The importance of being kinked: role of Pro residues in the selectivity of the helical antimicrobial peptide P5

Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against drug‐resistant bacteria. Many of them kill bacteria by perturbing their membranes but exhibit no significant toxicity towards eukaryotic cells. The identification of the features responsible for this selectivity is essential for their pharmacological development. AMPs exhibit few conserved features, but a statistical analysis of an AMP sequence database indicated that many α‐helical AMPs surprisingly have a helix‐breaking Pro residue in the middle of their sequence. To discriminate among the different possible hypotheses for the functional role of this feature, we designed an analogue of the antimicrobial peptide P5, in which the central Pro was deleted (analogue P5Del). Pro removal resulted in a dramatic increase of toxicity. This was explained by the observation that P5Del binds both charged and neutral membranes, whereas P5 has no appreciable affinity towards neutral bilayers. CD and simulative data provided a rationalization of this behavior. In solution P5, due to the presence of Pro, attains compact conformations, in which its apolar residues are partially shielded from the solvent, whereas P5Del is more helical. These structural differences reduce the hydrophobic driving force for association of P5 to neutral membranes, whereas its binding to anionic bilayers can still take place because of electrostatic attraction. After membrane binding, the Pro residue does not preclude the attainment of a membrane‐active amphiphilic helical conformation. These findings shed light on the role of Pro residues in the selectivity of AMPs and provide hints for the design of new, highly selective compounds. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Purification, crystallization, and preliminary x-ray diffraction studies of the flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607

The flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607 was purified by dye-ligand affinity chromatography. The protein was crystallized from solutions of high ionic strength, and one of the two crystal forms obtained has proven suitable for x-ray diffraction studies. Preliminary analysis showed that these crystals belong to the tetragonal space group 1422. The unit cell dimensions are a = b = 180.7 A; c = 127.9 A. The diffraction pattern extends to better than 3 A resolution. Crystal density measurements are consistent with one enzyme dimer of 2 x 69,000 Da comprising the asymmetric unit. Trypsin treatment of the native enzyme resulted in the removal of 157 amino acids at the N terminus. After purification, the remaining fragment (amino acids 158-631), which is still fully active in vitro, could be crystallized under the same conditions as native enzyme. Twinning problems, however, did not allow complete analysis of these crystals.     

Intergenic spacers of rRNA genes in three species of theCynareae (Asteraceae)

Intergenic spacers of the rRNA genes of three species of theCynareae tribe:Cynara cardunculus subsp.scolymus (artichoke),Onopordum acanthium, andO. illyricum were cloned in the plasmid pGEM-7zf(+). Detailed restriction mapping and partial sequencing of the IGSs were carried out. The structural analysis showed a clear diversity betweenCynara andOnopordum, while a high degree of homology was found between the twoOnopordum spp. In all three species a fragment of about 450 bp from the 5 end of 18S to the Acc I site with a high sequence homology was present. Nucleotide sequences upstream from the above mentioned Acc I site show a gradual decrease of homology betweenCynara andOnopordum.     

The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13.

Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.     

Studio preliminare sulla tipologia della vegetazione sommersa del Canale di Sicilia e isole vicine

Abstract

Preliminary studies on the marine phythobenthic communities in the Straits of Sicily and adjacent islands. — The results of algological researches carried out by the Gruppo di Algologia of the S.B.I. in the Straits of Sicily and adjacent islands are reported. 288 species have been collected, 275 of which have been identified: 160 were Rhodophyta, 62 Phaeophyta, 32 Chlorophyta, 16 Cyanophyta and 2 Angiospermae. The vegetational research leads to the following conclusions: 1 - the harbour areas (Pantelleria, Malta: loc. 1 and 11) show a nitrophile vegetation, particulary near La Valletta where an almost pure population of Pterocladia pinnata is found; 2 - there are important biogeographical differences between Pelagie islands (Lampedusa, Linosa) and Pantelleria. In fact the former ones have an algal flora composed mostly of eastern mediterranean species, whilst the latter shows strong affinities with the north-african basin, as is demonstrated by the rich well developed belt of Cystoseira sedoides; 3 - several species of atlantic origin, such as Callophyllis laciniata, Peyssonnelia coriacea, P. inamoena and Castagnea chordariaeformis, have been found on the banks (loc. 3, 6, 7).     

Increased GH responsiveness to dopamine receptor stimulation in alcohol addicts during the late withdrawal syndrome

In humans the release of growth hormone (GH) elicited by dopamine (DA) and DA agonists may represent a reliable model to assess change in sensitivity of DA receptors. We now report that in chronic alcoholics, 4–7 days after the suspension of alcohol consumption, the increase of GH response to DA infusion was higher than that seen in non alcoholic volunteers. The specificity of this GH response to DA administration was demonstrated by the use of domperidone, a novel peripheral antagonist of DA receptors. These results suggest the development of hyper-responsiveness of DA receptors involved in the control of GH secretion in chronic alcoholics during the later phases of the “withdrawal syndrome”.     

Sympathovagal Balance,Nighttime Blood Pressure,and QT Intervals in Normotensive Obese Women

Objective : We describe associations among the heart‐rate‐corrected QT (QTc) interval, QTc dispersion (QTc‐d), circadian BP variation, and autonomic function in obese normotensive women and the effect of sustained weight loss. Research Methods and Procedures : In 71 obese (BMI = 37.14 ± 2.6 kg/m²) women, 25 to 44 years of age, circadian BP variations (24‐hour ambulatory BP monitoring), autonomic function (power spectral analysis of RR interval oscillations), and cardiac repolarization times (QTc‐d and QTc interval) were recorded at baseline and after 1 year of a multidisciplinary program of weight reduction. Results : Compared with nonobese age‐matched women (n = 28, BMI = 23 ± 2.0 kg/m²), obese women had higher values of QTc‐d (p < 0.05) and QTc (p < 0.05), an altered sympathovagal balance (ratio of low‐frequency/high‐frequency power, p < 0.01), and a blunted nocturnal drop in BP (p < 0.01). In obese women, QTc‐d and the QTc interval correlated with diastolic nighttime BP (p < 0.01) and sympathovagal balance (p < 0.01). Waist‐to‐hip ratio, free fatty acids, and plasma insulin levels correlated with QT intervals and reduced nocturnal drops in both systolic and diastolic BP and sympathovagal balance (p < 0.01). After 1 year, obese women lost at least 10% of their original weight, which was associated with decrements of QTc‐d (p < 0.02), the QTc interval (p < 0.05), nighttime BP (p < 0.01), and sympathovagal balance (p < 0.02). Discussion : Sustained weight loss is a safe method to ameliorate diastolic nighttime BP drop and sympathetic overactivity, which may reduce the cardiovascular risk in obese women.     

Do environmental changes or juvenile competition act as mechanisms of species displacement in crayfishes?

The Big Creek Crayfish, Orconectes peruncus, is native to the St. Francis River drainage in Missouri, USA and is often absent where the introduced Woodland Crayfish, Orconectes hylas, has established. We performed a field experiment to determine whether effects of current abiotic conditions and interspecific competition with O. hylas were responsible for displacement of O. peruncus from parts of their former range. We examined growth and survival of juvenile male O. peruncus exposed to juvenile male O. hylas in enclosures at two sites in the former range of O. peruncus. Enclosures contained 8 (low density) or 16 individuals (high density) and had O. peruncus only (control) or both species (interspecific treatment). Juvenile O. peruncus were able to survive and grow in portions of their former range, implicating biotic versus abiotic factors in the displacement of O. peruncus. Survival rates of O. peruncus did not differ among treatments at either site. Orconectes peruncus showed significant growth in all treatments and interspecific effects were not greater than intraspecific effects on O. peruncus growth rates. High-density treatments showed significantly reduced O. peruncus growth rates compared to low-density treatments, except in Carver Creek interspecific treatments. When considered in the context of previous studies examining the effects of O. hylas on O. peruncus, results suggest that neither direct competition between juvenile males of the two species or abiotic change are responsible for the decreased range of O. peruncus. Additional research is required to determine the mechanism(s) driving the displacement of O. peruncus.     

A Multicenter,Randomized, Controlled Trial of Osteopathic Manipulative Treatment on Preterms

BackgroundDespite some preliminary evidence, it is still largely unknown whether osteopathic manipulative treatment improves preterm clinical outcomes.ResultsA total of 695 newborns were randomly assigned to either the study group (n= 352) or the control group (n=343). A statistical significant difference was observed between the two groups for the primary outcome (13.8 and 17.5 days for the study and control group respectively, p<0.001, effect size: 0.31). Multivariate analysis showed a reduction of the length of stay of 3.9 days (95% CI -5.5 to -2.3, p<0.001). Furthermore, there were significant reductions with treatment as compared to usual care in cost (difference between study and control group: 1,586.01€; 95% CI 1,087.18 to 6,277.28; p<0.001) but not in daily weight gain. There were no complications associated to the intervention.ConclusionsOsteopathic treatment reduced significantly the number of days of hospitalization and is cost-effective on a large cohort of preterm infants.