Involvement of phenylalanine 272 of DNA polymerase beta in discriminating between correct and incorrect deoxynucleoside triphosphates

DNA polymerase beta is a small monomeric polymerase that participates in base excision repair and meiosis [Sobol, R., et al. (1996) Nature 379, 183-186; Plug, A., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1327-1331]. A DNA polymerase beta mutator mutant, F272L, was identified by an in vivo genetic screen [Washington, S., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1321-1326]. Residue 272 is located within the deoxynucleoside triphosphate (dNTP) binding pocket of DNA polymerase beta according to the known DNA polymerase beta crystal structures [Pelletier, H., et al. (1994) Science 264, 1891-1893; Sawaya, M., et al. (1997) Biochemistry 36, 11205-11215]. The F272L mutant produces errors at a frequency 10-fold higher than that of wild type in vivo and in the in vitro HSV-tk gap-filling assay. F272L shows an increase in the frequency of both base substitution mutations and frameshift mutations. Single-enzyme turnover studies of misincorporation by wild type and F272L DNA polymerase beta demonstrate that there is a 4-fold decrease in fidelity of the mutant as compared to that of the wild type enzyme for a G:A mismatch. The decreased fidelity is due primarily to decreased discrimination between the correct and incorrect dNTP during ground-state binding. These results suggest that the phenylalanine 272 residue is critical for maintaining fidelity during the binding of the dNTP.     

Saposins and Their Interaction with Lipids

The lysosomal degradation of several sphingolipids requires the presence of four small glycoproteins called saposins, generated by proteolytic processing of a common precursor, prosaposin. Saposins share several structural properties, including six similarly located cysteines forming three disulfide bridges with the same cysteine pairings. Recently it has been noted that also other proteins have the same polypeptide motif characterized by the similar location of six cysteines. These saposin-like (SAPLIP) proteins are surfactant protein B (SP-B), Entamoeba histolytica poreforming peptide, NK-lysin, acid sphingomyelinase and acyloxyacyl hydrolase. The structural homology and the conserved disulfide bridges suggest for all SAPLIPs a common fold, called saposin fold. Up to now a precise fold, comprising five -helices, has been established only for NK-lysin. Despite their similar structure each saposin promotes the degradation of specific sphingolipids in lysosomes, e.g. Sap B that of sulfatides and Sap C that of glucosylceramides. The different activities of the saposins must reside within the module of the -helices and/or in additional specific regions of the molecule. It has been reported that saposins bind to lysosomal hydrolases and to several sphingolipids. Their structural and functional properties have been extensively reviewed and hypotheses regarding their molecular mechanisms of action have been proposed. Recent work of our group has evidenced a novel property of saposins: some of them undergo an acid-induced change in hydrophobicity that triggers their binding to phospholipid membranes. In this article we shortly review recent findings on the structure of saposins and on their interactions with lipids, with special attention to interactions with phospholipids. These findings offer a new approach for understanding the physiological role of saposins in lysosomes.     

ASPP1 and ASPP2: common activators of p53 family members

We recently showed that ASPP1 and ASPP2 stimulate the apoptotic function of p53. We show here that ASPP1 and ASPP2 also induce apoptosis independently of p53. By binding to p63 and p73 in vitro and in vivo, ASPP1 and ASPP2 stimulate the transactivation function of p63 and p73 on the promoters of Bax, PIG3, and PUMA but not mdm2 or p21(WAF-1/CIP1). The expression of ASPP1 and ASPP2 also enhances the apoptotic function of p63 and p73 by selectively inducing the expression of endogenous p53 target genes, such as PIG3 and PUMA, but not mdm2 or p21(WAF-1/CIP1). Removal of endogenous p63 or p73 with RNA interference demonstrated that (16) the p53-independent apoptotic function of ASPP1 and ASPP2 is mediated mainly by p63 and p73. Hence, ASPP1 and ASPP2 are the first two identified common activators of all p53 family members. All these results suggest that ASPP1 and ASPP2 could suppress tumor growth even in tumors expressing mutant p53.     

Concerted bis-alkylating reactivity of clerocidin towards unpaired cytosine residues in DNA

Clerocidin (CL) is a topoisomerase II poison, which cleaves DNA irreversibly at guanines (G) and reversibly at cytosines (C). Furthermore, the drug can induce enzyme-independent strand breaks at the G and C level. It has been previously shown that G-damage is induced by alkylation of the guanine N7, followed by spontaneous depurination and nucleic acid cleavage, whereas scission at C is obtained only after treatment with hot alkali, and no information is available to explain the nature of this damage. We present here a systematic study on the reactivity of CL towards C both in the DNA environment and in solution. Selected synthetic derivatives were employed to evaluate the role of each chemical group of the drug. The structure of CL–dC adduct was then characterized by tandem mass spectrometry and NMR: the adduct is a stable condensed ring system resulting from a concerted electrophilic attack of the adjacent carbonyl and epoxide groups of CL towards the exposed NH₂ and N3, respectively. This reaction mechanism, shown here for the first time, is characterized by faster kinetic rates than alkylation at G, due to the fact that the rate-determining step, alkylation at the epoxide, is an intramolecular process, provided a Schiff base linking CL and C can rapidly form, whereas the corresponding reaction of G N7 is intermolecular. These results provide helpful hints to explain the reversible/irreversible nature of topoisomerase II mediated DNA damage produced by CL at C/G steps.     

Paleoenvironmental analysis

New analysis has been carried out concerning the palaeoenvironmental reconstruction of some Italian sites dating from the Middle Pleistocene to the Bronze Age. Different aspects have been investigated on each site considering the data collected. The following sites have been analyzed: Isernia La Pineta (Molise); Visogliano and Caverna degli Orsi (Tieste); Toirano Caves (Liguria); Grotta Paglicci (Gargano); Riparo del Molare (Salerno); Grotta del Cavallo (Lecce); Castellaro Lagusello (Monzambano, Mantova).     

MUC3 and MCA serum levels and steroid receptor content in breast cancer

Mucins are an important class of complex glycoproteins expressed by many epithelial cells and their malignant counterparts. The aim of this study was to determine the serum levels of MUC3 and mucin-like carcinoma-associated antigen (MCA) in patients with primary breast cancer and to analyze the possible relationships between these two mucins and the steroid receptor status. The preoperative basal serum levels of MUC3 (ELISA assay with monoclonal antibody 1143/B7) and MCA (EIA assay with anti-MCA mouse monoclonal antibody b-12) were determined in 44 patients with breast cancer while estrogen receptor (ER) and progesterone receptor (PgR) levels were measured by the dextran-coated charcoal method in the cytosol of neoplastic tissue. MUC3 was expressed in 43/44 serum samples while high MCA serum levels were found in 16/44 only; the mean values of both markers did not correlate with menopausal status, tumor size, nodal involvement or ER. The only significant difference observed was a lower median value of MCA in patients with small tumors (T1-T2). No statistically significant correlation between MUC3 and MCA, MUC3 and ER or MCA and ER was observed; a statistically significant direct correlation between MUC3 and PgR+ status and a statistically significant inverse correlation between MCA and PgR+ were observed. Our results suggest that further investigations are necessary to establish whether progesterone can modulate MUC3 and MCA expression in breast cancer.     

Comparative genomics of rice and Arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot

Data mining methods have been used to identify 356 Cyt P450 genes and 99 related pseudogenes in the rice (Oryza sativa) genome using sequence information available from both the indica and japonica strains. Because neither of these genomes is completely available, some genes have been identified in only one strain, and 28 genes remain incomplete. Comparison of these rice genes with the 246 P450 genes and 26 pseudogenes in the Arabidopsis genome has indicated that most of the known plant P450 families existed before the monocot-dicot divergence that occurred approximately 200 million years ago. Comparative analysis of P450s in the Pinus expressed sequence tag collections has identified P450 families that predated the separation of gymnosperms and flowering plants. Complete mapping of all available plant P450s onto the Deep Green consensus plant phylogeny highlights certain lineage-specific families maintained (CYP80 in Ranunculales) and lineage-specific families lost (CYP92 in Arabidopsis) in the course of evolution.     

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.     

Impact of 1,5-disubstituted tetrazole ring on chelating ability of delta-selective opioid peptide

Complex formation between Cu(II) and three tetrazole analogues of opioid peptide-deltorphin I has been investigated. In potentiometric and spectroscopic (UV-Vis, CD and EPR) studies have been established the thermodynamic stability, speciation and structure of Cu(II) complexes with Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (L1), Tyr-Psi(CN4)-Gly-Phe-Asp-Val-Val-Gly-NH2 (L2), Tyr-Gly-Psi(CN4)-Phe-Asp-Val-Val-Gly-NH2 (L3) and Tyr-D-Ala-Psi(CN4)-Phe-Asp-Val-Val-Gly-NH2 (L4). The site of the insertion of tetrazole moiety Psi(CN4) into the peptide sequences has critical impact on their co-ordination ability. Comparison of the binding ability of the tetrazole analogues reveals that around physiological pH region the L3 and L4 are more effective ligands for copper(II) than L(1) and L(2). The peptide conformation changes achieved by Cu(II) co-ordination may be essential for binding of the tetrazole deltorphins at the opiate receptors.