Chitinase III in Euphorbia characias latex: Purification and characterization.

This paper deals with the purification of a class III endochitinase from Euphorbia characias latex. Described purification method includes an effective novel separation step using magnetic chitin particles. Application of magnetic affinity adsorbent noticeably simplifies and shortens the purification procedure. This step and the subsequently DEAE-cellulose chromatography enable to obtain the chitinase in homogeneous form. One protein band is present on PAGE in non-denaturing conditions and SDS-PAGE profile reveals a unique protein band of 36.5 ± 2 kDa. The optimal chitinase activity is observed at 50 °C, pH 5.0. E. characias latex chitinase is able to hydrolyze colloidal chitin giving, as reaction products, N-acetyl-D-glucosamine, chitobiose and chitotriose. Moreover, we observed that calcium and magnesium ions enhance chitinase activity. Finally, we cloned the cDNA encoding the E. characias latex chitinase. The partial cDNA nucleotide sequence contains 762 bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases.

Euphorbia characias latex: micromorphology of rubber particles and rubber transferase activity.

We have recently characterized a natural rubber in the latex of Euphorbia characias. Following that study, we here investigated the rubber particles and rubber transferase in that Mediterranean shrub. Rubber particles, observed by scanning electron microscopy, are spherical in shape with diameter ranging from 0.02 to 1.2 µm. Washed rubber particles exhibit rubber transferase activity with a rate of radiolabeled [(14)C]IPP incorporation of 4.5 pmol min(-1)mg(-1). Denaturing electrophoresis profile of washed rubber particles reveals a single protein band of 37 kDa that is recognized in western blot analysis by antibodies raised against the synthetic peptide whose sequence, DVVIRTSGETRLSNF, is included in one of the five regions conserved among cis-prenyl chain elongation enzymes. The cDNA nucleotide sequence of E. characias rubber transferase (GenBank JX564541) and the deduced amino acid sequence appear to be highly homologous to the sequence of several plant cis-prenyltransferases.

Domain mobility as probed by small-angle X-ray scattering may account for substrate access to the active site of two copper-dependent amine oxidases.

Amine oxidases are a family of dimeric enzymes that contain one copper(II) ion and one 2,4,5-trihydroxyphenyalanine quinone per subunit. Here, the low-resolution structures of two Cu/TPQ amine oxidases from lentil (Lens esculenta) seedlings and from Euphorbia characias latex have been determined in solution by small-angle X-ray scattering. The active site of these enzymes is highly buried and requires a conformational change to allow substrate access. The study suggests that the funnel-shaped cavity located between the D3 and D4 domains is narrower within the crystal structure, whereas in solution the D3 domain could undergo movement resulting in a protein conformational change that is likely to lead to easier substrate access.

Euphorbia characias latex amine oxidase and peroxidase: interacting enzymes?

This minireview deals the enzymatic transformation of some amino acids as arginine and ornithine, amines as tyramine, putrescine, spermine and spermidine, and other substances as nitric oxide and thiocyanate. These reactions, catalyzed by two proteins purified from the latex of Euphorbia characias, a copper/quinone containing amine oxidase and a cationic peroxidase, show enzymatic activity interactions probably occurring between these proteins in Euphorbia latex.

Analysis of the glycosylation pattern of plant copper amine oxidases by MALDI-TOF/TOF MS coupled to a manual chromatographic separation of glycans and glycopeptides.

The N-glycosylation in pea seedling amine oxidase and lentil seedling amine oxidase was analyzed in the present work. For that purpose, the enzymes were purified as native proteins from their natural sources. An enzymatic deglycosylation of pea seedling amine oxidase by endoglycosidase H under denaturing conditions combined with its proteolytic digestion by trypsin was carried out in order to analyze both N-glycans and "trimmed" N-glycopeptides with a residual N-acetylglucosamine attached at the originally occupied N-glycosylation sites. The released N-glycans were subjected to a manual chromatographic purification followed by MALDI-TOF/TOF MS. MS and MS/MS analyses were also performed directly on peptides and N-glycopeptides generated by proteolytic digestion of the studied enzymes. Sequencing of glycopeptides by MALDI-TOF/TOF MS/MS after their separation on a RP using a microgradient chromatographic device clearly demonstrated binding of paucimannose and hybrid N-glycan structures at Asn558. Such carbohydrates have been reported to exist in many plant N-glycoproteins, e.g. in peroxidases. Although high-mannose glycan structures were identified after the enzymatic deglycosylation, they could not be assigned to a particular N-glycosylation site. The presence of unoccupied glycosylation sites in several peptides was also confirmed from MS/MS results.

Nutrient availability links mitochondria, apoptosis, and obesity.

Mitochondria are the dominant source of the cellular energy requirements through oxidative phosphorylation, but they are also central players in apoptosis. Nutrient availability may have been the main evolutionary driving force behind these opposite mitochondrial functions: production of energy to sustain life and release of apoptotic proteins to trigger cell death. Here, we explore the link between nutrients, mitochondria and apoptosis with known and potential implications for age-related decline and metabolic syndromes.

Extraction and characterization of a natural rubber from Euphorbia characias latex.

A natural rubber was identified and characterized for the first time in the latex of the perennial Mediterranean shrub Euphorbia characias. Four different methods, i.e., acetone, acetic acid, trichloroacetic acid, and Triton® X-100, followed by successive treatments with cyclohexane/ethanol, were employed to extract the natural rubber. The rubber content was shown to be 14% (w/v) of the E. characias latex, a low content compared with that of Hevea brasiliensis (30-35%) but a similar content to other rubber producing plants. E. characias rubber showed a molecular weight of 93,000 with a M(w) /M(n) of 2.9. (1) H NMR, (13) C NMR, and FTIR analysis revealed the characteristic of the cis-1,4-polyisoprene typical of natural rubber. These results provided novel insight into latex components and will ultimately benefit the broader understanding of E. characias latex composition.

Nitric oxide, substrate of Euphorbia characias peroxidase, switches off the CN(-) inhibitory effect.

The oxidation of nitric oxide (NO) by Euphorbia characias latex peroxidase (ELP-Fe(III)), in the presence or in the absence of added calcium, has been investigated. The addition of hydrogen peroxide to the native enzyme leads to the formation of Compound I and serves to catalyse the NO oxidation. The addition of NO to Compound I leads to the formation of Compound II and, afterwards, to the native enzyme spectrum. Under anaerobic conditions, the incubation of the native enzyme (ELP-Fe(III))with NO leads to the formation of the stable complex, showing a characteristic absorption spectrum (ELP-Fe(II)-NO(+)). The rate of the formation of this complex is slower in the presence of calcium than in its absence, and the same applies to the rate of the formation of Compound II from Compound I, using NO as substrate. Finally, we demonstrate that NO protects ELP from the inactivation caused by CN(-) via a mechanism presumably requiring the formation of an enzyme-nitrosyl cyanide complex.

Bioseparation of Four Proteins from Euphorbia characias Latex: Amine Oxidase, Peroxidase, Nucleotide Pyrophosphatase/Phosphodiesterase, and Purple Acid Phosphatase.

This paper deals with the purification of four proteins from Euphorbia characias latex, a copper amine oxidase, a nucleotide pyrophosphatase/phosphodiesterase, a peroxidase, and a purple acid phosphatase. These proteins, very different in molecular weight, in primary structure, and in the catalyzed reaction, are purified using identical preliminary steps of purification and by chromatographic methods. In particular, the DEAE-cellulose chromatography is used as a useful purification step for all the four enzymes. The purification methods here reported allow to obtain a high purification of all the four proteins with a good yield. This paper will give some thorough suggestions for researchers busy in separation of macromolecules from different sources.

Body composition variations in ageing.

Age-related physiological variations of body composition concern both the fat-free mass (FFM) and the fat mass (FM). These variations expose the elderly person to the risk of malnutrition and could lead to conditions of disability. This paper aims to review the current state of knowledge on body composition in the aged population. The pattern of qualitative variations in body composition in old age is fairly well defined. In adulthood, the physiological variation of body mass involves a first increasing phase followed by a decreasing trend. The reduction is due mainly to the loss of fat-free mass, especially muscle mass. Total body water and bone mass also decrease. Fat mass tends to decrease and the reduction seems to be due mainly to the loss of subcutaneous fat. The quantitative aspects of the age of onset, rate and intensity of the physiological variations are still not completely clear. This poor quantitative definition is due to the variable and multifactorial phenomenology of ageing, the heterogeneity of assessment techniques and sampling models, and the limited number of empirical observations in oldest-old individuals.

Calcium ions and a secreted peroxidase in Euphorbia characias latex are made for each other.

This minireview deals of a protein, a class III secreted peroxidase, present as unique isoform in the latex of the perennial Mediterranean shrub Euphorbia characias. The paper reports on the molecular properties, on the structures (primary, secondary and tertiary), and on the catalytic mechanism of this enzyme. Here is also reported the extraordinary effect of calcium ions on the structure and on the enzyme activity of Euphorbia peroxidase. These ions can either enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to execute different metabolic pathways toward the same substrate. This review will give a valuable reference to the peroxidase fans and the general readers will find many thorough suggestions for future researches giving birth to new studies and important discoveries.

Alizarine derivatives as new dual inhibitors of the HIV-1 reverse transcriptase-associated DNA polymerase and RNase H activities effective also on the RNase H activity of non-nucleoside resistant reverse transcriptases.

HIV-1 reverse transcriptase (RT) has two associated activities, DNA polymerase and RNase H, both essential for viral replication and validated drug targets. Although all RT inhibitors approved for therapy target DNA polymerase activity, the search for new RT inhibitors that target the RNase H function and are possibly active on RTs resistant to the known non-nucleoside inhibitors (NNRTI) is a viable approach for anti-HIV drug development. In this study, several alizarine derivatives were synthesized and tested for both HIV-1 RT-associated activities. Alizarine analogues K-49 and KNA-53 showed IC(50) values for both RT-associated functions of ∼ 10 µm. When tested on the K103N RT, both derivatives inhibited the RT-associated functions equally, whereas when tested on the Y181C RT, KNA-53 inhibited the RNase H function and was inactive on the polymerase function. Mechanism of action studies showed that these derivatives do not intercalate into DNA and do not chelate the divalent cofactor Mg(2+) . Kinetic studies demonstrated that they are noncompetitive inhibitors, they do not bind to the RNase H active site or to the classical NNRTI binding pocket, even though efavirenz binding negatively influenced K-49/KNA-53 binding and vice versa. This behavior suggested that the alizarine derivatives binding site might be close to the NNRTI binding pocket. Docking experiments and molecular dynamic simulation confirmed the experimental data and the ability of these compounds to occupy a binding pocket close to the NNRTI site.

Euphorbia peroxidase catalyzes thiocyanate oxidation in two different ways, the distal calcium ion playing an essential role.

The oxidation of the pseudohalide thiocyanate (SCN(-)) by Euphorbia peroxidase, in the presence or absence of added calcium, is investigated. After incubation of the native enzyme with hydrogen peroxide, the formation of Compound I occurs and serves to catalyze the thiocyanate oxidation pathways. The addition of a stoichiometric amount of SCN(-) to Compound I leads to the native enzyme spectrum; this process clearly occurs via two electron transfers from pseudohalide to Compound I. In the presence of 10 mM calcium ions, the addition of a stoichiometric amount of SCN(-) to Compound I leads to the formation of Compound II that returns to the native enzyme after addition of a successive stoichiometric amount of SCN(-), indicating that the oxidation occurs via two consecutive one-electron transfer steps. Moreover, different reaction products can be detected when the enzyme-hydrogen peroxide-thiocyanate reaction is performed in the absence or presence of 10 mM Ca(2+) ions. The formation of hypothiocyanous acid is easy demonstrated in the absence of added calcium, whereas in the presence of this ion, CN(-) is formed as a reaction product that leads to the formation of an inactive species identified as the peroxidase-CN(-) complex. Thus, although monomeric, Euphorbia peroxidase is an allosteric enzyme, finely tuned by Ca(2+) ions. These ions either can enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to exploit different metabolic pathways toward the same substrate.

Assessment of nutritional status in the Amazigh children of Amizmiz (Azgour Valley, High Atlas and Morocco).

The Berbers of the High Atlas (Amazigh) live in very severe socio-economic and climatic conditions, which expose children to the risk of malnutrition. In this study we used anthropometry and bioelectrical impedance analysis for the assessment of nutritional status. Height, weight and bioelectrical parameters were taken on 71 children (28 boys and 43 girls). Height and BMI were standardized using the 2007 WHO reference. The results show that 36.6% of the children were classified as stunted and 8.5% as wasted. Based on the Bioelectrical Impedance Vector Analysis, children from the High Atlas had an adequate body cell mass, but a high risk of dehydration (42.3%).

Assessment of nutritional status in free-living elderly individuals by bioelectrical impedance vector analysis.

OBJECTIVE: The aim of the present research was to examine bioelectrical vector changes in relation to nutritional status in a sample of healthy free-living elderly people. METHODS: The study group consisted of 170 men and women 70 to 99 y of age. Anthropometric and bioelectrical (resistance and reactance, 50 kHz, 800 muA) measurements were taken. Bioelectrical impedance vector analysis was applied. Nutritional status was determined by the Mini-Nutritional Assessment. Bioelectrical characteristics of normal and undernourished individuals were compared statistically with Hotelling's T(2) test and graphically with 95% probability confidence ellipses. RESULTS: The impedance and multidimensional approaches showed a clear association. Undernourished subjects had a smaller phase angle (men 5.2 +/- 1.3 versus 5.7 +/- 1.0 degrees, P = 0.027; women 5.0 +/- 1.0 versus 5.4 +/- 0.9 degrees, P = 0.065) than normally nourished subjects. CONCLUSION: Bioelectrical impedance vector analysis represents a promising indicator of nutritional status, suitable in screening programs and clinical practice.

Interaction of methimazole with I2: X-ray crystal structure of the charge transfer complex methimazole-I2. implications for the mechanism of action of methimazole-based antithyroid drugs.

The antithyroid drug methimazole (MMI) reacts with molecular iodine to form, in a multistep process, 1-methylimidazole as final product. In this process, the charge transfer complex MMI-I 2 and the ionic disulfide [(C 4H 6N 2S-) 2] (2+) ( 1, dication MMI disulfide) have been isolated and their X-ray crystal structures solved. Dication MMI disulfide perchlorate acts effectively both in reducing I 2 to I (-) ions and in showing antioxidant properties in inactivating the enzyme lactoperoxidase compound I.

Town and gender effects on hair lead levels in children from three Sardinian towns (Italy) with different environmental backgrounds.

This study reports hair lead (PbH) levels measured in 2002 in 193 children from three Sardinian towns: Carbonia, Gonnesa, and Sinnai. Carbonia and Gonnesa are in a polluted area of Sardinia due to their vicinity to the industrial zone of Portovesme. As a consequence of its economy and location, Sinnai is not exposed to lead pollution. PbH concentrations were determined by inductively coupled plasma atomic absorption spectrometry. The aim of this study was to evaluate if hair is a reliable biomarker to determine different degrees of exposure of populations to lead pollution and if there is a tendency to higher accumulation by males or females. The girls of Carbonia had the highest mean PbH value (2.21 microg/g), followed by the Gonnesa girls (2.03 microg/g), Carbonia boys (1.86 microg/g), Gonnesa boys (0.91 microg/g), and finally the Sinnai boys (0.68 microg/g) and girls (0.50 microg/g). Two-way analysis of covariance, with age as covariate, revealed a significant effect of town and sex on log PbH. Spearman's rank correlation coefficient indicated a significant positive concordance between PbH levels and gender (score for males=1, females=2). The results suggest that hair is a reliable biomarker to determine different levels of exposure of populations to lead pollution, and they indicate that females tend to accumulate lead in the hair more than males of the same age.

Tyramine oxidation by copper/TPQ amine oxidase and peroxidase from Euphorbia characias latex.

Tyramine, an important plant intermediate, was found to be a substrate for two proteins, a copper amine oxidase and a peroxidase from Euphorbia characias latex. The oxidation of tyramine took place by two different mechanisms: oxidative deamination to p-hydroxyphenylacetaldehyde by the amine oxidase and formation of di-tyramine by the peroxidase. The di-tyramine was further oxidized at the two amino groups by the amino oxidase, whereas p-hydroxyphenylacetaldehyde was transformed to di-p-hydroxyphenylacetaldehyde by the peroxidase. Data obtained in this study indicate a new interesting scenario in the metabolism of tyramine.

Allosteric modulation of Euphorbia peroxidase by nickel ions.

A class III peroxidase, isolated and characterized from the latex of the perennial Mediterranean shrub Euphorbia characias, contains one ferric iron-protoporphyrin IX pentacoordinated with a histidine 'proximal' ligand as heme prosthetic group. In addition, the purified peroxidase contained 1 mole of endogenous Ca(2+) per mole of enzyme, and in the presence of excess Ca(2+), the catalytic efficiency was enhanced by three orders of magnitude. The incubation of the native enzyme with Ni(2+) causes reversible inhibition, whereas, in the presence of excess Ca(2+), Ni(2+) leads to an increase of the catalytic activity of Euphorbia peroxidase. UV/visible absorption spectra show that the heme iron remains in a quantum mechanically mixed-spin state as in the native enzyme after addition of Ni(2+), and only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ni(2+). In the presence of H(2)O(2) and in the absence of a reducing agent, Ni(2+) decreases the catalase-like activity of Euphorbia peroxidase and accelerates another pathway in which the inactive stable species accumulates with a shoulder at 619 nm. Analysis of the kinetic measurements suggests that Ni(2+) affects the H(2)O(2)-binding site and inhibits the formation of compound I. In the presence of excess Ca(2+), Ni(2+) accelerates the reduction of compound I to the native enzyme. The reported results are compatible with the hypothesis that ELP has two Ni(2+)-binding sites with opposite functional effects.