Radical Cations in Aromatic Hydrocarbon Carcinogenesis

Most carcinogens, including polycyclic aromatic hydrocarbons (PAH), require metabolic activation to produce the ultimate electrophilic species that bind covalently with cellular macromolecules to trigger the cancer process. Metabolic activation of PAH can be understood in terms of two main pathways: one-electron oxidation to yield reactive intermediate radical cations and monooxygenation to produce bay-region diol epoxides. The reason we have postulated that one-electron oxidation plays an important role in the activation of PAH derives from certain common characteristics of the radical cation chemistry of the most potent carcinogenic PAH. Two main features common to these PAH are: 1) a relatively low ionization potential, which allows easy metabolic removal of one electron, and 2) charge localization in the PAH radical cation that renders this intermediate specifically and efficiently reactive toward nucleophiles. Equally important, cytochrome P-450 and mammalian peroxidases catalyze one-electron oxidation. This mechanism plays a role in the binding of PAH to DNA. Chemical, biochemical and biological evidence will be presented supporting the important role of one-electron oxidation in the activation of PAH leading to initiation of cancer.     

Strobilomyces echinocephalus sp. nov. (Boletales) from south-western China, and a key to the genus Strobilomyces worldwide

Strobilomyces echinocephalus Gelardi & Vizzini sp. nov. is described as a new species, based on collections from Kunming (Yunnan Province, China). Macro-morphological and micro-morphological features, ecological data and analyses of RPB1 and ITS2 sequences support the establishment of this new taxon. Colour pictures of fresh basidiomata in habitat, scanning electron microscope (SEM) micro-photographs of the spores, and a line-drawing displaying the most relevant anatomical characters are provided in this paper. Strobilomyces zangii Gelardi nom. nov. is proposed to replace the name Heimiella nigricans M. Zang. A dichotomous key to the Strobilomyces species at global level is also provided at the end of this work.     

Involvement of Microorganisms in the Formation of Carbonate Speleothems in the Cervo Cave (L'Aquila-Italy)

Much is known about the bacterial precipitation of carbonate rocks, but comparatively little is known about the involvement of microbes in the formation of secondary mineral structures in caves. We hypothesized that bacteria isolated from calcareous stalactites, which are able to mediate CaCO₃ precipitation in vitro, play a role in the formation of carbonate speleothems. We collected numerous cultivable calcifying bacteria from calcareous speleothems from Cervo cave, implying that their presence was not occasional. The relative abundance of calcifying bacteria among total cultivable microflora was found to be related to the calcifying activity in the stalactites. We also determined the δ ¹³C and δ ¹⁸ O values of the Cervo cave speleothems from which bacteria were isolated and of the carbonates obtained in vitro to determine whether bacteria were indeed involved in the formation of secondary mineral structures. We identified three groups of biological carbonates produced in vitro at 11°C on the basis of their carbon isotopic composition: carbonates with δ ¹³C values (a) slightly more positive, (b) more negative, and (c) much more negative than those of the stalactite carbonates. The carbonates belonging to the first group, characterized by the most similar δ ¹³C values to stalactites, were produced by the most abundant strains. Most of calcifying isolates belonged to the genus Kocuria. Scanning electron microscopy showed that dominant morphologies of the bioliths were sherulithic with fibrous radiated interiors. We suggest a mechanism of carbonate crystal formation by bacteria.     

The multiple forms of bovine seminal ribonuclease: Structure and stability of a C-terminal swapped dimer

Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111–116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed.     

Astrocyte-Specific Overexpression of Insulin-Like Growth Factor-1 Protects Hippocampal Neurons and Reduces Behavioral Deficits following Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) survivors often suffer from long-lasting cognitive impairment that stems from hippocampal injury. Systemic administration of insulin-like growth factor-1 (IGF-1), a polypeptide growth factor known to play vital roles in neuronal survival, has been shown to attenuate posttraumatic cognitive and motor dysfunction. However, its neuroprotective effects in TBI have not been examined. To this end, moderate or severe contusion brain injury was induced in mice with conditional (postnatal) overexpression of IGF-1 using the controlled cortical impact (CCI) injury model. CCI brain injury produces robust reactive astrocytosis in regions of neuronal damage such as the hippocampus. We exploited this regional astrocytosis by linking expression of hIGF-1 to the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, effectively targeting IGF-1 delivery to vulnerable neurons. Following brain injury, IGF-1Tg mice exhibited a progressive increase in hippocampal IGF-1 levels which was coupled with enhanced hippocampal reactive astrocytosis and significantly greater GFAP levels relative to WT mice. IGF-1 overexpression stimulated Akt phosphorylation and reduced acute (1 and 3d) hippocampal neurodegeneration, culminating in greater neuron survival at 10d after CCI injury. Hippocampal neuroprotection achieved by IGF-1 overexpression was accompanied by improved motor and cognitive function in brain-injured mice. These data provide strong support for the therapeutic efficacy of increased brain levels of IGF-1 in the setting of TBI.     

Phylogeny and morphology of the <Emphasis Type="Italic">Peziza ammophila</Emphasis> complex (Pezizales,Ascomycota), with description of two new species and a new form

This study was focused on species of Peziza belonging to the “P. ammophila” complex, using both morphological and molecular approaches. Molecular and morphological analyses showed that several taxa are hidden under the name “ammophila” and, as a consequence, two additional species and one form are established: P. hellenica sp. nov., P. oceanica sp. nov. and P. ammophila f. megaspora f. nov. On the basis of the data that we have, these taxa seem to be related to some geographical areas; P. ammophila appears to be prevalent in northern, central and southern Europe, its f. megaspora is distributed in France and in the Netherlands, P. hellenica comes from Greece and P. oceanica from New Zealand. A cryptic species, not yet circumscribed satisfactorily, is described as P. deceptiva ad int. Moreover, the problem concerning the identification of P. ammophila is raised, the species is epitypified with a sequenced collection from Sicily (Italy) and a key to the species of the complex is provided.     

Evolutionary Rate and Genetic Heterogeneity of Human T-Cell Lymphotropic Virus Type II (HTLV-II) Using Isolates from European Injecting Drug Users

Seven new Italian and two new British HTLV-II isolates were obtained from injecting drug users and the entire long terminal repeat (LTR) region was sequenced. Restriction analysis showed that all the Italian isolates are of the IIb subtype, whereas the British isolates are of the IIa subtype. To understand whether the further differentiation of each two principal HTLV-II subtypes in several subgroups could be statistically supported by phylogenetic analysis, the neighbor-joining, parsimony, and maximum likelihood methods were used. The separation between IIa and IIb is very well supported by all three methods. At least two phylogenetic subgroups exist within the HTLV-IIa and at least three within the HTLV-IIb subtype. In the present analysis, no statistical support was obtained for additional phylogroups. Two particular subgroups seem interesting because they include all European and North American injecting drug user strains within the IIa and IIb subtypes, respectively. These data confirm that European HTLV-II infection among drug users is probably derived from North America. They also suggest that though a certain differentiation by restriction analysis in different subgroups is possible, carefully interpreted phylogenetic analyses remain necessary. Using the likelihood ratio test, a molecular clock for the drug user strains was calibrated. A fixation rate between 1.08 × 10⁻⁴ and 2.7 × 10⁻⁵ nucleotide substitutions per site per year was calculated for the IIa and IIb injecting drug user strains. This is the lowest fixation rate so far reported for RNA viruses, including for HIV, which typically range between 10⁻² and 10⁻⁴.     

Formation of dopamine quinone-DNA adducts and their potential role in the etiology of Parkinson's disease

The neurotransmitter dopamine is oxidized to its quinone (DA-Q), which at neutral pH undergoes intramolecular cyclization by 1,4-Michael addition, followed by oxidation to form leukochrome, then aminochrome, and finally neuromelanin. At lower pH, the amino group of DA is partially protonated, allowing the competitive intermolecular 1,4-Michael addition with nucleophiles in DNA to form the depurinating adducts, DA-6-N3Ade and DA-6-N7Gua. Catechol estrogen-3,4-quinones react by 1,4-Michael addition to form the depurinating 4-hydroxyestrone(estradiol)-1-N3Ade [4-OHE1(E2)-1-N3Ade] and 4-OHE1(E2)-1-N7Gua adducts, which are implicated in the initiation of breast and other human cancers. The effect of pH was studied by reacting tyrosinase-activated DA with DNA and measuring the formation of depurinating adducts. The most adducts were formed at pH 4, 5, and 6, and their level was nominal at pH 7 and 8. The N3Ade adduct depurinated instantaneously, but N7Gua had a half-life of 3 H. The slow loss of the N7Gua adduct is analogous to that observed in previous studies of natural and synthetic estrogens. The antioxidants N-acetylcysteine and resveratrol efficiently blocked formation of the DA-DNA adducts. Thus, slightly acidic conditions render competitive the reaction of DA-Q with DNA to form depurinating adducts. We hypothesize that formation of these adducts could lead to mutations that initiate Parkinson's disease. If so, use of N-acetylcysteine and resveratrol as dietary supplements may prevent initiation of this disease.     

The short stature homeodomain protein SHOX induces cellular growth arrest and apoptosis and is expressed in human growth plate chondrocytes

Mutations in the homeobox gene SHOX cause growth retardation and the skeletal abnormalities associated with Léri-Weill, Langer, and Turner syndromes. Little is known about the mechanism underlying these SHOX-related inherited disorders of bone formation. Here we demonstrate that SHOX expression in osteogenic stable cell lines, primary oral fibroblasts, and primary chondrocytes leads to cell cycle arrest and apoptosis. These events are associated with alterations in the expression of several cellular genes, including pRB, p53, and the cyclin kinase inhibitors p21(Cip1) and p27(Kip1). A SHOX mutant, such as seen in Léri-Weill syndrome patients, does not display these activities of the wild type protein. We have also shown that endogenous SHOX is mainly expressed in hypertrophic/apoptotic chondrocytes of the growth plate, strongly suggesting that the protein plays a direct role in regulating the differentiation of these cells. This study provides the first insight into the biological function of SHOX as regulator of cellular proliferation and viability and relates these cellular events to the phenotypic consequences of SHOX deficiency.