GJB2 c.35del variant up-regulates GJA1 gene expression and affects differentiation of human stem cells.

Pathogenic DNA alterations in GJB2 are present in nearly half of non-syndromic hearing loss cases with autosomal recessive inheritance. The most frequent variant in GJB2 causing non-syndromic hearing loss is the frameshifting c.35del. GJB2 encodes Cx26, a protein of the connexin family that assembles hemichannels and gap junctions. The expression of paralogous proteins is believed to compensate for the loss of function of specific connexins. As Cx26 has been involved in cell differentiation in distinct tissues, we employed stem cells derived from human exfoliated deciduous teeth (SHEDs), homozygous for the c.35del variant, to assess GJB2 roles in stem cell differentiation and the relationship between its loss of function and the expression of paralogous genes. Primary SHED cultures from patients and control individuals were compared. SHEDs from patients had significantly less GJB2 mRNA and increased amount of GJA1 (Cx43), but not GJB6 (Cx30) or GJB3 (Cx31) mRNA. In addition, they presented higher induced differentiation to adipocytes and osteocytes but lower chondrocyte differentiation. Our results suggest that GJA1 increased expression may be involved in functional compensation for GJB2 loss of function in human stem cells, and it may explain changes in differentiation properties observed in SHEDs with and without the c.35del variant.

Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells.

Thiol peroxidases (TP) are ubiquitous and abundant antioxidant proteins of the peroxiredoxin and glutathione peroxidase families that can catalytically and rapidly reduce biologically relevant peroxides, such as hydrogen peroxide and peroxynitrite. However, the TP catalytic cycle is complex, depending on multiple redox reactions and partners, and is subjected to branching and competition points that may limit their peroxide reductase activity in vivo. The goals of the present study were to demonstrate peroxynitrite reductase activity of TP members in live cells in real time and to evaluate its catalytic characteristics. To these ends, we developed a simple fluorescence assay using coumarin boronic acid (CBA), exploiting that fact that TP and CBA compete for peroxynitrite, with the expectation that higher TP peroxynitrite reductase activity will lower the CBA oxidation. TP peroxynitrite reductase activity was evaluated by comparing CBA oxidation in live wild type and genetically modified Δ8 (TP-deficient strain) and Δ8+TSA1 (Δ8 strain that expresses only one TP member, the TSA1 gene) Saccharomyces cerevisiae strains. The results showed that CBA oxidation decreased with cell density and increased with increasing peroxynitrite availability. Additionally, the rate of CBA oxidation decreased in the order Δ8 > Δ8+TSA1 > WT strains both in control and glycerol-adapted (expressing higher TP levels) cells, showing that the CBA competition assay could reliably detect peroxynitrite in real time in live cells, comparing CBA oxidation in strains with reduced and increased TP expression. Finally, there were no signs of compromised TP peroxynitrite reductase activity during experimental runs, even at the highest peroxynitrite levels tested. Altogether, the results show that TP is a major component in the defense of yeast against peroxynitrite insults under basal and increasing stressful conditions.

Oxidative stress response in sugarcane

A resposta ao estresse oxidativo näo é bem conhecida em plantas como em bactérias, leveduras e humanos. Por exemplo, assume-se que óxido nítrico tem várias funções em plantas apesar do gene que codificaria para óxido nítrico sintetase nunca ter sido isolado. Este trabalho descreve os resultados de uma busca no banco de dados de seqüências expressas de cana de açúcar (SUCEST) de genes envolvidos na resposta ao estresse oxidativo. Eu näo encontrei genes similares a óxido nítrico no banco do dados do SUCEST, mas uma via alternativa para a produçäo deste radical livre pode ser proposta. Eu também encontrei vários genes envolvidos na defesa antioxidante, como quelantes de metais, antioxidantes de baixo peso molecular, enzimas antioxidantes e sistemas de reparo. Ascorbato (vitamina C) é um importante antioxidante em plantas porque é encontrado em altas concentrações em células vegetais e porque é substrato de ascorbato peroxidase, uma enzima que eu encontrei em diferentes isoformas no banco de dados do SUCEST. Eu também encontrei várias enzimas envolvidas na biossíntese de antioxidantes de baixo peso molecular que podem ser alvos para manipulaçäo genética. A obtençäo de plantas modificadas geneticamente que sintetizariam vitaminas C e E em altos níveis poderiam melhorar o valor nutritional e a tolerância a estresses de cana de açúcar. Os diversos componentes do sistema de defesa antioxidante interagem entre si e as suas sínteses devem ser muito bem reguladas. Fatores de transcriçäo envolvidos na regulaçäo da resposta ao estresse oxidativo de bactérias, leveduras e de humanos diferem consideravelmente entre si e quando foram utilizados para buscas no banco de dados do SUCEST, somente genes com similaridades fracas foram encontrados, sugerindo que estas proteínas näo säo muito conservadas. O envolvimento de espécies reativas de oxigênio e nitrogênio na defesa de plantas contra patógenos também é discutido neste trabalho.