Clinical, cytogenetic and molecular-cytogenetic characterization of a patient with a de novo tandem proximal-intermediate duplication of 16q and review of the literature.

Partial trisomy 16 is rare and most of the reported cases are secondary to chromosome rearrangements resulting in concurrent monosomies or trisomies of a second chromosome. Only a few patients survive the neonatal period and the duplication of the long arm seems to be mainly responsible for the prenatal lethality of the full trisomy 16. The reported patients with a partial 16q trisomy have a wide spectrum of congenital anomalies that include dysmorphic features, central nervous system malformations, failure to thrive, and club feet. The patients with duplications of proximal 16q frequently have short stature, developmental delay, speech delay, learning difficulties, and mild to severe behavioral problems. Here we describe a patient with an inverted de novo tandem duplication of 16q with breakpoints evaluated in detail by molecular-cytogenetic techniques. Main clinical features include postural, motor and speech delay with severe learning difficulties and behavioral problems, obesity, microcephaly, and mild dysmorphic features. In the report we attempt to classify the few reported patients with pure partial duplications of 16q in more narrow and homogeneous groups: proximal, proximal-intermediate, intermediate, and intermediate-distal duplications. Moreover, we emphasize the importance of proper cytogenetic investigation and complete molecular cytogenetic refinement in all cases with a suspected chromosomal anomaly.

Protein isoaspartate methyltransferase prevents apoptosis induced by oxidative stress in endothelial cells: role of Bcl-Xl deamidation and methylation.

BACKGROUND: Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal alpha-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s). METHODOLOGY/PRINCIPAL FINDINGS: Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H(2)O(2); 0.1-0.5 mM range). Results show that A) Cells overexpressing "wild type" human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H(2)O(2) concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H(2)O(2) treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase. CONCLUSION/SIGNIFICANCE: Apoptosis can be modulated by "on/off" switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein.

Matrix metalloproteinases and their inhibitors as biomarkers for metal toxicity in vitro.

Exposure to nickel and chromium, and their compounds, has been associated with adverse health effects. These metals are two human carcinogens whose pathogenesis involves active extracellular matrix degradation and remodelling. In this work we have compared the effects of in vitro exposure to nickel and chromium of a keratinocyte cell line (HaCat). The modulation of matrix metalloproteinase genes was used as biomarker of chemical damage. Confluent cells were constantly exposed to subtoxic chromium and nickel concentrations (10(-5) and 10(-7)M) up to 72 h. Total RNA was extracted and specific matrix metalloproteinase, and inhibitor, gene expression was analyzed by RT-PCR. Moreover, cell cycle alterations were evaluated by flow cytometry. Nickel and chromium showed different results, with an upregulation of MMP-2 mRNA production in nickel-treated cells while chromium exposure down-regulated MMP-2 mRNA production. This result could be correlated to the precocious (6h) over-expression of tissue inhibitor-1 (TIMP-1) mRNA in chromium-treated cells. Cell cycle analysis showed and increase of cells with 4N DNA. These results could be explained as a survival response of cells that escape metal induced apoptosis through the anti-apoptotic effects of TIMP-1. These cells that encompass the genotoxic insult may have a selective proliferation advantage, and therefore represent the precursor pool from which degenerating variants may emerge. To study if the chemical damage was reversible, subconfluent cells were stimulated only for 24 h, then the medium was replaced without metal. Cells were able to recover from nickel exposure, showing only weak alterations in specific mRNA expression and cell cycle alteration respect to control. Chromium-induced damage was irreversible. Our results demonstrated that there is an association between metal toxicity and expression of MMPs and their inhibitors. These biomarkers could be potentially useful to elaborate a prediction model of chemical toxicity.

Hydroxytyrosol, a natural antioxidant from olive oil, prevents protein damage induced by long-wave ultraviolet radiation in melanoma cells.

Previous studies showed that long-wave ultraviolet (UVA) radiation induces severe skin damage through the generation of reactive oxygen species and the depletion of endogenous antioxidant systems. Recent results from our laboratory indicate a dramatic increase of both lipid peroxidation products (TBARS) and abnormal L-isoaspartyl residues, marker of protein damage, in UVA-irradiated human melanoma cells. In this study, the effects of hydroxytyrosol (DOPET), the major antioxidant compound present in olive oil, on UVA-induced cell damages, have been investigated, using a human melanoma cell line (M14) as a model system. In UVA-irradiated M14 cells, a protective effect of DOPET in preventing the uprise of typical markers of oxidative stress, such as TBARS and 2'7'-dichlorofluorescein (DCF) fluorescence intensity, was observed. In addition, DOPET prevents the increase of altered L-isoAsp residues induced by UVA irradiation. These protective effects are dose dependent, reaching the maximum at 400 microM DOPET. At higher concentrations, DOPET causes an arrest of M14 cell proliferation and acts as a proapoptotic stimulus by activating caspase-3 activity. In the investigated model system, DOPET is quantitatively converted into its methylated derivative, endowed with a radical scavenging ability comparable to that of its parent compound. These findings are in line with the hypothesis that the oxidative stress plays a major role in mediating the UVA-induced protein damage. Results suggest that DOPET may exerts differential effects on melanoma cells according to the dose employed and this must always be taken into account when olive oil-derived large consumer products, including cosmetics and functional foods, are employed.

Folate treatment and unbalanced methylation and changes of allelic expression induced by hyperhomocysteinaemia in patients with uraemia.

BACKGROUND: Hyperhomocysteinaemia occurs in several genetically determined and acquired disorders and is highly prevalent in patients with uraemia. In these disorders, homocysteine precursor S-adenosylhomocysteine, a powerful competitive inhibitor of S-adenosylmethionine-dependent methyltransferases, is increased, suggesting unbalanced methylation. We aimed to investigate whether DNA hypomethylation is present in patients with uraemia who also have hyperhomocysteinaemia and whether regulation of specific classes of genes, dependent on DNA methylation, is compromised. METHODS: We selected men with hyperhomocysteinaemia and uraemia who were having standard haemodialysis treatment, and compared them with healthy male controls. We measured the homocysteine concentration from plasma samples and obtained DNA and RNA samples from peripheral mononuclear cells. DNA methylation was assessed by cytosine extension assay and by Southern blotting. Allelic expression of pseudoautosomal and imprinted genes was investigated by analysis of suitable restriction fragment length polymorphisms. FINDINGS: Total DNA hypomethylation was higher in patients than in controls (z score -4.593, p=0.0006) and allelic expression was changed in both sex-linked and imprinted genes. The shift from monoallelic to biallelic expression was dependent on homocysteine concentrations. Folate therapy, a common method to reduce hyperhomocysteinaemia, restored DNA methylation to normal levels and corrected the patterns of gene expression. INTERPRETATION: Our results suggest that hyperhomocysteinaemia affects epigenetic control of gene expression, which can be reverted by folate treatment. Our data support the hypothesis that the toxic action of homocysteine can be mediated by macromolecule hypomethylation.