Two thermophilic and thermostable enzymes, isolated from Sulfolohus solfataricus, S-adenosylhomocysteine hydrolase and 5'-methylthioadenosine phosphorylase, were exposed to 10.4 GHz microwave radiation in order to discriminate between thermal and non-thermal microwave effects. The exposure causes a non-thermal, irreversible and time-dependent inactivation of both enzymes; the inactivation rate is related to the energy absorbed and is independent of the enzyme concentration. The influence of salts on enzyme inactivation has also been investigated. Conformational changes of 5-adenosylhomocysteine hydrolase, detected by fluorescence and circular dichroism techniques, suggest that microwaves induce protein structural rearrangements not related to temperature.

S-Adenosylhomocysteine/5'-methylthioadenosine nucleosidase (EC 3.2.2.9) was purified to homogeneity from Escherichia coli to a final specific activity of 373 ,umol of 5'-methylthioadenosine cleaved/min per mg of protein. Affinity chromatography on S-formycinylhomocysteine-Sepharose is the key step of the purification procedure. The enzyme, responsible for the cleavage of the glycosidic bond of both Sadenosylhomocysteine and 5'-methylthioadenosine, was partially characterized. The apparent Km for 5'-methylthioadenosine is 0.4 /LM, and that for S-adenosylhomocysteine is 4.3 /tM. The maximal rate of cleavage of S-adenosylhomocysteine is approx. 40% of that of 5'-methylthioadenosine. Some 25 analogues of the two naturally occurring thioethers were studied as potential substrates or inhibitors of the enzyme. Except for the analogues modified in the 5'-position of the ribose moiety or the 2-position of the purine ring, none of the compounds tested was effective as a substrate. Moreover, 5'-methylthioformycin, 5'-chloroformycin, S-formycinylhomocysteine, 5'-methylthiotubercidin and S-tubercidinylhomocysteine were powerful inhibitors of the enzyme activity. The results obtained allow the hypothesis of a mechanism of enzymic catalysis requiring as a key step the protonation of N-7 of the purine ring.

The structure of 5-deoxy-5-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) has been determined alone, as ternary complexes with sulfate plus substrates 5-deoxy-5-methylthioadenosine, adenosine, or guanosine, or with the noncleavable substrate analog Formycin B and as binary complexes with phosphate or sulfate alone. The structure of unliganded SsMTAP was refined at 2.5-Å resolution and the structures of the complexes were refined at resolutions ranging from 1.6 to 2.0 Å. SsMTAP is unusual both for its broad substrate specificity and for its extreme thermal stability. The hexameric structure of SsMTAP is similar to that of purine-nucleoside phosphorylase (PNP) from Escherichia coli, however, only SsMTAP accepts 5-deoxy-5-methylthioadenosine as a substrate. The active site of SsMTAP is similar to that of E. coli PNP with 13 of 18 nearest residues being identical. The main differences are at Thr 89 , which corresponds to serine in E. coli PNP, and Glu 163 , which corresponds to proline in E. coli PNP. In addition, a water molecule is found near the purine N-7 position in the guanosine complex of SsMTAP. Thr 89 is near the 5-position of the nucleoside and may account for the ability of SsMTAP to accept either hydrophobic or hydrophilic substituents in that position. Unlike E. coli PNP, the structures of SsMTAP reveal a substrate-induced conformational change involving Glu 163 . This residue is located at the interface between subunits and swings in toward the active site upon nucleoside binding. The high-resolution structures of SsMTAP suggest that the transition state is stabilized in different ways for 6-amino versus 6-oxo substrates. SsMTAP has optimal activity at 120°C and retains full activity after 2 h at 100°C. Examination of the three-dimensional structure of SsMTAP suggests that unlike most thermophilic enzymes, disulfide linkages play a key in role in its thermal stability. 5Ј-Deoxy-5Ј-methylthioadenosine phosphorylase (EC 2.4.2.28)from Sulfolobus solfataricus (SsMTAP) 1 functions in the purine salvage pathway where it catalyzes the phosphorolysis of 5Ј-deoxy-5Ј-methylthioadenosine (MTA) (1), a sulfur-containing nucleoside generated as a by-product of polyamine biosynthesis (2). The products of the MTA cleavage reaction are adenine and 5-methylthio-D-ribose 1-phosphate. S. solfataricus is a thermophilic microorganism belonging to Archaea, the third primary domain (3). The biosynthesis of the symmetrical polyamines, sym-norspermine and sym-norspermine, is highly operative in thermophilic archaebacteria (4), suggesting an important role for MTAP in the catabolism of large quantities of MTA in these organisms (Scheme 1).SsMTAP has been classified as a hyperthermophilic enzyme since its optimum temperature (120°C) is higher than the boiling point of water (1). The enzyme is also characterized by extreme thermostability, demonstrating full activity after 2 h at 100°C and showing an apparent melting temperature of 132°C. In addition, the enzyme remains stable in the presence of protein ...

Head and neck carcinoma (HNC) is a heterogeneous disease encompassing a variety of tumors according to the origin. Laryngeal cancer (LC) represents one of the most frequent tumors in the head and neck region. Despite clinical studies and advance in treatment, satisfactory curative strategy has not yet been reached. Therefore, there is an urgent need for the identification of specific molecular signatures that better predict the clinical outcomes and markers that serve as suitable therapeutic targets. Long non-coding RNAs (lncRNA) are reported as important regulators of gene expression and represent an innovative pharmacological application as molecular biomarkers in cancer. The purpose of this review is to discuss the most relevant epigenetic and histological prognostic biomarkers in HNC, with particular focus on LC. We summarize the emerging roles of long non-coding RNAs in HNC and LC development and their possible use in early diagnosis.

Among nutraceuticals, polyphenols represent the most intriguing and studied class of compounds that can be therapeutics for a large spectrum of the most common diseases, including cancer. Although polyphenols are well known as potent antioxidants, a pro-oxidant effect has been associated with a pro-apoptotic function of these compounds in various types of tumor cells. Annurca apple, a southern Italian variety, is characterized by an extremely high content of polyphenols and displays a stronger antioxidant activity compared with other varieties. In the present study we explored the antiproliferative effect of Annurca apple polyphenol extract (APE) in human breast cancer MCF-7 cells and we investigated the underlying mechanisms. Results showed that at 500 µM catechin equivalent (EqC) APE acts as a pro-oxidant increasing thiobarbituric acid-reactive species cell content of approximately 6-fold more than the untreated cells. We found that APE strongly inhibits the proliferation of MCF-7 cells by inducing G2/M cell cycle arrest and apoptosis. Immunoblot analysis demonstrated that APE treatment increases the levels of p53 and p21, downregulates the expression of the cell cycle regulatory protein cyclin D1, and inhibits ERK1/2 phosphorylation. Moreover, APE treatment caused a marked increase of pro-apoptotic Bax/Bcl-2 ratio paralleled by caspase-9, -6, -7, and poly(ADP ribose) polymerase cleavage. Altogether our data indicate that APE, at elevated concentrations, acts as a potent pro-oxidant and antiproliferative agent able to downregulate ERK1/2 pathway leading to cell cycle inhibition and apoptosis and provides a rationale for its potential use in the development of novel therapeutics towards breast cancer.