Gennaro Raimo

Antioxidant Activity and Chemical Components as Potential Anticancer Agents in the Olive Leaf (Olea europaea L. cv Leccino.) Decoction

Epidemiological studies have shown that a reduced risk of chronic diseases such as cancer and cardiovascular diseases is correlated with a regular consumption of fruits and vegetable, many of which are rich in polyphenols. The additive and synergistic effect of phytochemicals in fruits and vegetables may reduce chronic diseases related to oxidative stress in human body. Olea europaea L. leaf are rich in phenolic components, which have been proposed to play a role in cancer prevention. The purpose of this study was to identify the main components in the Olea europaea L. leaf (cv. Leccino) preserved during the decoction preparation, in order to delineate the antioxidant activities of the crude extracts and its isolated compounds by using different in vitro assays including DPPH radicalscavenging capacity, total antioxidant capacity (TAC), xanthine oxidase (XO) inhibitory effect and the ability to delay the linoleic acid peroxidation process (ALP). The aqueous decoction was partitioned obtaining four extracts and the n-butanol extract showed the highest antioxidant activity and the highest total phenolic content. Phytochemical investigation leads to the isolation of thirteen secondary metabolites including simple phenolics, flavonoids, secoiridoids whose structures were elucidated by spectroscopic data (1D and 2D NMR) and spectrometric techniques. A significant free radical scavenging effect against DPPH has been evidenced in fraxamoside (1) (EC50 62.6 µM) and taxifolin (5) (EC50 50.0 µM), isolated for the first time from the water decoction. The most active compound in the TAC evaluation, was the 3,4 dihydro-phenyl glycol (8) (0.90 caffeic acid equiv.) while taxifolin and fraxamoside resulted as the most efficient inhibitors of XO activity (IC50 2.7 and 5.2 µM, respectively). Secoxyloganin (4), oleuropein (2) and tyrosol (6) showed the highest ALP activity. This study adds to the growing body of data supporting the bioactivities of phytochemicals and their potential impact on human health.

Molecular, functional and structural properties of an archaebacterial elongation factor 2.

The elongation factor 2 (aEF-2) from the extreme thermo-acidophilic archaebacterium Sulfolobus solfataricus, is the only cytosolic target protein which is ADP-ribosylated by diphtheria toxin in presence of NAD. Once ADP-ribosylated, aEF-2 is no longer able to sustain poly(Phe) synthesis in vitro. aEF-2 displays a great thermoresistance: at the growth temperature of the archaebacterium, 87 degrees C, its half-life is 3 h. The amino acid sequence of the N-terminal region of aEF-2 has been determined up to residue 22. In the first 15 positions such a sequence is identical to that of EF-2 from Sulfolobus acidocaldarius and very similar to that of EF-2 from other archaebacteria or eukaryotes. The same is true for the primary structure of the peptide containing the ADP-ribosylation site. The fact that the primary structure of EF-2 at the ADP-ribosylation site is highly conserved ensures either the correct recognition of the histidine residue by the enzymes involved in its modification to diphthamide, or the proper interaction with the diphtheria toxin.

Archaeal elongation factor 1β is a dimer. Primary structure, molecular and biochemical properties

The elongation factor 1 beta (EF-1 beta), that in eukarya and archaea promotes the replacement of GDP by GTP on the elongation factor 1 alpha x GDP complex, was purified to homogeneity from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1 beta). Its primary structure was established by sequenced Edman degradation of the entire protein or its proteolytic peptides. The molecular weight of SsEF-1 beta was estimated as about 10000 or 20000 under denaturing or native conditions respectively; this finding suggests that the native protein exists as a dimer. The peptide chain of SsEF-1 beta is much shorter than that of its eukaryotic analogues and homology is found only at their C-terminal region; no homology exists between SsEF-1 beta and eubacterial EF-Ts. At 50 degrees C, at a concentration of SsEF-1 beta 5-fold higher than that of SsEF-1 alpha x [3H]GDP the rate of the exchange of [3H]GDP for GTP becomes about 160-fold faster. An analysis of the values of the energetic parameters indicates that in the presence of SsEF-1 beta the GDP/GTP exchange is entropically favoured. At 100 degrees C the half-life of SsEF-1 beta is about 4 h.

Resistance of archaebacterial aEF-1α.GDP against denaturation by heat and urea

Abstract The elongation factor aEF-1α, isolated as aEF-1α· GDP from the thermoacidophilic archaebacterium Sulfolobus solfataricus, exchanges GDP for [3H]GDP at a rate which reaches a maximum at 95°C. The rate constants at different temperatures of the heat inactivation of aEF-1α· GDP are considerably lower compared to those referred to Escherichia coli EF-Tu· GDP. The Tm values determined for both aEF-1α· GDP and EF-Tu· GDP are 97 and 53°C, respectively. The addition of GDP during the heat treatment protects significantly EF-Tu· GDP but only slightly aEF-1α· GDP. The ability of aEF-1α· GDP to exchange GDP for [3H]GDP is impaired at 70°C by urea at concentrations which are greater compared to those required to inactivate E. coli EF-Tu· GDP at 45°C; apparently both factors are hot protected by GDP against inactivation by urea.

Stability against temperature of Sulfolobus solfataricus elongation factor 1α, a multi-domain protein

The elongation factors (EF-Tu/EF-1 alpha) are universal proteins, involved in protein biosynthesis. A detailed characterization of the stability against temperature of SsEF-1 alpha, a three-domain protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus is presented. Thermal denaturation of both the GDP-bound (SsEF-1 alpha*.GDP) and the ligand-free (nfSsEF-1 alpha) forms was investigated by means of circular dichroism and fluorescence measurements, over the 4.0-7.5 pH interval. Data indicate that the unfolding process is cooperative with no intermediate species and that the few inter-domain contacts identified in the crystal structure of SsEF-1 alpha play a role also at high temperatures. Finally, it is shown that the enzyme exhibits two different interchangeable thermally denatured states, depending on pH.

Heterologous expression in Escherichia coli of the gene encoding an archaeal thermoacidophilic elongation factor 2. Properties of the recombinant protein

The gene encoding the elongation factor 2 from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-2) was expressed in Escherichia coli using the pT7-7 expression vector. The synthesis of the heterologous product did not increase upon addition of isopropyl-beta-thiogalactopyranoside. The amount of purified intact recombinant SsEF-2 (SsEF-2rec) was about 3 mg from 60 g of transformed wet cells. Recombinant and naturally occurring SsEF-2 showed identical electrophoretic mobility, immunological properties and the N-terminal amino acid sequence; both were lacking the initial methionine. Differently from SsEF-2, SsEF-2rec did not undergo post-translational modification of His603 into diphthamide, as indicated by its inability to be ADP-ribosylated. SsEF-2rec appeared indistinguishable from SsEF-2 in the fulfillment of its biological functions; in fact, it was fully capable to support poly(Phe) synthesis, to bind GDP and to display either the intrinsic or the ribosome-dependent GTPase. Finally, SsEF-2rec was endowed with the same heat stability as SsEF-2. Altogether these findings proved that SsEF-2rec was functionally active as SsEF-2. The used expression system could allow to produce mutated forms of SsEF-2 obtained by mutagenesis of the corresponding gene.

The first nucleotide sequence of an archaeal elongation factor 1β gene

Abstract An archaeal elongation factor 1β gene has been isolated for the first time from a Sulfolobus solfataricus genomic library. The sequenced clone (869 bp) contained two open reading frames, one coding for a protein made of 91 amino acid residues (SsEF-1β), the other one encoding a nonidentified product (ORF 115). The amino acid sequences of segments at the N-and C-terminal of the translated SsEF-1β were identical to those determined for the native protein. Northern and Southern analyses showed that the SsEF-1β gene is represented in S. solfataricus by a unique sequence. Compared to eubacterial or eukaryal corresponding genes the SsEF-1β is much shorter.

Molecular and functional characterization of polynucleotide phosphorylase from the antarctic eubacterium Pseudoalteromonas haloplanktis.

Polyribonucleotide phosphorilase from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis (PhPNPase) has been purified. This enzyme catalyzes both the RNA polymerisation and degradation reaction, showing the highest activity at temperatures below 40 degrees C. PhPNPase is quite sensitive to heat treatment and it is endowed with remarkable halotolerance.

The site for GTP hydrolysis on the archaeal elongation factor 2 is unmasked by aliphatic alcohols

An appropriate mixture of ethylene glycol and BaCl2 enhanced the otherwise very low intrinsic GTPase activity of the elongation factor 2 isolated from the archaeon Sulfolobus solfataricus (SsEF-2). The enzymatic activity became up to 300-fold higher than that of the SsEF-2 GTPase measured in the absence of any stimulator, but remained 20-fold lower than that stimulated by ribosome. The stimulatory effect of ethylene glycol/Ba2+ was attributed to the increased affinity for GTP, probably related to a conformational change occurring in a hydrophobic region near the catalytic site.

Studies on the Polypeptide Elongation Factor 2 from Sulfolobus solfataricus INTERACTION WITH GUANOSINE NUCLEOTIDES AND GTPase ACTIVITY STIMULATED BY RIBOSOMES

The elongation factor 2 from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-2) binds [3H]GDP at 1:1 molar ratio. The bound [3H]GDP is displaced by GTP or its nonhydrolyzable analogue guanyl-5′-yl imidodiphosphate (Gpp(NH)p) but not by ATP, thus indicating that only the two guanosine nucleotides compete for the same binding site. The affinity of SsEF-2 for [3H]GDP is higher than that for GTP and Gpp(NH)p. On the contrary, in the presence of ribosomes the affinity of SsEF-2 for GDP is lower than that for Gpp(NH)p. SsEF-2 is endowed with an intrinsic hardly detectable GTPase activity that is stimulated by ribosomes up to 2000-fold. The ribosome-stimulated SsEF-2 GTPase (GTPase) reaches a maximum at pH 7.8 and is not affected by ATP but is competitively inhibited by either GDP or Gpp(NH)p. Both K for [-P]GTP and k of GTPase increase with increasing temperature, and the highest catalytic efficiency is reached at 80°C. The ADP-ribosylation of SsEF-2 does not significantly affect either the binding of GDP and GTP or the kinetics of the GTPase. A hypothesis on the stimulation by ribosome of SsEF-2 GTPase is proposed.