The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.

Rationale: NADPH oxidases are a major source of superoxide (O 2 ؊ ) in the cardiovascular system. The function of Nox4, a member of the Nox family of NADPH oxidases, in the heart is poorly understood. Objective: The goal of this study was to elucidate the role of Nox4 in mediating oxidative stress and growth/death in the heart. Methods and Results: Expression of Nox4 in the heart was increased in response to hypertrophic stimuli and aging.Neither transgenic mice with cardiac specific overexpression of Nox4 (Tg-Nox4) nor those with catalytically inactive Nox4 (Tg-Nox4-P437H) showed an obvious baseline cardiac phenotype at young ages. Tg-Nox4 gradually displayed decreased left ventricular (LV) function with enhanced O 2 ؊ production in the heart, which was accompanied by increased apoptosis and fibrosis at 13 to 14 months of age. On the other hand, the level of oxidative stress was attenuated in Tg-Nox4-P437H. Although the size of cardiac myocytes was significantly greater in Tg-Nox4 than in nontransgenic, the LV weight/tibial length was not significantly altered in Tg-Nox4 mice. Overexpression of Nox4 in cultured cardiac myocytes induced apoptotic cell death but not hypertrophy. Nox4 is primarily localized in mitochondria and upregulation of Nox4 enhanced both rotenone-and diphenyleneiodonium-sensitive O 2 ؊ production in mitochondria. Cysteine residues in mitochondrial proteins, including aconitase and NADH dehydrogenases, were oxidized and their activities decreased in Tg-Nox4. Key Words: reactive oxygen species Ⅲ oxidative stress Ⅲ superoxide Ⅲ hypertrophy Ⅲ apoptosis Ⅲ aging R eactive oxygen species (ROS), such as superoxide (O 2 Ϫ ) and H 2 O 2 , play an important role in regulating cell growth and death of cardiac myocytes. [1][2][3] In the heart under pathological conditions, mitochondria are the major source of ROS, which are generated primarily through electron leakage from the electron transport chain. 4 The leakage of electrons is a passive process caused by damage and/or downregulation of mitochondrial proteins, and does not appear to be tightly regulated. 5 ROS are also produced through O 2 Ϫ -producing enzymes, such as NADPH oxidases and xanthine oxidase. Although NADPH oxidases are the major source of O 2 Ϫ production, their contribution to overall increases in ROS and myocardial responses under stress is not fully understood. Thus far, seven members of the NADPH oxidase (Nox) family of proteins (Nox1 to Nox5 and Duox1 and 2) have been identified. 6 -8 All Nox proteins possess 6 membranespanning domains and a cytoplasmic region containing NAD(P)H-and FAD-binding domains in their C-terminal regions. Nox1, -2, -3 and -4 form a heterodimer with p22 phox , another catalytic core component of NADPH oxidases which stabilizes Nox proteins. Nox proteins accept electrons from either NADPH or NADH 8,9 and transfer them to molecular oxygen to generate O 2 Ϫ .Nox4 is ubiquitously expressed in various cell types and tissues, including kidneys, the heart, and blood vessels. 10,11 Distinct from other members of...

Although originally considered toxic, hydrogen sulfide (H2S) has been implicated in mediating various biological processes. Nevertheless, its cellular targets and mode of action are not well understood. Protein Tyrosine Phosphatases (PTPs), which regulate numerous signal transduction pathways, utilize an essential Cys residue at the active site, which is characterized by a low pKa and is susceptible to reversible oxidation. Here, we report that PTP1B, the founding member of this enzyme family, was reversibly inactivated by H2S, in vitro and in vivo, via sulfhydration of the active site Cys residue. Unlike oxidized PTP1B, the sulfhydrated enzyme was preferentially reduced by thioredoxin in vitro, compared to glutathione or dithiothreitol. Sulfhydration of the active site Cys in PTP1B in cells required the presence of cystathionine-γ-lyase (CSE), a critical enzyme in H2S production, and resulted in inhibition of phosphatase activity. Suppression of CSE decreased H2S production and decreased the phosphorylation on Tyr619, and activation, of PERK [protein kinase-like endoplasmic reticulum (ER) kinase] in response to ER stress. PERK, which phosphorylates the eukaryotic translational initiation factor 2 (eIF2α) leading to attenuation of protein translation, was a direct substrate of PTP1B. In addition, CSE knockdown also led to activation of SRC, previously shown to be mediated by PTP1B. These effects of suppressing H2S production on the response to ER stress were abrogated by a small molecule inhibitor of PTP1B. Together, these data define a signaling function for H2S in inhibiting PTP1B activity and thereby promoting PERK activity during the response to ER stress.

Transnitrosylation and denitrosylation are emerging as key post-translational modification events in regulating both normal physiology and a wide spectrum of human diseases.