, mutation of aspartic acid 121 to alanine disrupts the timing of active-site dynamics, the product-release step, and global conformational changes, indicating that D121 plays a significant role in coordinating the dynamic events in RNase A. In addition, this mutation results in 90% loss of catalytic activity despite the absence of direct participation of D121 in the chemical reaction or in interactions with the substrate. These data suggest that one role of this conserved residue is to facilitate important millisecond protein dynamics.     

Cost-effective large-scale expression of proteins for NMR studies

We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and ¹³C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling of ²H, ¹³C and ¹⁵N using auto-induction or isopropyl-β-d-1-thiogalactopyranoside-induction are described, with primary focus on minimal isotope consumption and high reproducibility of protein expression. These protocols are based on high cell-density fermentation, but the key procedures are easily transferred to shake flask cultures.