Purification, characterization and cytotoxicity assessment of Ageritin: The first ribotoxin from the basidiomycete mushroom Agrocybe aegerita.

BACKGROUND: Several species belonging to Ascomycota phylum produce extracellular ribonucleases, known as ribotoxins, which exhibit RNase activity through the cleavage of a single phosphodiester bond, located at the universally conserved sarcin/ricin loop of the large rRNA leading to inhibition of protein biosynthesis. Clarifying the structure-function relationship in ribotoxins is interesting for their use in human tumour therapy and in construction of pest resistant transgenic plants. RESULTS: The ribotoxin Ageritin has been isolated for the first time from the Basidiomycetes class. The enzyme, characterized by means of its amino acid composition, N-terminal sequence and a circular dichroism, structurally differs from Ascomycota ribotoxin prototype, although it was able, as α-sarcin, to release a specific α-fragment. However, it does not display aspecific ribonucleolytic activity. Ageritin exerts cytotoxicity and cell death promoting effects towards CNS model cell lines (SK-N-BE(2)-C, U-251 and C6), as vinblastine, a plant alkaloid used in cancer therapy. Moreover, our results indicate that Ageritin initially activates caspase-8, whereas caspase-9 cleavage was not detected, demonstrating the involvement of an extrinsic apoptotic pathway. CONCLUSIONS: Our findings show that Ageritin is the earliest diverging member of the Ascomycota ribotoxin family, suggesting that ribotoxins are more widely distributed among fungi than previously believed. GENERAL SIGNIFICANCE: Ageritin, structurally different from the widely known Ascomycota ribotoxins, with promising anti-cancer properties vs. aggressive brain tumours, has been found from the basidiomycete fungus Agrocybe aegerita. Finally, this finding highlights that the ribotoxin family has divergent members in Basidiomycota phylum, whose structural and functional characterization can give new information on ribotoxin or ribonuclease superfamilies.

Molecular characterization of myoglobin from Sciurus vulgaris meridionalis: Primary structure, kinetics and spectroscopic studies.

Myoglobins (Mbs) are heme-proteins involved in dioxygen storage necessary for metabolic respiration. Mbs are intensely investigated as archetype to investigate structure/function relationship in globular proteins. In this work, the myoglobin from Sciurus vulgaris meridionalis has been for the first time isolated and purified with a high yield and homogeneity. The primary structure characterization has been performed by applying a strategy based on high resolution tandem mass spectrometry. Proximal (position 93, α-helix F8) and distal (position 64, α-helix E7) histidinyl residues as well as most of the amino acid residues (i.e., Leu29, Lys45, Thr67, Val68) involved in the autoxidation mechanism are conserved in the squirrel Mb. The structural and dynamical properties of the squirrel Mb have been also deeply investigated by CD, NMR. Furthermore, molecular dynamics studies of Mbs from different species have been performed. In addition, the functional properties of squirrel Mb have been characterized by determining its autoxidation kinetic and thermal stability in comparison with crested porcupine and reindeer Mbs. Interestingly, a higher autoxidation rate was revealed for squirrel Mb with respect to reindeer and crested porcupine Mbs. Even considering the very similar structural fold, molecular dynamics data show a higher conformational mobility of squirrel Mb with respect to reindeer and crested porcupine.

A new active antimicrobial peptide from PD-L4, a type 1 ribosome inactivating protein of Phytolacca dioica L.: A new function of RIPs for plant defence?

We investigated the antimicrobial activity of PD-L4, a type 1 RIP from Phytolacca dioica. We found that this protein is active on different bacterial strains both in a native and denatured/alkylated form and that this biological activity is related to a cryptic peptide, named PDL440-65, identified by chemical fragmentation. This peptide showed the same antimicrobial activity of full-length protein and possessed, similarly to several antimicrobial peptides, an immunomodulatory effect on human cells. It assumes an alpha-helical conformation when interact with mimic membrane agents as TFE and likely bacterial membranes are a target of this peptide. To date PDL440-65 is the first antimicrobial peptide identified in a type 1 RIP.

Valle Agricola lentil, an unknown lentil (Lens culinaris Medik.) seed from Southern Italy as a novel antioxidant and prebiotic source.

In order to promote 'Valle Agricola' lentil, an autochthonous lentil of the Campania Region, a thorough investigation of its biochemical and nutritional properties has been carried out. The macronutrient content (proteins, carbohydrates and lipids), free and total amino acids, and unsaturated fatty acids were determined. The antioxidant capability of raw 'Valle Agricola' lentils, as well as of boiled ones, was estimated in terms of their total phenol content (TPC), ORAC value, and free radical scavenging capacities using DPPH and ABTS assays. The data obtained evidenced that the boiling process slightly decreased Valle Agricola lentil's antioxidant power. Furthermore, when trypsin and chymotrypsin inhibitory activities were measured, a large decrease of the levels of anti-nutritional factors was estimated. In order to have a phytochemical overview of this autochthonous lentil seed, LC-ESI-MS/MS analysis was applied to raw and boiled lentil extracts. Flavonol glycosides and free flavanols, as well as typical seed prebiotic saccharides, were the most representative constituents.

An improved UPLC method for the detection of undeclared horse meat addition by using myoglobin as molecular marker.

In 2013, following the scandal of the presence of undeclared horse meat in various processed beef products across the Europe, several researches have been undertaken for the safety of consumer health. In this framework, an improved UPLC separation method has been developed to detect the presence of horse myoglobin in raw meat samples. The separation of both horse and beef myoglobins was achieved in only seven minutes. The methodology was improved by preparing mixtures with different composition percentages of horse and beef meat. By using myoglobin as marker, low amounts (0.50mg/0.50g, w/w; ∼0.1%) of horse meat can be detected and quantified in minced raw meat samples with high reproducibility and sensitivity, thus offering a valid alternative to conventional PCR techniques.

Cytotoxic activity of chimeric protein PD-L4UWSCI(tr) does not appear be affected by specificity of inhibition mediated by anti-protease WSCI domain.

In a previously study, a type 1 ribosome inactivating protein (PD-L4) and a wheat subtilisin/chymotrypsin inhibitor (WSCI) were engineered into a chimeric protein (PD-L4UWSCI) that presented in addition to the same properties of both domains an intriguing selective cytotoxic action on murine tumor cells. This finding supported the idea that the protection of C-terminal region of PD-L4 could amplify its cytotoxic action by virtue of a greater resistance to proteases. Several authors indeed revealed that the cytotoxicity of RIPs depends not only on the intracellular routing, but also on the intrinsic resistance to proteolysis. In this regard in the present work we have produced a variant of chimeric protein, named PD-L4UWSCI(tr), changing the inhibitory specificity of WSCI domain. The purpose of this approach was to check if the cytotoxicity of the chimeric protein was altered depending on the properties of protease inhibitor domain or by a different fold of whole protein. Data collected supposedly indicate that WSCI domain contributes to cytotoxicity of chimeric protein exclusively from a structural point of view.

Chemical composition, nutritional value and antioxidant properties of autochthonous Prunus avium cultivars from Campania Region.

During a screening program aimed at the evaluation of antioxidative and antiproliferative properties, as well as nutritional properties of local edible plants, two endemic sweet cherry cultivars ('Del Monte' and 'Della Recca') were of interest. Macronutrient components (proteins, carbohydrates and lipids) of both the cherry cultivars were determined as well as free and total amino acids. Pomological traits were defined. HPLC-ESI/MSn analysis, carried out on phenolic extracts properly prepared by extractive techniques from freeze dried fruits of both the cherry cultivars, showed that investigated cultivars differed in their colorless phenolic composition. Hydroxycinnamoyl quinic acid derivatives were present in both the cherry cultivars. 'Della Recca' cv. was particularly rich in 4-O-coumaroyl quinic and 5-O-caffeoylquinic acid, whereas quercetin-3-O-rutinoside was the main phenol compound of 'Del Monte' cultivar. The antiradical properties of the extracts were investigated by DPPH and ABTS methods. 'Della Recca' cv. cherries exhibited a pronounced antiradical activity: at 62.5µg/mL dose level ABTS radical cation was converted in its reduced form by 88.7% and DPPH radical was reduced by 75.3%. The antiproliferative efficacy of 'Della Recca' and 'Del Monte' extracts were evaluated towards five cancer cell lines (HepG2, A549, HeLa, SK-B-NE(2)-C, and SH-SY5Y) through MTT assay. 'Della Recca' phenol extract showed a dose-dependent inhibiting activity towards cervical cancer HeLa cell line.

Myoglobin as marker in meat adulteration: a UPLC method for determining the presence of pork meat in raw beef burger.

The identification of meat animal species used in raw burgers is very important with respect to economic and religious considerations. Therefore, international supervisory bodies have implemented procedures to control the employed meat species. In this paper we propose myoglobin as a powerful molecular marker to evaluate the presence of non-declared meat addition in raw beef burgers by using ultra-performance liquid chromatography (UPLC) for the separation and identification of edible animal species (beef, chicken, horse, ostrich, pig and water buffalo). Meat samples were pre-treated with sodium nitrite to transform oxymyoglobin and deoxymyoglobin to the more stable metmyoglobin. The developed method was validated, preparing mixtures with different percentages of pork and beef minced meat. The obtained results show that using myoglobin as marker, 5% (25 mg/500 mg) of pork or beef meat can be detected in premixed minced meat samples.

Metabolic effects of intensified insulin therapy.

Intensified insulin therapy is usually carried out either with multiple subcutaneous insulin injections (ICT: intensified conventional therapy) or with continuous subcutaneous insulin infusion (CSII) by minipumps. For two years we have been studying two matched groups of type I diabetic patients, treated with 3 daily insulin injections (ICT) and with CSII ( Microjet , Miles), respectively. Blood glucose control, as assessed by integrated mean blood glucose (MBG), was similar in both groups, but a better metabolic stability ('M' index of Schlichtkrull ) was evident in the CSII group. From the 24-hour profiles of plasma 'free' IRI and metabolites (glucose, beta-OH-butyrate, lactate, pyruvate, alanine), both the hepatic and peripheral underinsulinization and related metabolic alterations were still evident in both groups of patients. The number of hypoglycaemic episodes, recorded by home blood glucose monitoring, was similar in both groups of patients, while the perception of symptomatic hypoglycaemia seemed to be reduced in the patients treated with CSII.

Kinetics and metabolic activity of biosynthetic NPH insulin evaluated by the glucose clamp technique.

The glucose clamp technique has been used to evaluate the metabolic activity of NPH biosynthetic insulin in diabetic subjects free from anti-insulin antibodies. After overnight blood glucose normalization with a glucose-controlled insulin infusion system (Biostator), an s.c. injection of NPH insulin was given in the abdominal region. The insulin dose (0.236 +/- 0.05 U/kg body wt) was related to the usual intermediate-acting insulin requirement in the morning. Glucose was clamped at 100 mg/dl by a feedback i.v. glucose infusion. The end of the action of s.c. injected insulin considered conventionally to be the time of the spontaneous rise of blood glucose to 110 mg/dl. Free insulin levels were higher and the length of action was longer after NPH porcine than after NPH biosynthetic human insulin (BHI) (area under the free insulin curve: porcine 1423 +/- 556 mU/L/h; BHI 1045 +/- 338 mU/L/h, P less than 0.05; length of action: porcine 16.0 +/- 3.2 h; BHI 13.7 +/- 0.9 h, P less than 0.05); the glucose requirement was higher after porcine (76.8 +/- 13.5 g) than after BHI (58.5 +/- 14.6 g) without reaching statistical significance. However, the metabolic activity of the bioavailable insulin (index of plasma free insulin activity) was similar for the two insulins (porcine 381 +/- 77.4, BHI 342.8 +/- 54.2 mU/L/g of glucose/h). We conclude that a difference in pharmacokinetics exists between NPH BHI and porcine NPH insulin, which makes the latter metabolically more active. The different behavior does not seem to be related to the insulin molecule itself but could be a consequence of the unequal content of protamine in the two pharmacologic preparations.

[Use of the endocrine artificial pancreas (GCIIS; Biostator) in heart surgery]. / Applicazione del pancreas artificiale endocrino (GCIIS; Biostator) nella cardiochirurgia.

It is well known that deep hypothermia used in open-heart surgery is usually associated with a marked reduction in carbohydrate tolerance, especially dangerous in the diabetic patient, since it might result in severe metabolic complications, namely ketosis and hyperosmolar coma. In order to prevent the occurrence of such complications we treated an insulin-dependent diabetic patient undergoing cardiovascular surgery, with a feed-back insulin infusion operated by an artificial pancreas (GCIIS, Biostator). The Biostator was then used to perform a continuous (minute by minute) blood glucose monitoring in 2 more patients, a type II (non insulin-dependent) diabetic and a non diabetic. Blood samples were drawn sequentially in order to determine plasma free insulin concentration. The glycemic profile observed in the insulin-dependent diabetic under artificial pancreas treatment was similar to that in the non-diabetic. Plasma free insulin levels dropped near to zero during by-pass cooling, returning toward basal level during the rewarming phase. Such results are then discussed by the Authors and given a pathogenetic interpretation.

Improvement of artificial endocrine pancreas (Biostator; GCIIS) performance combining feedback controlled insulin administration with a pre-programmed insulin infusion.

It is well known that the glycemic control accomplished by the artificial endocrine pancreas (Biostator; GCIIS), requires an excessive insulin consumption. In order to improve the Biostator performance, we have studied 5 insulin dependent diabetic patients on anticipated automatic pre-programmed insulin infusion starting at the beginning of breakfast. Such an operating method allowed us to obtain a significant reduction in insulin requirements (from 17,3 +/- 2,2 U to 13,3 +/- 2,2 U), and a better glycemic control (MBG 98,7 +/- 5,4 vs 111,1 +/- 5,6; M 2,34 +/- 0,9 vs 6,96 +/- 1,7; delta G 43,8 +/- 3 vs 73,4 +/- 9,7), as compared to those observed under conventional feed-back control in relation to breakfast.