Sirtuin 3, a New Target of PGC-1α, Plays an Important Role in the Suppression of ROS and Mitochondrial Biogenesis

Background

Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1α induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.

Results

Here we show that PGC-1α strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1α led to decreased Sirt3 gene expression. PGC-1α activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (−407/−399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRα bound to the identified ERRE and PGC-1α co-localized with ERRα in the mSirt3 promoter. Knockdown of ERRα reduced the induction of Sirt3 by PGC-1α in C₂C₁₂ myotubes. Furthermore, Sirt3 was essential for PGC-1α-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1α in C₂C₁₂ myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1α on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1α on mitochondrial biogenesis in C₂C₁₂ myotubes.

Conclusion

Our results indicate that Sirt3 functions as a downstream target gene of PGC-1α and mediates the PGC-1α effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease.     

Streptomyces sp. 173, an insecticidal micro-organism from marine

AIMS: To find new insecticidal antibiotics from marine micro-organisms. METHODS AND RESULTS: Strains isolated from seawater and sea sediments from Beidiahe and Dagang of the east coast of China were screened for their insecticidal qualities. The screening was carried out using bioassay of brine shrimp and the insect pest Helicoverpa armigera. The fermentation, preliminary extraction and isolation of Streptomyces sp.173 were carried out. CONCLUSIONS: In total 331 isolates were examined through bioassay of brine shrimp and 40 isolates (12.08%) showed potential insecticidal activities. Of the 40 isolates, one isolate, designated Streptomyces sp.173, was found to have strong insecticidal activity against both brine shrimp and H. armigera, similar to that of avermectin B1. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated Streptomyces sp.173 has great insecticidal potency. This work indicated that marine micro-organisms could be an important source of insecticidal antibiotics and the improved anti-brine shrimp bioassay is suitable for primary screening.     

Highly sensitive near-simultaneous assay of multiple "lean meat agent" residues in swine urine using a disposable electrochemiluminescent immunosensors array

Nowadays, β(2)-agonists are abused illegally as "lean meat agents" for food-producing animals, and cause increasing food-safety accidents in some countries. Due to their hazard to the human health, "lean meat agents" are banned in most countries and required to be routinely monitored. We herein report a disposable electrochemiluminescent immunosensors array for near-simultaneous assay of multiple β(2)-agonist residues in swine urine, by using ractopamine and salbutamol as the models. In this investigation, a screen-printed carbon electrodes array was assembled and acted as the substrate of the immunosensors array. Then the immunosensors array was constructed by site-selectively immobilizing the antigens of ractopamine and salbutamol on the working electrodes of array. After the competitive immuno-binding, with the aid of a homemade single-pore-four-throw switch, the electrochemiluminescent signals of the two β(2)-agonists were sequentially detected using a non-array detector. The limits of detection for ractopamine and salbutamol were 8.5 and 17pg/mL, respectively, which were much lower than those of the most previous reports. Compared with other routine methods based on chromatography and ELISA, this method is more suitable for screening of multiple β(2)-agonists in quantities of samples, owing to its merits of low cost, user-friendliness and high throughput, and shows great promise in food safety and agonist surveillance.     

Integrin-associated molecules and signalling cross talking in osteoclast cytoskeleton regulation

In the ageing skeleton, the balance of bone reconstruction could commonly be broken by the increasing of bone resorption and decreasing of bone formation. Consequently, the bone resorption gradually occupies a dominant status. During this imbalance process, osteoclast is unique cell linage act the bone resorptive biological activity, which is a highly differentiated ultimate cell derived from monocyte/macrophage. The erosive function of osteoclasts is that they have to adhere the bone matrix and migrate along it, in which adhesive cytoskeleton recombination of osteoclast is essential. In that, the podosome is a membrane binding microdomain organelle, based on dynamic actin, which forms a cytoskeleton superstructure connected with the plasma membrane. Otherwise, as the main adhesive protein, integrin regulates the formation of podosome and cytoskeleton, which collaborates with the various molecules including: c-Cbl, p130^Cas, c-Src and Pyk2, through several signalling cascades cross talking, including: M-CSF and RANKL. In our current study, we discuss the role of integrin and associated molecules in osteoclastogenesis cytoskeletal, especially podosomes, regulation and relevant signalling cascades cross talking.     

β3受体激动剂对培养的大鼠心肌细胞搏动频率和细胞内环-磷酸腺苷水平的影响

目的：观察β3受体激动剂(BRL-37344)对培养的大鼠心肌细胞搏动频率和细胞内环-磷酸腺苷(cAMP)水平的影响,以探讨β3受体在心肌细胞中的作用。方法：分离培养乳鼠心肌细胞,随机分为八组：对照组、ISO组、Nadolol ISO组、BRL组、PTX BRL组、L-NAME BRL、Nadolol BRL组和Buprandol BRL组,观察心肌细胞搏动频率,并应用酶联免疫方法测定cAMP含量,逆转录多聚酶链反应(RT-PCR)方法测β3受体mRNA表达。结果：ISO(非选择性β受体激动剂)可显著增加心肌细胞搏动频率和升高cAMP水平,这种作用可被Nadolol(为β1,β2受体抑制剂)阻断。BRL-37344可显著降低心肌细胞搏动频率和cAMP含量,这种作用可被PTX(Gi蛋白抑制剂)和Bupranolol(非选择性β受体阻滞荆)完全阻断,同时可被L-NAME(一氧化氮合酶抑制剂)部分阻断,不受Badolol影响。RT-PCR方法测出心肌细胞中有β3受体mRNA表达。结论：心肌细胞中存在β3受体,它在心肌表现为负性变力作用,β3受体的效应不受β1,β2受体抑制剂影响。心脏β3受体信号途径中可能有Gi蛋白的参与,并且经过一氧化氮合酶途径发挥其作用。     

Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.     

Resonance Rayleigh-scattering method for the determination of proteins with gold nanoparticle probe

Gold nanoparticles with a 12-nm diameter were used as probes for the determination of proteins by resonance Rayleigh-scattering techniques. In weak acidic solution, large amounts of citrate anions will self-assemble on the surface of positively charged gold nanoparticles to form supermolecular compounds with negative charges. Below the isoelectric point, proteins with positive charges such as human serum albumin (HSA), bovine serum albumin (BSA), and ovalbumin (Ova) can bind gold nanoparticles to form larger volume products (the diameter of the binding product of gold nanoparticles with HSA is 23 nm.) through electrostatic force, hydrogen bonds, and hydrophobic effects, which can result in a red shift of the maximum absorption wavelength, the remarkable enhancement of the resonance Rayleigh-scattering intensity (RRS), and the appearance of the RRS spectra. At the same time, the second-order-scattering (SOS) and frequency-doubling-scattering (FDS) intensities are also enhanced. The binding products of gold nanoparticles with different proteins have similar spectral characteristics and the maximum wavelengths are located near 303 nm for RRS, 540 nm for SOS, and 390 for FDS, respectively. The scattering enhancement (DeltaI) is directly proportional to the concentration of proteins. Among them, the RRS method has the highest sensitivity and the detection limits are 0.38 ng/ml for HSA, 0.45 ng/ml for BSA, and 0.56 ng/ml for Ova, separately. The methods have good selectivity. A new RRS method for the determination of trace proteins using a gold nanoparticle probe has been developed. Because gold nanoparticle probes do not need to be modified chemically in advance, the method is very simple and fast.