Reversible two-step unfolding of heme-human serum albumin: A 1H-NMR relaxometric and circular dichroism study

Human serum albumin (HSA) participates in heme scavenging, the bound heme turning out to be a reactivity center and a powerful spectroscopic probe. Here, the reversible unfolding of heme-HSA has been investigated by H-1-NMR relaxometry, circular dichroism, and absorption spectroscopy. In the presence of 6 equiv of myristate ( thus fully saturating all available fatty acid binding sites in serum heme-albumin), 1.0 M guanidinium chloride induces some unfolding of heme-HSA, leading to the formation of a folding intermediate; this species is characterized by increased relaxivity and enhanced dichroism signal in the Soret region, suggesting a more compact heme pocket conformation. Heme binds to the folding intermediate with K-d = (1.2 +/- 0.1) x 10(-6) M. In the absence of myristate, the conformation of the folding intermediate state is destabilized and heme binding is weakened [K-d = (3.4 +/- 0.1) x 10(-5) M]. Further addition of guanidinium chloride ( up to 5 M) brings about the usual denaturation process. In conclusion, myristate protects HSA from unfolding, stabilizing a folding intermediate state in equilibrium with the native and the fully unfolded protein, envisaging a two-step unfolding pathway for heme-HSA in the presence of myristate

Comparison between in vitro chemical and ex vivo biological assays to evaluate antioxidant capacity of botanical extracts

The anti-oxidative activity of plant-derived extracts is well-known and confers health-promoting effects on functional foods and food supplements. Aim of this work is to evaluate the capability of two different assays to predict the real biological antioxidant efficiency. At this purpose, extracts from five different plant-derived matrices and commercial purified phytochemicals were analyzed for their anti-oxidative properties by using well-standardized in vitro chemical method (TEAC) and an ex vivo biological assay. The biological assay, a cellular membrane system obtained from erythrocytes of healthy volunteers, is based on the capability of phytochemicals treatment to prevent membrane lipid peroxidation under oxidative stress by UV-B radiation. Plant extracts naturally rich in phenols with different structure and purified phytochemicals showed different in vitro and ex vivo antioxidant capacities. A high correlation between phenolic contents of the plant-derived extracts and their ability to prevent oxidative injuries in a biological system was found, thus underlying the relevance of this class of metabolites in preventing oxidative stress. On the other hand, a low correlation between the antioxidant capacities was shown between in vitro and ex vivo antioxidant assay. Moreover, data presented in this work show how food complex matrices are more effective in preventing oxidative damages at biological level than pure phytochemicals, even if for these latter, the antioxidant activity was generally higher than that observed for food complex matrices

Hα3: an Hα imaging survey of HI selected galaxies from ALFALFA. VI. The role of bars in quenching star formation from z = 3 to the present epoch

A growing body of evidence indicates that the star formation rate per unit stellar mass (sSFR) decreases with increasing mass in normal main-sequence star-forming galaxies. Many processes have been advocated as being responsible for this trend (also known as mass quenching), e.g., feedback from active galactic nuclei (AGNs), and the formation of classical bulges. In order to improve our insight into the mechanisms regulating the star formation in normal star-forming galaxies across cosmic epochs, we determine a refined star formation versus stellar mass relation in the local Universe. To this end we use the Hα narrow-band imaging follow-up survey (Hα3) of field galaxies selected from the HI Arecibo Legacy Fast ALFA Survey (ALFALFA) in the Coma and Local superclusters. By complementing this local determination with high-redshift measurements from the literature, we reconstruct the star formation history of main-sequence galaxies as a function of stellar mass from the present epoch up to z = 3. In agreement with previous studies, our analysis shows that quenching mechanisms occur above a threshold stellar mass Mknee that evolves with redshift as ∝ (1 + z)2. Moreover, visual morphological classification of individual objects in our local sample reveals a sharp increase in the fraction of visually classified strong bars with mass, hinting that strong bars may contribute to the observed downturn in the sSFR above Mknee. We test this hypothesis using a simple but physically motivated numerical model for bar formation, finding that strong bars can rapidly quench star formation in the central few kpc of field galaxies. We conclude that strong bars contribute significantly to the red colors observed in the inner parts of massive galaxies, although additional mechanisms are likely required to quench the star formation in the outer regions of massive spiral galaxies. Intriguingly, when we extrapolate our model to higher redshifts, we successfully recover the observed redshift evolution for Mknee. Our study highlights how the formation of strong bars in massive galaxies is an important mechanism in regulating the redshift evolution of the sSFR for field main-sequence galaxies

Proteomics of saliva: personal experience

The salivary proteome is a complex protein mixture resulting from the activity of salivary glands with the contribution of other components that form the oral environment such as oral tissues and micro-organisms. For diagnosis purposes, saliva collection has the great advantage of being an easy and non-invasive technique. Human saliva proteomics have proven to be a novel approach in the search for protein biomarkers for detection of different local and systemic diseases. Currently, more than 1400 salivary proteins have been identified. In the last few years, our research group has extensively studied the salivary proteomics in order to analyse the salivary composition, investigating the major families of proteins present in human and mammalian saliva, the post-translational modifications, the different contributions of glands, the physiological and pathological modifications of saliva. The aim of this report is to present our personal experience in salivary proteomics. In conclusion, salivary proteome analysis represents an important field both for diagnosis and monitoring of various diseases and could be considered a novel approach to prevention of various pathological conditions

Reaction rate of NaOCl in contact with bovine dentine: effect of activation, exposure time, concentration and pH

Abstract Macedo RG, Wesselink PR, Zaccheo F, Fanali D, van der Sluis LWM. Reaction rate of NaOCl in contact with bovine dentine: effect of activation, exposure time, concentration and pH. International Endodontic Journal. Aim To determine the influence of activation method (ultrasound or laser), concentration, pH and exposure time on the reaction rate (RR) of NaOCl when in contact with dentinal walls. Methodology The walls from standardized root canals in bovine incisors were exposed to a standardized volume of sodium hypochlorite (NaOCl) with different concentrations (2% and 10%), pH (5 and 12) and exposure times (1 and 4 min). Two irrigation protocols were tested: passive ultrasonic irrigation or laser activated irrigation with no activation as the control. The activation interval lasted 1 min followed by a rest interval of 3 min with no activation. The RR was determined by measuring the iodine concentration using an iodine/thiosulfate titration method. Results Exposure time, concentration and activation method influenced the reaction rate of NaOCl whereas pH did not. Conclusions Activation is a strong modulator of the reaction rate of NaOCl. During the rest interval of 3 min, the consumption of available chlorine increased significantly. This effect seems to be more pronounced after irrigant activation by laser. pH did not affect the reaction rate of 2% NaOCl

Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography

Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-β-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of l-MTX and its enantiomeric impurity d-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10−7 M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of l-MTX. Furthermore, l-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples

Characterization of the binding sites of the anticancer ruthenium(III) complexes KP1019 and KP1339 on human serum albumin via competition studies

Indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019) and its Na+ analogue (KP1339) are two of the most prominent non-platinum antitumor metal complexes currently undergoing clinical trials. After intravenous administration, they are known to bind to human serum albumin (HSA) in a noncovalent manner. To elucidate their HSA binding sites, displacement reactions with the established site markers warfarin and dansylglycine as well as bilirubin were monitored by spectrofluorimetry, ultrafiltration-UV-vis spectrophotometry, and/or capillary zone electrophoresis. Conditional stability constants for the binding of KP1019 and KP1339 to sites I and II of HSA were determined, indicating that both Ru(III) compounds bind to both sites with moderately strong affinity (log K (1)' = 5.3-5.8). No preference for either binding site was found, and similar results were obtained for both metal complexes, demonstrating low influence of the counter ion on the binding event

Aloe barbadensis: how a miraculous plant becomes reality

Aloe barbadensis Miller is a plant that is native to North and East Africa and has accompanied man for over 5,000 years. The aloe vera plant has been endowed with digestive, dermatological, culinary and cosmetic virtues. On this basis, aloe provides a range of possibilities for fascinating studies from several points of view, including the analysis of chemical composition, the biochemistry involved in various activities and its application in pharmacology, as well as from horticultural and economic standpoints. The use of aloe vera as a medicinal plant is mentioned in numerous ancient texts such as the Bible. This multitude of medicinal uses has been described and discussed for centuries, thus transforming this miracle plant into reality. A summary of the historical uses, chemical composition and biological activities of this species is presented in this review. The latest clinical studies involved in vivo and in vitro assays conducted with aloe vera gel or its metabolites and the results of these studies are reviewed