Purification of human plasma fibronectin by double affinity chromatography on gelatin and heparin-Trisacryl

In this work the human plasma fibronectin was purified by affinity chromatography using a tandem column system. The first affinity column was filled with gelatin-Trisacryl whereas the second one contained heparin-Trisacryl. This double affinity chromatography demonstrated its high efficiency in term of purity and yield. Several analytical methods (electrophoresis, immunoelectrophoresis, F.P.L.C. and adhesion assay on cultured eucaryotic cells) evidenced in fact the high purity of the preparation as well as its biological behaviour in term of cell adhesion and spreading. The performances of the sorbents used facilitate the scaling up when large quantities of FNP are needed.     

Properties of succinylated wheat-germ agglutinin.

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 +/- 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 microgram/ml with both lectins and the concentrations of N-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins. The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins. The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.     

Functional integrity of intravenous immunoglobulin following irradiation with a virucidal dose of gamma radiation.

Although intravenous immunoglobulins (IVIG) and other plasma therapeutics have had a relatively good safety record, improved methods for viral clearance are constantly being evaluated and incorporated into new manufacturing processes. Gamma irradiation has been used routinely to assure sterility of healthcare products and medical devices, but it has not been applied successfully as a viral inactivation method for biologics. We examine whether virucidal doses of gamma irradiation (50 kGy) can be delivered to a manufacturing intermediate form of IVIG, a fractionated plasma paste, with negligible effect on structural and functional integrity of purified IgG product. Immunoglobulins from paste were examined for radiation-induced damage by SDS-PAGE and ELISAs utilizing viral antigens specific for rubella, CMV and mumps. Fc domain integrity was assessed by immunoblotting, quantitatively comparing the binding of irradiated and non-irradiated materials to cell surface Fcgamma receptors, and by employing quantitative RT-PCR to study the kinetics of accumulation of mRNA for the immune modulatory cytokines IL-1alpha, IL-1beta, IL-4, IL-8, IFNgamma, and TNFalpha. The results demonstrate that Fab and Fc domains of IVIG remain essentially intact and functional after gamma irradiation to virucidal doses, suggesting that this method could be used to enhance the safety of IVIG products.     

High temporal and individual variation in the prevalence and intensity of chytrid infection in the southernmost Leaf Frog of the genus Pithecopus (Anura,Phyllomedusidae)

Hydrobiologia - Pithecopus rusticus is an endemic amphibian restricted to the type locality, in southern Brazil, and possibly endangered to extinction, due to habitat degradation. However, an...     

Antioxidant effects of different extracts from Melissa officinalis, Matricaria recutita and Cymbopogon citratus

Considering the important role of oxidative stress in the pathogenesis of several neurological diseases, and the growing evidence of the presence of compounds with antioxidant properties in the plant extracts, the aim of the present study was to investigate the antioxidant capacity of three plants used in Brazil to treat neurological disorders: Melissa officinalis, Matricaria recutita and Cymbopogon citratus. The antioxidant effect of phenolic compounds commonly found in plant extracts, namely, quercetin, gallic acid, quercitrin and rutin was also examined for comparative purposes. Cerebral lipid peroxidation (assessed by TBARS) was induced by iron sulfate (10 μM), sodium nitroprusside (5 μM) or 3-nitropropionic acid (2 mM). Free radical scavenger properties and the chemical composition of plant extracts were assessed by 1′-1′ Diphenyl-2′ picrylhydrazyl (DPPH) method and by Thin Layer Chromatography (TLC), respectively. M. officinalis aqueous extract caused the highest decrease in TBARS production induced by all tested pro-oxidants. In the DPPH assay, M. officinalis presented also the best antioxidant effect, but, in this case, the antioxidant potencies were similar for the aqueous, methanolic and ethanolic extracts. Among the purified compounds, quercetin had the highest antioxidant activity followed by gallic acid, quercitrin and rutin. In this work, we have demonstrated that the plant extracts could protect against oxidative damage induced by various pro-oxidant agents that induce lipid peroxidation by different process. Thus, plant extracts could inhibit the generation of early chemical reactive species that subsequently initiate lipid peroxidation or, alternatively, they could block a common final pathway in the process of polyunsaturated fatty acids peroxidation. Our study indicates that M. officinalis could be considered an effective agent in the prevention of various neurological diseases associated with oxidative stress.     

Protocol for the purification of proteins from biological extracts for identification by mass spectrometry

When a protein signal is selected by mass spectrometry as being a potential biomarker, it is necessary to formally identify it. This process involves separation of the protein in question and its identification by either peptide fingerprinting or tandem mass spectrometry sequencing. In the following pages, a simple and rapid protocol is described. Basically, the protocol consists of an initial rational selection of a few sorbents followed by alignment of these as a series of columns to obtain the purified target protein. This preparation is then submitted to electrophoresis, the band is excised and the trypsin digest is analyzed by either mass spectrometry (mass fingerprinting approach) or by LC-MS/MS (sequencing). The development of the process takes only a few days. Experimental data for the isolation and identification of proteins are discussed and two examples are shown.     

Effects of Self‐Conditioning Techniques (Self‐Hypnosis) in Promoting Weight Loss in Patients with Severe Obesity: A Randomized Controlled Trial

Objective

The usefulness of the rapid‐induction techniques of hypnosis as an adjunctive weight‐loss treatment has not been defined. This randomized controlled trial evaluated whether self‐conditioning techniques (self‐hypnosis) added to lifestyle interventions contributed to weight loss (primary outcome), changes in metabolic and inflammatory variables, and quality of life (QoL) improvement (secondary outcomes) in severe obesity.

Methods

Individuals (with BMI = 35‐50 kg/m²) without organic or psychiatric comorbidity were randomly assigned to the intervention (n = 60) or control arm (n = 60). All received exercise and behavioral recommendations and individualized diets. The intervention consisted of three hypnosis sessions, during which self‐hypnosis was taught to increase self‐control before eating. Diet, exercise, satiety, QoL, anthropometric measurements, and blood variables were collected and measured at enrollment and at 1 year (trial end).

Results

A similar weight loss was observed in the intervention (?6.5 kg) and control (?5.6 kg) arms (β = ?0.45; 95% CI: ?3.78 to 2.88; P = 0.79). However, habitual hypnosis users lost more weight (?9.6 kg; β = ?10.2; 95% CI: ?14.2 to ?6.18; P < 0.001) and greatly reduced their caloric intake (?682.5 kcal; β = ?643.6; 95% CI: ?1064.0 to ?223.2; P = 0.005) in linear regression models. At trial end, the intervention arm showed lower C‐reactive protein values (β = ?2.55; 95% CI: ?3.80 to ?1.31; P < 0.001), higher satiety (β = 19.2; 95% CI: 7.71‐30.6; P = 0.001), and better QoL (β = 0.09; 95% CI: 0.02‐0.16; P = 0.01).

Conclusions

Self‐hypnosis was not associated with differences in weight change but was associated with improved satiety, QoL, and inflammation. Indeed, habitual hypnosis users showed a greater weight loss.