Rocco Rossano

Extracting and purifying R-phycoerythrin from Mediterranean red algae Corallina elongata Ellis & Solander

R-Phycoerythrin (R-PE) is a protein acting as a photosynthetic accessory pigment in red algae (Rodophyta). This protein has gained importance in many biotechnological applications in food science, immunodiagnostic, therapy, cosmetics, protein and cell labelling, and analytical processes. In this paper we report on a new, one step procedure for the extraction and purification of R-PE from a new source: the Mediterranean red algae Corallina elongata Ellis & Solander. This red algae contains mainly R-PE and is suitable for the production in culture. No other contaminating phycobiliproteins could be detected in the extracts. The method we propose for the purification is based on the use of hydroxyapatite, a chromatographic resin that can be produced in the laboratory at very low cost and can be used batch-wise with large amounts of extracts, alternative to chromatography, and therefore can be scaled up. Both the yield and the purity of R-PE are very good.

Nutrition Facts in Multiple Sclerosis

The question whether dietary habits and lifestyle have influence on the course of multiple sclerosis (MS) is still a matter of debate, and at present, MS therapy is not associated with any information on diet and lifestyle. Here we show that dietary factors and lifestyle may exacerbate or ameliorate MS symptoms by modulating the inflammatory status of the disease both in relapsing-remitting MS and in primary-progressive MS. This is achieved by controlling both the metabolic and inflammatory pathways in the human cell and the composition of commensal gut microbiota. What increases inflammation are hypercaloric Western-style diets, characterized by high salt, animal fat, red meat, sugar-sweetened drinks, fried food, low fiber, and lack of physical exercise. The persistence of this type of diet upregulates the metabolism of human cells toward biosynthetic pathways including those of proinflammatory molecules and also leads to a dysbiotic gut microbiota, alteration of intestinal immunity, and low-grade systemic inflammation. Conversely, exercise and low-calorie diets based on the assumption of vegetables, fruit, legumes, fish, prebiotics, and probiotics act on nuclear receptors and enzymes that upregulate oxidative metabolism, downregulate the synthesis of proinflammatory molecules, and restore or maintain a healthy symbiotic gut microbiota. Now that we know the molecular mechanisms by which dietary factors and exercise affect the inflammatory status in MS, we can expect that a nutritional intervention with anti-inflammatory food and dietary supplements can alleviate possible side effects of immune-modulatory drugs and the symptoms of chronic fatigue syndrome and thus favor patient wellness.

Effect of Cryopreservation on Sea Bass Sperm Proteins

Abstract In the present study we used two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to verify whether the protein expression of sea bass sperm was affected by the cryopreservation procedure. The protein profiles differed between fresh and frozen-thawed semen as revealed by visual inspection and by image analysis software. We identified 163 spots in fresh sperm; among these, 13 were significantly decreased and 8 were absent in two-dimensional gel obtained with cryopreserved sperm. Five of these spots were analyzed with MALDI-TOF, but only three showed a significant match in the databases used in bio-informatics analysis (PeptIdent, Mascot, and MS-Fit). In particular, spot 5 showed homology with a novel protein of zebrafish (similar to SKB1 of human and mouse), spot 13 showed homology with amphibian G1/S-specific cyclin E2, and spot 20 showed homology with the hypothetical protein DKFZp566A1524 of Brachidanio rerio. The present work shows that the use of the cryopreservation procedure causes the degradation of sperm proteins and among these, two could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm.

Spectrophotometric assay using o-phtaldialdehyde for the determination of transglutaminase activity on casein

In this work, the possibility of using a simple and quick method was tested for determining transglutaminase activity on casein using a spectrophotometric assay. The enzyme activity was estimated on the basis of the decrease of o-phthaldialdehyde reactive e-amino groups of lysine following the formation of isopeptide bonds. The lysine residues involved in the formation of isopeptide bonds when the reaction reaches its plateau are equal to 0.126 μmol per mg of casein. This value results as equal to 0.205 μmol per mg of casein when N-carbobenzoxy-glutaminyl-glycine is added to the reaction medium as a small size acyl group donor. The electrophoretic analysis of the reaction products emphasised a different kinetic formation of casein polymers with the two substrate solutions used. This proposed method has resulted as accurate, with a mean coefficient of variation of 4.6%.

Inhibitory Effect of Polyunsaturated Fatty Acids on MMP-9 Release from Microglial Cells—Implications for Complementary Multiple Sclerosis Treatment

We investigated whether polyunsaturated fatty acids (PUFA), which might be a useful complementary therapy among patients with multiple sclerosis (MS), are able to modulate matrix metalloproteinase (MMP) production in microglial cultures. MMPs are myelinotoxic factors. Primary cultures of rat microglia were treated with different doses of omega-3 (ω-3) PUFA or purified fish oil, containing a mixture of ω-3 and ω-6 PUFA, and simultaneously activated by exposure to lipopolysaccharide (LPS). Culture supernatants were subjected to zymography and Western blot analysis for the assessment of MMP-2 and MMP-9 levels. Increased amounts of MMP-9, but not of the constitutively expressed MMP-2, were observed in supernatants from LPS-treated microglia in comparison with non-treated control cells. The treatment with both ω-3 PUFA and fish oil dose-dependently inhibited the LPS-induced production of MMP-9. Our results suggest that a low fat diet supplemented with ω-3 PUFA may become recommended for the well being of MS patients under therapy.

May Diet and Dietary Supplements Improve the Wellness of Multiple Sclerosis Patients? A Molecular Approach

Multiple sclerosis is a complex and multifactorial neurological disease, and nutrition is one of the environmental factors possibly involved in its pathogenesis. At present, the role of nutrition is unclear, and MS therapy is not associated to a particular diet. MS clinical trials based on specific diets or dietary supplements are very few and in some cases controversial. To understand how diet can influence the course of MS and improve the wellness of MS patients, it is necessary to identify the dietary molecules, their targets and the molecular mechanisms involved in the control of the disease. The aim of this paper is to provide a molecular basis for the nutritional intervention in MS by evaluating at molecular level the effect of dietary molecules on the inflammatory and autoimmune processes involved in the disease.

Diversity of stress responses in dairy thermophilic streptococci.

Exponential and stationary phase cells of 56 strains of the dairy Streptococcus species S. thermophilus, S. macedonicus and S. salivarius, were exposed to acid, osmotic, oxidative and heat stresses to investigate the diversity of their responses. Three strains of dairy related streptococci, Lactococcus lactis ATCC11454, Enterococcus faecium DSM20477 and Enterococcus faecalis DSM20478, were included for comparison purposes. Acid and heat adaptation and cross-protection to stress were studied in ten strains with different stress response patterns. Cell death and the changes in protein expression were evaluated by plate counts and Sodium Dodecyl Sulfate Polyacrilamide Gel Electrophoresis, respectively. All strains of all species were highly tolerant of osmotic stress. With a few exceptions, acid and oxidative treatments reduced the number of viable cells by >5 log units but responses to heat stress were more variable. For some, but not all strains, stationary phase cells were more resistant to some or all stresses. Matrix cluster analysis was used to group strains on the basis of their pattern of stress response in seven clusters. Significant associations between the sources of strains and stress resistance were found for acid and oxidative stresses. Adaptation to stress during the exponential phase enhanced the survival of acid and heat stressed cells from 1 to 60,000-folds, but a detrimental effect of adaptation on cell viability was evident for oxidative and osmotic stresses for three strains. Adaptation and entry into the stationary phase resulted in significant changes of protein bands whose estimated molecular masses corresponded with those of proteins (DnaK, GprE, GroEL, and GroES) involved in the general stress response but no statistically significant correlation between stress response and band intensity was evident.

Extraction and immobilization in one step of two β-glucosidases released from a yeast strain of Debaryomyces hansenii

Abstract An extracellular, constitutive, and nonglucose repressed β-glucosidase from a yeast strain of Debaryomyces hansenii was purified and immobilized using a one-step procedure on hydroxyapatite (HTP). Analysis of purified enzyme gave two bands both on SDS gel electrophoresis, native gel electrophoresis, and capillary electrophoresis. The two bands on SDS gels were positive for carbohydrate staining. Their apparent molecular mass was estimated to be 122 and 96 kDa with carbohydrates, and 109 and 81 kDa after carbohydrate removal, respectively. Amino acid analysis of electroblotted bands revealed that the n -terminus was blocked in both cases. Gel slices corresponding to the two bands, as obtained after native gel electrophoresis, were found to be reactive when incubated separately with p-nitro-phenyl-β- d -glucopyranoside (pNPG) as substrate. The Km of the two forms coeluted from HTP in the same fractions was 3.68 ± 0.06 m m . The optimum pH was 5. The immobilized enzyme exhibited a lower activity than the purified free enzymes, but both were much more stable than the enzymes in cell-free supernatant. The two enzyme isoforms in the mixture were only active against few glycosides with β-linkage configuration. Since the HTP-bound enzyme was found to be active, stable, easily separable from the substrate, and reusable, it could be potentially used in its immobilized form for the release of specific-bound aroma in wine and fruit juices.

Effect of inactivation of stress response regulators on the growth and survival of Streptococcus thermophilus Sfi39.

Streptococcus thermophilus is an important dairy starter used for the manufacture of fermented milks and cheeses. As for many other lactic acid bacteria, its survival during propagation and preservation of starter cultures and its performance in dairy fermentations are significantly affected by its ability to cope with many environmental stresses. This, in turn, is dependent on the expression of a several genes, which is often controlled by negative response regulators, and has complex relationships with the control of metabolism. In this paper we describe the effect of growth phase, adaptation and inactivation of genes for stress response regulators (hrcA, ctsR and rr01) on the growth, acid production and stress resistance and intracellular protein patterns in the yoghurt strain S. thermophilus Sfi39. Inactivation of stress response regulators significantly affected growth, acid production in milk and stress tolerance. Although mutants showed an increased resistance during the exponential phase compared to the wild type strain, the effect of the mutations was complex and in some cases mutants were unable to mount an acid- or heat-shock response after exposure to mild heat and acid stresses. Significant differences in intracellular protein patterns analyzed by SDS-PAGE and 2-DE/mass spectrometry were found as a function of growth phase, adaptation and mutation, but a detailed transcriptomic and proteomic study would be needed to provide a mechanistic interpretation of the effect of the mutations and to identify a strategy for the construction of food-grade mutants with improved growth and performances.

Structure-Dependent Inhibition of Gelatinases by Dietary Antioxidants in Rat Astrocytes and Sera of Multiple Sclerosis Patients

We investigated whether polyphenols modulate the expression and activity of the enzymes gelatinases A (MMP-2) and B (MMP-9), involved in the pathogenesis of multiple sclerosis (MS). LPS-activated primary rat astrocytes were treated with the flavonoids quercetin (QRC) and cathechins [green tea extract (GTE)] and the non-flavonoids resveratrol (RSV) and tyrosol/hydroxytyrosol (Oliplus). As assessed by zymography and RT-PCR, RSV and Oliplus, but not QRC and GTE, dose-dependently inhibited the LPS-induced levels and mRNA expression of MMP-2 and MMP-9. By contrast, in cell-free systems direct inhibition of gelatinase activity in MS sera was determined by QRC and GTE, but not by RSV. Oliplus was only partially effective. Our results indicate that the flavonoids and non-flavonoids tested exert their inhibitory effect on MMPs, displaying different mechanisms of action, possibly related to their structure. Therefore, their combined use may represent a powerful tool for the down-regulation of MMPs in the course of MS.