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Palmieri V.O.,University of Bari | De Rasmo D.,University of Bari | Signorile A.,University of Bari | Sardanelli A.M.,University of Bari | And 7 more authors.
Nutrition | Year: 2011

Objectives: Different nuclear genes are thought to be involved in the regulation of the complex phenotype of metabolic syndrome (MS) and their number is increasing. A mutation in mitochondrial DNA (mtDNA), T4291C in transfer RNA isoleucine (tRNAile), has been associated with MS in a large American family. In addition, a mtDNA T16189C variant, already known to be associated with insulin resistance and type 2 diabetes mellitus in Caucasians, seems to underlie susceptibility to MS in the Chinese population. Our aim was to verify the T4291C and T16189C variants in subjects affected by different phenotypes of MS. Methods: Seventy patients with MS and 35 healthy individuals were investigated for the presence of the mtDNA variants by polymerase chain reaction-restriction fragment length polymorphism analysis. Results: The T4291C variant was absent in patients and in controls. The T16189C variant was more frequent in patients with MS than in control subjects (21.4% versus 5.7%, P < 0.04) and was associated with hypertension (P = 0.01), waist circumference (P = 0.02), body mass index (P = 0.009), visceral fat thickness (P = 0.04), homeostasis model assessment (P = 0.03), and the number of MS diagnostic criteria (P = 0.01). Conclusion: The mtDNA T16189C variant is associated with MS and its different clinical expressions. Prospective studies are warranted to establish the clinical relevance of this association. © 2011 Elsevier Inc.


Panelli D.,University of Bari | Lorusso F.P.,University of Bari | Trentadue R.,University of Bari | Stella A.,University of Bari | And 3 more authors.
Biochimie | Year: 2012

In this study the impact of hUPF1 and hUPF2 knockdown on alternative splicing (AS) isoforms of different genes encoding subunits of respiratory chain complex I and complex IV is described. As expected, loss of both hUPF1 and hUPF2 led to impairment of nonsense-mediated mRNA decay (NMD) and accumulation of PTC-containing NMD substrates generated by both complex I and complex IV genes. The levels of some complex I splice variants, which did not contain PTC as well as the level of some complex I canonical transcripts were, however, affected only by hUPF1 knockdown. This finding confirms that NMD plays a role in the maintenance of the transcriptome integrity and reveals a specific impact of hUPF1 on the regulation of complex I genes. © 2012 Elsevier Masson SAS. All rights reserved.


Panelli D.,University of Bari | Lorusso F.P.,University of Bari | Papa F.,University of Bari | Panelli P.,University of Bari | And 5 more authors.
Biochimica et Biophysica Acta - Gene Regulatory Mechanisms | Year: 2013

A study is presented on the regulation of alternative splicing (AS) of the Ndufb11 gene of complex I of the mitochondrial respiratory chain and the impact on this process of rotenone treatment in neuroblastoma cells. In physiological conditions the Ndufb11 gene produces at high level a short transcript isoform encoding for a 153 aa protein. This subunit is essential for the assembly of a functional and stable mammalian complex I. The gene produces also, at low level, a longer transcript isoform encoding for a 163 aa protein whose role is unknown. Evidence is presented here showing that the level of the two isoforms is regulated by three DGGGD ESS elements located in exon 2 which can bind the hnRNPH1 protein. In neuronal cells rotenone treatment affects the Ndufb11 alternative splicing pathway, with the increase of the 163/153 mRNAs ratio. This effect appears to be due to the down-regulation of the hnRNPH1 protein. Since rotenone induces apoptosis in neuronal cells, the post-transcriptional regulation of the Ndufb11 gene can be involved in the programmed cell death process. © 2012 Elsevier B.V.


Volpicella M.,University of Bari | Leoni C.,University of Bari | Costanza A.,University of Bari | de Leo F.,Institute of Biomembranes and Bioenergetics | And 2 more authors.
Current Protein and Peptide Science | Year: 2011

Plant protease inhibitors (PIs) are generally small proteins present in high concentrations in storage tissues (tubers and seeds), and to a lower level in leaves. Even if most of them are active against serine and cysteine proteases, PIs active against aspartic proteases and carboxypeptidases have also been identified. Inhibitors of serine proteases are further classifiable in several families on the basis of their structural features. They comprise the families known as Bowman-Birk, Kunitz, Potato I and Potato II, which are the subject of review articles included in this special issue. In the present article we aim to give an overview of other families of plant PIs, active either against serine proteases or other class of proteases, describing their distribution, activity and main structural characteristics. © 2011 Bentham Science Publishers Ltd.


Guaragnella N.,Institute of Biomembranes and Bioenergetics | Giannattasio S.,Institute of Biomembranes and Bioenergetics | Moro L.,Institute of Biomembranes and Bioenergetics
Biochemical Pharmacology | Year: 2014

Mitochondrial dysfunction has been associated with cancer development and progression. Recent evidences suggest that pathogenic mutations or depletion of the mitochondrial genome can contribute to development of chemoresistance in malignant tumors. In this review we will describe the current knowledge on the role of mitochondrial dysfunction in the development of chemoresistance in cancer. We will also discuss the significance of this research topic in the context of development of more effective, targeted therapeutic modalities and diagnostic strategies for cancer patients, with a particular focus on the potential use of PARP inhibitors in cancer patients displaying mitochondrial DNA mutations. We will discuss recent studies highlighting the importance of the cross-talk between the tumor microenvironment and mitochondrial functionality in determining selective response to certain chemotherapeutic drugs. Finally, owing to the similarities between cancer and yeast cell metabolism, we will point out the use of yeast as a model system to study cancer-related genes and for anti-cancer drugs screening. © 2014 Elsevier Inc. All rights reserved.


Moro L.,Institute of Biomembranes and Bioenergetics | Guaragnella N.,Institute of Biomembranes and Bioenergetics | Giannattasio S.,Institute of Biomembranes and Bioenergetics
Journal of Visualized Experiments | Year: 2015

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 °C instead of the conventional 70-100 °C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other 'large' proteins like BRCA2 should be readily achievable. © 2015 Journal of Visualized Experiments.


PubMed | Institute of Biomembranes and Bioenergetics
Type: Journal Article | Journal: Biochemical pharmacology | Year: 2014

Mitochondrial dysfunction has been associated with cancer development and progression. Recent evidences suggest that pathogenic mutations or depletion of the mitochondrial genome can contribute to development of chemoresistance in malignant tumors. In this review we will describe the current knowledge on the role of mitochondrial dysfunction in the development of chemoresistance in cancer. We will also discuss the significance of this research topic in the context of development of more effective, targeted therapeutic modalities and diagnostic strategies for cancer patients, with a particular focus on the potential use of PARP inhibitors in cancer patients displaying mitochondrial DNA mutations. We will discuss recent studies highlighting the importance of the cross-talk between the tumor microenvironment and mitochondrial functionality in determining selective response to certain chemotherapeutic drugs. Finally, owing to the similarities between cancer and yeast cell metabolism, we will point out the use of yeast as a model system to study cancer-related genes and for anti-cancer drugs screening.


PubMed | Institute of Biomembranes and Bioenergetics
Type: | Journal: Journal of visualized experiments : JoVE | Year: 2015

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 C instead of the conventional 70-100 C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other large proteins like BRCA2 should be readily achievable.

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