Dysregulation of mitochondrial function and biogenesis modulators in adipose tissue of obese children.

BACKGROUND/OBJECTIVES: We aimed to evaluate mitochondrial biogenesis (MB), structure, metabolism and dysfunction in abdominal adipose tissue from male pediatric patients with obesity. SUBJECTS/METHODS: Samples were collected from five children with obesity (percentile ⩾95) and five eutrophic boys (percentile ⩾5/⩽85) (8-12 years old) following parental informed consent. We analyzed the expression of key genes involved in MB (sirtuin-1 (SIRT1), peroxisome proliferator-activated receptor-γ (PPARγ), PPARγ coactivator-1α (PGC1α), nuclear respiratory factors 1 and 2 (NRF1, NRF2) and mitochondrial transcription factor A (TFAM) and surrogates for mitochondrial function/structure/metabolism (porin, TOMM20, complex I and V, UCP1, UCP2, SIRT3, SOD2) by western blot. Citrate synthase (CS), complex I (CI) activity, adenosine triphosphate (ATP) levels, mitochondrial DNA (mtDNA) content and oxidative stress end points were also determined. RESULTS: Most MB proteins were significantly decreased in samples from children with obesity except complex I, V and superoxide dismutase-2 (SOD2). Similarly, CS and CI activity showed a significant reduction, as well as ATP levels and mtDNA content. PPARγ, PGC1α, complex I and V and SOD2 were hyperacetylated compared with lean samples. Concurrently, in samples from children with obesity, we found decreased SOD2 activity and redox state imbalance highlighted by decreased reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and significant increases in protein carbonylation. CONCLUSIONS: Adipose tissue from children with obesity demonstrates a dysregulation of key modulators of MB and organelle structure, and displays hyperacetylation of key proteins and altered expression of upstream regulators of cell metabolism.

An oligoarray for the detection of human papillomavirus type 16 variants.

On the basis of human papillomavirus (HPV) E6 gene mutations, there are more than five variants of HPV 16. We applied a sensitive and specific stacking hybridization assay using an oligoarray for the detection of Asian-American (AA) and European (E) (E350G) HPV 16 variants. A simple glass slide was coated with capture probes consisting of short oligonucleotide DNA sequences (7-9 mers) specific for AA and E variants. Two different regions of the E6 HPV 16 gene were amplified with a set of two primers, which were used as target DNA. These targets were preannealed with auxiliary labeled oligonucleotides and hybridized to the oligoarray in the presence of specific and complementary capture probes. Our designed array based on shorter capture probes successfully discriminated between HPV 16 AA and E variants. The present DNA oligoarray system could be useful as a reliable technique for HPV 16 detection and does not require specialized equipment; nevertheless, further intra- and interlaboratory studies are needed.

Differentially expressed genes between high-risk human papillomavirus types in human cervical cancer cells.

Cervical carcinoma (CC) is one of the most common cancers among women worldwide and the first cause of death among the Mexican female population. Human papillomavirus (HPV) infection is the most important etiologic factor for CC. Of the oncogenic types, HPV16 and HPV18 are found in 60-70% of invasive CCs worldwide. HPV18 appears to be associated with a more aggressive form of cervical neoplasia than HPV16 infection. At present, there are no studies on differentially expressed cellular genes between transformed cells harboring HPV16 and HPV18 sequences. Based on previous complementary DNA microarray data from our group, 13 genes were found to be differentially overexpressed between HPV16- and HPV18-transformed cells. These genes were as follows: E6BP, UBE4A, C20orf14, ATF7, ABCC8, SLC6A12, WASF3, SUV39H1, SPAG8, CCNC, E2FFE, BIRC5, and DEDD. Differential expression of six selected genes was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). All real-time RT-PCRs confirmed differential expression between HPV18 and HPV(-) samples. The present work identifies genes from signaling pathways triggered by HPV transformation that could be differentially deregulated between HPV16(+) and HPV18(+) samples.