Roberto Leoncini

Proteins from Mucuna pruriens and enzymes from Echis carinatus venom: Characterization and cross-reactions

Mucuna pruriens seeds have been widely used against snakebite in traditional medicine. The antivenin property of a water extract of seeds was assessed in vivoin mice. The serum of mice treated with extract was tested for its immunological properties. Two proteins of Echis carinatusvenom with apparent molecular masses of 25 and 16 kDa were detected by Western blot analysis carried out using IgG of mice immunized with extract or its partially purified protein fractions. By enzymatic in-gel digestion and electrospray ionization-mass spectrometry/mass spectrometry analysis of immunoreactive venom proteins, phospholipase A2, the most toxic enzyme of snake venom, was identified. These results demonstrate that the observed antivenin activity has an immune mechanism. Antibodies of mice treated with non-lethal doses of venom reacted against some proteins ofM. pruriens extract. Proteins of E. carinatusvenom and M. pruriens extract have at least one epitope in common as confirmed by immunodiffusion assay.

Enzyme activities controlling adenosine levels in normal and neoplastic tissues

Adenosine is known to be associated with effects such as inhibition of immune response, coronary vasodilation, stimulation of angiogenesis, and inhibition of inflammatory reactions. Some authors suggest that adenosine may also have similar functions in tumor tissues. Tissue levels of adenosine are under close regulation by different enzymes acting at different levels. Adenosine is produced from AMP by the action of 5′-nucleotidase (5′-NT) and is converted back into AMP by adenosine kinase (AK) or into inosine by adenosine deaminase (ADA). Inosine is converted into purine catabolites by purine nucleoside phosphorylase (PNP), whereas AMP is converted into ADP and ATP by adenylate kinase (MK).The aim of this study was to analyze the activities of the above enzymes in fragments of neoplastic and apparently normal mucosa, obtained less than 5 cm and at least 10 cm from tumors, in 40 patients with colorectal cancer.The results showed much higher activities of ADA, AK, 5′-NT, and PNP in tumor tissue than in neighboring mucosa (p>0.01 for ADA, AK, and PNP; p>0.05 for 5′-NT), suggesting that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissue. The simultaneous increase in ADA and 5′-NT activities might be a physiological attempt by cancer cells to provide more substrate to accelerate salvage pathway activity.

Studies on possible protection against snake venom using Mucuna pruriens protein immunization

Immunization against cobra venom using Mucuna pruriens derived serum immunoglobulins was carried out in a mouse model. The neutralizing ability of circulating antibodies was assessed by challenging the immunized mice with a minimum lethal dose of purified venom after 4, 24, 72 and 168 h. The single injection of the antibody preparation produced a high and sustained immune response with high survival rates of treated animals.

Adenosine Kinase Gene Expression in Human Colorectal Cancer

Real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate gene expression of adenosine kinase, a key enzyme in adenosine metabolism, in human intestinal biopsy specimens of 10 colorectal cancer patients. Quantitative mRNA expression levels were normalized against the reference gene β -actin. The results showed that adenosine kinase gene expression was significantly higher in cancer than in normal-appearing tissue, in line with our previous measurements of adenosine kinase enzyme activities in colorectal tumor samples.

Determination of urinary methylated purine pattern by high-performance liquid chromatography

We describe the group selective separation and quantification of unmodified and modified purines in human urine by high-performance reverse phase liquid chromatography. The pattern of oxypurines and methylated purines: hypoxanthine (Hx), xanthine (X), 1-methyl hypoxanthine (1-MHx), 1-methyl guanine (1-MG), 3-methyl guanine (3-MG), 7-methyl guanine (7-MG), 1-methyl xanthine (1-MX), 3-methyl xanthine (3-MX), 7-methyl xanthine (7-MX), 1,7-dimethyl guanine (1,7-dMG), 1,3-dimethyl xanthine (1,3-dMX), 1,7-dimethyl xanthine (3,7-dMX) and 1,3,7-trimethyl xanthine (1,3,7-tMX) were determined in a single run in urine of a healthy subject and a gout patient before and after treatment with allopurinol. This method may be useful to investigate the urinary pattern of methylated bases in diseases involving purine metabolism.

Proteomic analysis of the pathophysiological process involved in the antisnake venom effect of Mucuna pruriens extract

Previously, we reported the antisnake venom properties of a Mucuna pruriens seed extract (MPE) and tested its in vivo efficacy against Echis carinatus venom (EV) in short‐ (1 injection) and long‐term (three weekly injections) treatments. The aim of the present study was to investigate plasma proteome changes associated with MPE treatments and identify proteins responsible for survival of envenomated mice (CHALLENGED mice). Six treatment groups were studied. Three control groups: one saline, one short‐term and one long‐term MPE treatment. One group received EV alone. Two test groups received EV with either a short‐term or long‐term MPE treatment (CHALLENGED mice). The plasma from each group was analysed by 2‐DE/MALDI‐TOF MS. The most significant changes with treatment were: albumin, haptoglobin, fibrinogen, serum amyloid A and serum amyloid P. Most of these changes were explained by EV effects on coagulation, inflammation and haemolysis. However, MPE treatments prevented the EV‐induced elevation in HPT. Consequently, HPT levels were similar to controls in the plasma of CHALLENGED mice. The plasma of CHALLENGED mice showed substantial proteomic modifications. This suggests the mechanism of MPE protection involves the activation of counterbalancing processes to compensate for the imbalances caused by EV.

Double inhibition ofl-threonine dehydratase by aminothiols

The inhibition of highly purified rat liver L-threonine dehydratase (L-threonine hydro-lyase (deaminating), EC 4.2.1.16) by aminothiols (L-cysteine, D-cysteine, cysteamine) has been studied. Single inhibition experiments evaluated by Lineweaver-Burk and Dixon plots showed, in a given concentration range, partially (parabolic) competitive inhibitions, indicating two binding sites for each inhibitor. Double inhibition experiments revealed that the inhibition was antagonistic, the two inhibitors weakening each others effect. Formation of EI1 and EI2 binary complexes, and ESI1, ESI2 and EI1I2 ternary complexes was demonstrated, while formation of the quaternary complex ESI1I2 was ruled out. It is assumed that one inhibitor-binding site coincides with the substrate-binding center while the second inhibitor-binding (allosteric, regulatory) site may comprise the pyridoxal-phosphate-binding SH group(s). The comparison between Km and Ki values and the evaluation of intracellular concentrations of L-threonine, L-cysteine and cysteamine suggest a possible physiological role of the inhibition.

Inhibition and regulation of rat liver L-threonine dehydrogenase by different fatty acids and their derivatives

Rat liver L-threonine dehydrogenase is a mitochondrial enzyme which transforms L-threonine either into aminoacetone or into acetyl-CoA. We show that it is inhibited by several fatty acids and their derivatives: short chain fatty acids, L-2-hydroxybutyrate and D-3-hydroxybutyrate, long chain fatty acids, such as lauric acid, myristic acid, palmitic and stearic acids, bicarboxylic acids such as malonic acid and its derivatives methyl- and hydroxymalonic acids. The inhibition occurs at low and physiological concentrations of such compounds, which are normally present and metabolized in mitochondria. It presumably plays a role in the physiology of acetyl-CoA-dependent formation of fatty acids and ketobodies, in L-threonine-dependent gluconeogenesis, and in the regulation of L-threonine metabolism by L-threonine dehydrogenase and L-threonine deaminase.

Effects of snake venom proteases on human fibrinogen chains

BACKGROUND Proteomic approach is an effective method to study changes in human plasma proteome. Coagulopathies are commonly encountered in victims of viper envenomation which were treated with an administration of immunoglobulin. Unfortunately, this treatment shows significant risk to the patient due to an anaphylactic reaction. Since Echis carinatus Venom (EV) toxins mainly acts both directly and indirectly on fibrinogen, we planned to establish a suitable analysis of its beta (FIBB) e gamma (FIBG) chains. This study will help us to understand the mechanism of envenomation and to find alternative treatments other than the common treatment with the administration of IgG. STUDY DESIGN AND METHODS We evaluated the EV proteolytic activity on whole human plasma proteome from the blood of an healthy volunteer. Two-dimensional electrophoresis (2-DE) using mini-gel was performed to analyse EV effects on the differents fibrinogen chains. RESULTS Changes in whole plasma proteome were focused on fibrinogen beta and gamma chains after EV incubation. Protein spots were detected and analyzed using ImageMaster 2D Platinum software. Results were represented as mean +/- standard deviation (mean+/-SD) with p<0.05 as a statistically significant value. 2-DE gel analysis showed that some spots of FIBB disappeared and some spots of FIBG decreased. CONCLUSION We found that the proteomic approach is a valid method in studying in-depth causes of different diseases, in particular those are involved in coagulopathies linked with proteins like fibrinogen from victims of viper envenomation.

Erythrocyte Sedimentation Rate measurement by VES Matic Cube 80 in relation to inflammation plasma proteins

Westergren method is considered as the reference procedure to measure Erythrocyte Sedimentation Rate (ESR) by the International Council for Standardization in Haematology. However, a closed automated method, VES Matic Cube 80 (DIESSE S.p.A., Siena, Italy), has been introduced as a new ESR measurement instrument. In this article, we report two different studies: first, we compared the two methods (Westergren and VES Matic Cube 80) and second, we correlated the inflammatory state of 248 patients with their ESR values. Total protein, albumin, C‐reactive protein, and other inflammatory proteins were detected in each sample. The results obtained using VES Matic Cube 80demonstrated a good correlation with those obtained using the Westergren method (Ordinary linear regression: y=0.955x–0.205, r2=0.816, P<0.05; Passing–Bablock regression equation: y=0.9153x−0.5763; Bland–Altman analysis: bias 1.2; limits of agreement −17.4–19.9) and with the inflammatory protein levels (CRP: r=0.554 and r=0.498 and Fibrinogen: r=0.699 and r=0.663 for Ves Matic Cube 80 and Westergren, respectively), supporting the hypothesis that VES Matic Cube 80 offers a fast and safe ESR determination, ensuring precision and a very good correlation with the reference method. J. Clin. Lab. Anal. 25:198–202, 2011.