Heterologous Expression of the Alba Protein from the Hyperthermophilic Archaeon Aeropyrum Pernix

Nucleic acid binding proteins have important roles in DNA and RNA packaging, stabilisation and repair, and in gene regulation, and they are therefore essential for all organisms. All of the known hyperthermophiles have at least one DNA sequence encoding for the Alba proteins. The Alba proteins are small (approximately 10 kDa), DNA-binding, basic proteins that appear to partly compensate for the lack of histones in the archaea Aeropyrum pernix and other hyperthermophiles. Two sequences of these potential histone counterparts, the Alba proteins, were identified in the Aeropyrum pernix genome (APE1832.1 and APE1823). By using a wide range of experimental techniques and by examining several combinations of expression systems the expression of recombinant Alba1 and Alba2 proteins was optimized. Co-expression of both of the Alba proteins was needed when isolating recombinant Alba2. The purification of both recombinant Alba1 and Alba2 His-tagged proteins were simplyfied in satisfactory yield. The electrophoretic mobility shift assay demonstrated the ability of the Alba1 and Alba2 proteins from Aeropyrum pernix to bind DNA. (doi: 10.5562/cca1772

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

AbstractOstreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins

Interaction of α-Synuclein with Negatively Charged Lipid Membranes Monitored by Surface Plasmon Resonance

Aggregation of presynaptic protein α-synuclein is implicated in the development of Parkinson’s disease. Interaction of α-synuclein with lipid membranes appears to be critical for its physiological and pathological roles. Anionic lipids trigger conformational transition of α-synuclein from its natively disordered into an α-helical structure. Here we used surface plasmon resonance (SPR) to determine the affinities of α-synuclein for the small unilamellar vesicles composed of anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) or 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. α-Synuclein bound in a concentration dependent manner to equimolar mixtures of POPC/POPS and POPC/DPPG vesicles. The affinity of α-synuclein for POPC/POPS was ~3-fold higher than for POPC/DPPG. These results indicate that headgroup charge is not the only factor contributing to α-synuclein-membrane association. This work is licensed under a Creative Commons Attribution 4.0 International License

EFFECTS OF pH ON HUE ANGLE AND VISIBLE ABSORPTION MAXIMA OF CYANIDIN

As a major sub-group of flavonoids, anthocyanins are water soluble plant pigments responsible for the blue, purple and red color of many plant tissues. They occur primarily as glycosides of their respective aglycone anthocyanidin. Cyanidin is the most abundant anthocyanidin in fruits and vegetables (50%). In recent years, the interest in the properties and stability of anthocyanin extracts has increased. However, there remain little data in the literature relating to the properties and stability of pure anthocyanins, and especially of the anthocyanidins. The aim of the study was to compare under various pH conditions visible absorbance maxima (λmax-vis) with the corresponding hue angle (hab) values of cyanidin as a color reference. Thus cyanidin aqueous solution was subjected to spectroscopic and colorimetric study to examine influence of pH value on hab and λmax-vis. The cyanidin had reddish nuances at the lowest pH values. By stepwise pH increase the colour of cyanidin is gradually changed toward magenta and lilac tones, and then with further pH increase to more reddish nuances. In alkaline region cyanidin showed yellow green tones, which is gradually changed toward yellow nuances with further pH increases.&nbsp

PHYSICO-CHEMICAL PARAMETERS OF FRESH STANDARD AND LIGHT MAYONNAISE

Mayonnaise is an oil-in-water (O/W) emulsion and is widely consumed as a traditional seasoning due to its creamy mouth feel and special flavor. The standard mayonnaise contains 65%–80% fat, which contributes to its texture, appearance, flavor, and shelf life. However, demand for low-fat mayonnaise has been rising. To produce light mayonnaise, fat from the basic formula for the preparation of mayonnaise shall be replaced by other ingredients while preserving its viscosity, texture, mouth feeling, taste and flavour to a highest possible degree. The sensory and physiochemical properties of mayonnaise are significantly affected by the elimination of fat. The objective of this study was to investigate the relationship between the type of mayonnaise and physico-chemical parameters - pH value, acid value and water activity. The experiment is based on analysis of mayonnaise of three Serbian manufacturers, standard and light mayonnaise for each of them. The samples of the fresh mayonnaise coming from the same manufacturer and of different mayonnaise type (standard and light) did not differ in terms of their pH values. Each manufacturer had specific pH value of mayonnaise. Type of mayonnaise had a very high impact on acid values and water activities. Acid values of fresh light mayonnaises were higher than of standard one regardless of the manufacturer. Fresh standard mayonnaises from all three manufacturers had similar water activities. The same was true for light mayonnaise. Fresh light mayonnaise had higher values of water activity compared to standard mayonnaise regardless of the manufacturer

White Hop Shoot Production in Slovenia: Total Phenolic, Microelement and Pesticide Residue Content in Five Commercial Cultivars

Berba bijelih izdanaka hmelja mogla bi biti opravdana ako bi se dokazalo njihovo pozitivno djelovanje na zdravlje ljudi. Svrha je ovoga rada bila odrediti utjecaj kultivara i godine uzgoja hmelja na ukupni udjel fenola, antioksidacijski potencijal, te udjele mikroelemenata i ostataka pesticida. Biomasa biljaka bitno se razlikovala ovisno o kultivaru (3,1-7,1 g suhe tvari biljke) i godini uzgoja (2009.-2001.). Ukupni maseni udjel fenola, izražen u ekvivalentima klorogene kiseline u suhoj masi, bio je od 0,60 do 1,80 mg/g, ovisno o kultivaru i godini uzgoja. Sposobnost uklanjanja slobodnih radikala uzoraka ubranih u razdoblju od 2010. do 2012. iznosila je od 11 do 19 µg ekvivalenata klorogene kiseline. Reduktivna sposobnost ispitana FRAP metodom bila je <0,01; a bitni utjecaj kultivara (pC≤0,05) i godine uzgoja (py≤0,05) opažen je samo u uzorcima iz 2012. godine. U bijelim izdancima kultivara hmelja Humulus lupulus ‘Dana’ ispitana je prisutnost tragova mikroelemenata i potencijalno aktivnih spojeva zaostalih nakon primjene pesticida. Udjel je cinka u suhoj masi izdanaka hmelja bio vrlo malen (4 mg/kg), a u tlu ga je bilo ispod granice detekcije. Udjel bakra je u izdancima također bio vrlo malen (2,3 mg/kg), dok ga je u tlu bilo 100 mg/kg, što je ispod kritične razine (300 mg/kg). Udjeli svih 182 aktivnih sastojaka iz ostataka pesticida upotrijebljenih u prethodnim sadnjama bili su ispod granica detekcije. Iz rezultata možemo zaključiti da bijeli izdanci hmelja imaju bolja antioksidacijska svojstva od šišarki i lišća hmelja, te da ne sadržavaju ostatke pesticida.Harvesting of white hop shoots might be justified if they can be shown to be beneficial to human health. The aim of the present study is to determine the effects of hop cultivars and year of production on total phenolics, antioxidant potential, microelements and pesticide residues. Biomass per plant was highly variable among the cultivars (3.1-7.1 g dry mass per plant) and depended on hop cultivar and year (2009-2011). Total phenolics as chlorogenic acid equivalents (CAE) on dry mass basis varied from 0.60 to 1.80 mg/g, and showed significant effects across hop cultivar and year. The radical scavenging activities of the samples collected in years 2010-2012 ranged from 11 to 19 μg CAE. Ferric reducing activity was <0.01, with significantly different effects across hop cultivars (pC≤0.05) and year (py≤0.05) observed only in 2012. Traces of microelements and potentially active compounds from the use of pesticides in white hop shoots of Humulus lupulus ‘Dana’ were analysed. The content of zinc in the hop shoots on dry mass basis was very low (4 mg/kg), and it was below the limit of detection in the soil. The content of copper in the hop shoots was also very low (2.3 mg/kg), while in the soil it was below the critical emission (100 vs 300 mg/kg, respectively). All 182 active ingredients from the residues of the previously used pesticides were below the limits of detection. It can be concluded that these white hop shoots are better antioxidants than hop cones and hop leaves, and that they do not contain any pesticide residues

Characterization of ciprofloxacin binding to the linear single- and doublestranded DNA

Abstract The binding of ciprofloxacin to natural and synthetic polymeric DNAs was investigated at different solvent conditions using a combination of spectroscopic and hydrodynamic techniques. In 10 mM cacodylate buffer (pH 7.0) containing 108.6 mM Na + , no sequence preferences in the interaction of ciprofloxacin with DNA was detected, while in 2 mM cacodylate buffer (pH 7.0) containing only 1.7 mM Na + , a significant binding of ciprofloxacin to natural and synthetic linear double-stranded DNA was observed. At low ionic strength of solution, ciprofloxacin binding to DNA duplex containing alternating AT base pairs is accompanied by the largest enhancement in thermal stability (e.g

Effects of selected essential oils on the growth and production of ochratoxin A by Penicillium verrucosum

Eterična olja origana (Origanum vulgare L.), mete (Mentha piperita L.), janeža (Foeniculum vulgare Mill.), in borovih (Abies alba Mill.) iglic in storžev, ter njihove sestavine timol, karvakrol, mentol in anisaldehid smo testirali kot snovi z protiglivno aktivnostjo proti plesnim vrste Penicillium verrucosum. Najnižje minimalne inhibitorne koncentracije (MICs) smo določili eteričnemu olju origana, ter karvakrolu, timolu, in mentolu. Rast plesni vrste P. verrucosum je bila popolnoma inhibirana po 21 dneh inkubacije v prisotnosti ½ MIC timola (0,0625 mg mL-1), medtem ko so eterično olje origana (½ MIC, ¼ MIC), timol (¼ MIC) in karvakrol (½ MIC, ¼ MIC) inhibirali rast plesni od 8,3 % do 70.8 %. Dodatek mentola v koncentracijah ¼ MIC in ½ MIC (0.1875 mg mL-1 in 0.3750 mg mL-1) pa ni imel inhibitornega vpliva na rast plesni vrste P. verrucosum. Popolno inhibicijo tvorbe ohratoksina A smo določili le v primeru, ko je bila tudi rast teh plesni popolnoma inhibirana (pri ½ MIC timola). Največjo inhibicijo tvorbe ohratoksina A (96,9 %) po 21 dnevih inkubacije smo določili pri ¼ MIC timola (0.0313 mg mL-1). Pospešeno tvorbo ohratoksina A (od 13,9 % do 28,8 %) pa smo določili pri ¼ MIC eteričnega olja origana (0.2930 mg mL-1) in ½ MIC karvakrola (0.1953 mg mL-1). Protiglivni vplivi so bili odvisni od testirane snovi, njene koncentracije ter časa interakcij med protiglivno snovjo in plesnimi vrste P. verrucosum. Rezultati nakazujejo možnosti uporabe eteričnega olja origana v živilih namesto kemijskih konzervansov. Ker pa so živila kompleksna okolja v katerih je potrebno upoštevati več dejavnikov, so potrebne nadaljnje raziskave.Essential oils from oregano (Origanum vulgare L.), mint (Mentha piperita L.), fennel (Foeniculum vulgare Mill.), and pine (Abies alba Mill.) needles and cones, and their active substances thymol, carvacrol, menthol, and anisaldehyde were tested for antifungal activity against Penicillium verrucosum. The lowest minimal inhibitory concentrations (MICs) were achieved for essential oil of oregano, followed by carvacrol, thymol, and menthol. These antifungal components were further investigated, as the main aim of our study was to elucidate the effect of natural antifungals on ochratoxin A production. During 21 days of exposure, the growth of P. verrucosum, and subsequently the production of ochratoxin A, was fully inhibited by thymol at ½ MIC (0.0625 mg mL-1), but menthol at ¼ and ½ MIC (0.1875 and 3750 mg mL-1) showed no growth inhibition. After 21 days of incubation, the greatest inhibitory effect on ochratoxin production (inhibition was 96.9 %) was also achieved with thymol at ¼ MIC (0.0313 mg mL-1). Essential oil of oregano (¼ MIC, 0.2930 μL mL-1) and carvacrol (½ MIC, 0.1953 μL mL-1) stimulate production of ochratoxin A at 13.9 % to 28.8 %, respectively. The observed antifungal effects depended on the agent, the concentration used, and the time of interaction between the agent and P. verrucosum. Our results indicate the possibility of using oregano essential oil as a substitute for artificial preservatives in certain foods, but further research is needed

Haloarchaeal communities in the crystallizers of two adriatic solar salterns

Abstract: Solar salterns operate only for short dry periods of the year in the north shore of the Adriatic Sea because of its relatively humid and cold Mediterranean climate. In a previous paper, we showed that the NaCl precipitation ponds (crystallizers) of Northern Adriatic Seovlje salterns have different haloarchaeal populations from those typically found in dry and hot climates such as Southern Spain. To check whether there is a common pattern of haloarchaeal diversity in these less extreme conditions, diversity in crystallizers of other Adriatic solar salterns in Ston, Croatia was ascertained by molecular and culture methods. In addition, the cultivation approach was used to further describe haloarchaeal diversity in both salterns. Over the period of two solar salt collection seasons, isolates related to species of the genera Haloferax, Haloarcula, and Haloterrigena were recovered from both salterns. Within the same sampling effort, relatives of the genus Halorubrum and a Natrinema-like isolate were cultivated from Slovenian Seovlje salterns while Halobacterium related isolates were obtained from the Croatian Ston salterns. Concurrent with our previous findings, a library of Croatian saltern crystallizer PCR-amplified 16S rRNA genes was dominated by sequences related to the genus Halorubrum. The microbial community structure was similar in both salterns but diversity indices showed greater values in Slovenian salterns when compared with Croatian salterns. Key words: 16S rRNA, Haloarchaea, saltern, hypersaline, halophiles. Résumé : Les salines solaires ne fonctionnent que pendant de brèves périodes de temps sec dans l&apos;année sur la rive nord de la mer Adriatique à cause de son climat méditerranéen relativement humide et froid. Dans un article précédent, nous avons démontré que les bassins de précipitation de NaCl (cristalliseurs) des salines de Seovlje de l&apos;Adriatique Nord renferment des populations haloarchéenes différentes de celles normalement retrouvées dans des climats chauds et secs tels que le sud de l&apos;Espagne. Pour vérifier s&apos;il existe une configuration commune à la diversité haloarchéene dans ces conditions moins extrêmes, nous avons décrit la diversité dans les cristalliseurs d&apos;autres salines solaires adriatiquesles salines Ston en Croatie -par des méthodes moléculaires et de culture. De plus, l&apos;approche de culture fut employée afin de décrire davantage la diversité haloarchéene des deux salines. Sur une période de deux saisons de collecte de sel solaire, des isolats apparentés à des espèces des genres Haloferax, Haloarcula et Haloterrigena ont été recueillis des deux salines. À l&apos;issue d&apos;un même échantillonnage, des parents du genre Halorubrum et un isolat semblable à Natrinema ont été cultivés à partir de la saline slovène de Seovlje alors que des isolats apparentés à Halobacterium ont été obtenus des salines Croates de Ston. En conformité avec nos découvertes précédentes, la banque de gènes de ARNr 16S amplifiés par PCR du cristalliseur salin Croate était dominée par des séquences apparentées au genre Halorubrum. La structure de la communauté microbienne était semblable dans les deux salines mais les indices de diversité ont démontré des valeurs supérieures dans les salines slovènes comparativement aux salines croates

Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR)

This study aimed to design and functionally characterize peptide mimetics of the nanobody (Nb) related to the β2-adrenergic receptor (β2-AR) (nanobody-derived peptide, NDP). We postulated that the computationally derived and optimized complementarity-determining region 3 (CDR3) of Nb is sufficient for its interaction with receptor. Sequence-related Nb-families preferring the agonist-bound active conformation of β2-AR were analysed using the informational spectrum method (ISM) and β2-AR:NDP complexes studied using protein-peptide docking and molecular dynamics (MD) simulations in conjunction with metadynamics calculations of free energy binding. The selected NDP of Nb71, designated P3, was 17 amino acids long and included CDR3. Metadynamics calculations yielded a binding free energy for the β2-AR:P3 complex of ΔG = (−7.23 ± 0.04) kcal/mol, or a Kd of (7.9 ± 0.5) μM, for T = 310 K. In vitro circular dichroism (CD) spectropolarimetry and microscale thermophoresis (MST) data provided additional evidence for P3 interaction with agonist-activated β2-AR, which displayed ~10-fold higher affinity for P3 than the unstimulated receptor (MST-derived EC50 of 3.57 µM vs. 58.22 µM), while its ability to inhibit the agonist-induced interaction of β2-AR with β-arrestin 2 was less evident. In summary, theoretical and experimental evidence indicated that P3 preferentially binds agonist-activated β2-AR. © 2019, The Author(s)